CHITTENDEN, Ph.D., Professor of Physiological Chemistry.

ON THE PROTEOLYTIC ACTION OF BROMELIN,THE FERMENT OF PINEAPPLE JUICE. BY R. H.CHITTENDEN, Ph.D., Professor of Physiological Chemistry.

(From the Sheffield Biological Laboratory of Yale University.)

IN a previous paper' the writer has reported the results of a preliminarystudy of the proteolytic action of pineapple juice, giving some accountof the conditions, especially with reference to temperature and reaction,which modify the proteid-dissolving power of the fluid, and also severalmethods for the partial isolation of the ferment.

The more important results there recorded may be briefly sum-marized as follows:-Fresh pineapple juice is strongly proteolytic, andits proteid-digesting power may be manifested, on some proteids atleast, in a neutral acid, and even faintly alkaline reacting fluid, thusindicating that the ferment resembles trypsin rather than pepsin. Withcoagulated egg-albumin, the neutralized juice is most active, whilewith blood-fibrin, juice of the natural acidity is more active than theneutralized fluid.

The products of digestion are apparently the same as those formedby trypsin, viz.: proteoses and true peptone, together with leucin andtyrosin.

Neutralized pineapple juice exerts its maximuim digestive power atabout 600 C., but it is quite active at even 120 C. and 700 C.

Associated with the proteolytic ferment is a milk-curdling orrennet-like ferment, which can be separated from the fresh juice,together with the proteolytic ferment, by saturation of the fluid withammonium sulphate.

The proteolytic ferment appears to be associated with a peculiar

1 "On the Ferments contained in the juice of the pineapple (Ananassa sativa), togetherwith some observations on the composition and proteolytic action of the juice." Tranm.Conn. Acad. Vol. viii. Dec. 1891.

PH. XV. '17

R. H. CHITTENDEN.

proteose-like body, which is more or less completely separated frompineapple juice by saturation of the solution with sodium chloride,ammonium sulphate, and magnesium sulphate.

These statements indicate in a general way the character of theresults previously obtained where the fresh juice itself was moreespecially studied. In the present investigation the above preliminarywork has been extended and particular attention paid to the nature ofthe isolated ferment and its action on the three typical proteids,blood-fibrin, egg-albumin, and myosin, with a special study of theresultant products.

1. Nature of the Ferment.

(From experiments made by Theodore S. Hart, B.A.)

As is well known, the pineapple is an exceedingly juicy fruit, anaverage sized one frequently yielding nearly a litre of juice. The latter,when filtered, is a clear yellow fluid of strong acid reaction, the averageacidity expressed in terms of HCI being equivalent to 0-45 per cent.Neutralization of the acid fluid fails to produce any precipitatewhatever, thus showing the entire absence of acid-albumin. Aspreviously stated, fresh pineapple juice filtered clear and of averageacidity subjected to fractional heat-precipitation grows slightly turbidat about 60O620 C., the turbidity increasing gradually as the tempera-ture is raised until 75-780 C. is reached, when a small flockyprecipitate results. The filtrate from this separation, on being furtherheated shows signs of turbidity at about 820 C., increasing with tilerise in temperature, without, however, any distinct signs of flockinguntil the boiling point is reached. As the fluid commences to boil, butsometimes only after persistent boiling, a fine flocky precipitateseparates which on filtration leaves a perfectly clear fluid. Withneutralized pineapple juice the initial turbidity makes its appearanceat about 72-740 C., with separation of flocks at 82-830 C. Thefiltrate from this precipitate remains clear, even when the solutionis boiled, but a drop or two of acetic acid added to the hot fluidproduces a turbidity which on further heating eventually changes toa flocculent precipitate. Both of these heat-precipitates are more orless soluble in dilute acid (2l0 per cent. HCI) and in dilute alkali,especially with the aid of heat.

While these observations favour the view that pineapple juicecontains two distinct proteids precipitable by heat, the one at about

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PROTEOLYTIC ACTION OF BROMELIN.

750 C. in an acid soluition or 820 C. in a neutral fluid, the other at1000 C. in an acid fluid, it may be that these reactions are due to asingle proteid incompletely precipitated at temperatures below theboiling point. As previously stated, the total amount of proteid matterin pineapple juice is comparatively small, and apparently the greaterportion of it is separable from the acid juice by boiling. 100 c.c. offiltered juice of natural acidity yields on an average about 0-027 gramof proteid (dried at 1100 C.), or less than 0-025 per cent. by simplyboiling the solution.

(NH14)2S04 Precipitation:-As previously stated, saturation of pine-apple juice with amnmonium sulphate results in a complete precipitationof the contained proteids, together with the proteolytic and rennet-likeferments. The latter bodies are likewise completely precipitated, asis evident from the fact that the filtrate, after removal of theammonium salt by dialysis, fails to show any proteolytic action onblood-fibrin, or any curdling action on milk.

This ammonium sulphate precipitate, when thrown down from aneutral fluid, is readily soluble in water and after being washed with asaturated solution of ammonium sulphate, is best purified by dialyzingthe aqueous solution in running water. When the dialysis has beencontinued until the ammonium salt is entirely removed, the solutionusually becomes quite turbid; little if any precipitate can be filteredoff, but the fluid has more or less of a milky appearance from thepartial separation of the contained proteid. On simply warming thesolution, the turbidity disappears until at 700 C. the fluid again becomesturbid. This turbidity increases with the rise of temperature, but doesnot pass into a flocculent precipitate even on boiling, until a drop ofacetic acid is added. In these heat-precipitations, however, thereaction usually changes to alkaline, which fact doubtless interferessomewhat with a distinct separation. It is very evident, however, thatthe proteid or proteids separated by ammonium sulphate and purifiedby dialysis are very incompletely precipitated from an aqueous solutionby heat; in fact, the greater the degree of purification the less completeappears the heat-precipitation.

The turbidity of the above dialyzed solution is also cleared up byaddition of a little salt solution, likewise by the addition of 0-2 percent. hydrochloric acid and 0 5 per cent. sodium carbonate.

By evaporation at 400 C. of the filtered dialyzed solution of the am-monium sulphate precipitate, the ferment with its associated proteidscan be obtained as a dry flaky residue readily soluble in water. So

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B. H. CHITTENDEN.

prepared the ferment has a marked proteolytic action on blood-fibrinespecially in a slightly acid (HC1) fluid, and a milk-curdling action inneutral solution. Made into a concentrated solution with a little waterand then treated with a large excess of strong alcohol, the proteidmatter with the associated ferments is completely precipitated. Evenby three weeks contact, however, with strong alcohol, the proteids arenot coagulated; i.e. the precipitate is still soluble in water and thesolution shows proteolytic action; a fact which certainly favours theview that the ferment, or the proteid associated with the ferment, isa proteose-like body rather than a true globulin, or an ordinaryvegetable albumin.

NaCl Precipitation :-Neutralized pineapple juice saturated withsodium chloride yields a distinct flocculent precipitate of proteid mattercontaining the greater portion of the proteolytic ferment, together withthe milk-curdling ferment'. In preparing the ferment by this method,the NaCl precipitate is thoroughly washed with saturated salt-solution,then dissolved in a small amount of water, filtered from any insolubleresidue and dialyzed in running water until the chloride is entirelyremoved. At the end of the dialysis, the fluid is generally quite turbidand frequently contains more or less sediment. Heat alone, or theaddition of a little salt solution, partially removes this turbidity, but thefluid is completely cleared up only on the addition of 0-2 per cent.hydrochloric acid or 0-5 per cent. sodium carbonate. The addition ofa single drop of dilute acid may produce a precipitate, but this at oncedisappears on the addition of a drop or two more of acid.

The dialyzed solution gives good proteid reactions with Millon'sreagent, and with cupric sulphate and potassium hydroxide. Subjectedto fractional heat-precipitation, the turbid neutral fluid clears greatlyat about 400 C. with a reappearance of cloudiness at 700 C., but no flocksappear even when the solution is boiled. Boiling renders the solutionstrongly alkaline. Addition of a drop or two of acetic acid to the hotfluid and further boiling usually produces a light flocculent precipitate.

By cooling the dialyzed solution of the sodium chloride precipitateon ice the separation of insoluble matter can be somewhat increased.Under such conditions, the sediment (a), at the most very small inamount, can be removed by filtration, while the more soluble matter

1 When the salt used in saturating the pineapple juice contains much calcium, theremay be in addition to the above considerable calcium citrate, etc. in the precipitate.Again, with some samples of fruit there is considerable extraneous matter precipitatedwith the proteids on saturation of the neutralized juice with sodium chloride.

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remains in the clear filtrate (b). The slight precipitate (a) thusobtained is soluble in 5 per cent. salt solution, yielding a somewhatturbid fluid. The latter becomes quite turbid at 550 C. with formationof flocks at 700 0. The filtrate from this precipitate usuallyflocks again at 800 C. and also at 860 C., the solution becoming distinctlyalkaline on boiling. Dilute acetic, hydrochloric or nitric acid added tothe sodium chloride solution of the precipitate (a) produces a heavyturbidity, changing to flocks when the mixture is heated. The sodiumchloride solution of this proteid curdles milk and shows proteolyticaction, especially in neutral and faintly acid solutions. Subjectedagain to dialysis the proteid separates out, in part at least, and is stillsoluble in salt solution, dilute acid and alkali carbonate. Againseparated from salt solution by dialysis, it still retains the aboveproperties, excepting that it may no longer show proteolytic actioneither in neutral, acid or alkaline solutions, although it will still curdlemilk. Such heat-precipitation as can be obtained by boiling the saltsolution of the proteid is difficultly, if at all, soluble in dilute acid andalkali. When dissolved in salt solution, the proteid is precipitated, inpart at least, by nitric acid, but the precipitate is insoluble on heatingand in an excess of the acid. It is likewise precipitated by cupricsulphate and basic lead acetate.

The filtrate (b) containing the proteid or proteids more soluble inwater, i.e. the bulk of the sodium chloride precipitate, shows pronouncedproteolytic action on blood-fibrin in neutral solution, and likewisecurdles milk. The aqueous solution, diluted with an equal volume of10 per cent. solution of sodium chloride, thus making a fluid comparableto the 5 per cent. salt-solution of the precipitate (a), grows turbid at550 C., followed by a flocculent precipitate at 850 C. This precipitate,like the one in (a), is only slowly, if at all, soluble in dilute acid andalkali. The greater portion of the more soluble proteid matter isapparently not precipitable by heat, or else very incompletely so, for itseems quite probable that such heat-precipitation as is obtained is dueto the presence of the above globulin-like body, only a portion of whichhas been separated by dialysis. Addition of alcohol to the suitablyconcentrated solution of the more soluble proteids (b), precipitates allof the albuminous matter present together with the ferment. Thisprecipitate, even after two to three weeks of contact with strongalcohol, is still soluble in water and preserves its proteolytic power;hence, in the more soluble proteid we evidently have a true proteose-like body, rather than a globulin or albumin.

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It is thus seen that the precipitate of proteids produced by saturat-ing neutralized pineapple juice with sodium chloride may possibly be amixture of at least two distinct proteids, one of which is in part atleast insoluble in water, but readily dissolved by a little salt soluition,or by very dilute acid or alkali carbonate. This body is present onlyin very small amount and apparently when purified by repeateddialysis from all traces of the more soluble proteid is devoid ofproteolytic power, but usually shows more or less curdling action onmilk. This proteid is evidently a globulin, or possibly a form ofvegetable heteroproteose, only a portion of which is separable from theoriginal sodium chloride precipitate by the above method of treatment.The main bulk of the proteolytic ferment is evidently associated witha more soluble proteid, which from its reactions resembles proto-albumose. At the same time we have never been able to obtain thisbody entirely free from substances precipitable by heat. We mayassume quite plausibly a mixture of protoalbumose and heteroalbumose,with a small amount of true globulin as composing the original sodiumchloride precipitate. There is nothing antagonistic in the observedreactions to such an assumption, and it is well known that it isextremely difficult to completely separate a small amount of hetero-albumose from the related bodies, especially protoalbumose. If ourexperience in the separation of these substances from pineapple juicehad been limited to one or two trials, doubtless we should be far morepronounced in our statements. But frequent repetition, in an attemptto acquire more positive knowledge, has resulted in so many slightlydivergent results that we are loath to make a positive statementregarding the chemical nature of the proteid or proteids with whichthe proteolytic ferment is associated in the sodium chloride precipitate.We are indeed forced to the opinion that the proteids present in thejuice of the ripe fruit are somewhat variable in their nature, theirexact chemical character being dependent upon the age or ripeness ofthe fruit. At the same time, all physiologists who are at all familiarby practical experience with proteids are well aware that the ordinarylines of demarcation between the different groups of proteid bodies,especially in the vegetable kingdom, are in many cases very indistinct,and further that this indistinctness is rapidly growing inore pronouncedas our knowledge increases. For example, a typical globulin has longbeen considered as a body insoluble in water, but soluble in solutionsof sodium chloride, yet recently Osboriel has called attention to the

1 "(Crystallized vegetable proteids." Amer. Chem. Jour., Vol. xxv. p. 674.

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fact that the crystallized globulin obtainable in large quantity fromhemp-seed may be dissolved, when in a pure crystallized form, indistilled water to a considerable extent. Hence, on the basis of thisreaction alone, it might be asserted that the above hemp-seed globulinis a mixture of a water-soluble proteose or albumin with an insolubleglobulin, yet from other reactions we know that such is not the case.Again, it is questionable how far heat-precipitation at definite tempera-tures can be trusted as an indication of individuality, or taken as ameans of complete separation, especially with the vegetable proteids.Heat-precipitation is in many cases exceedingly slow and incomplete,and the same may be equally true of heat-coagulation. Thus, in thestudy of a crystallized vegetable globulin, at present being carried onin this laboratory, persistent boiling (30 minutes) of a 5 or 10 per cent.sodium chloride solution of the globtulin results in a little over 50 percent. of the body being precipitated as an insoluble coagulum. Bydialysis of the filtrate from this coagulum, the remainder of the globulinhowever, separates in the characteristic crystalline form apparently un-altered, and on being dissolved in salt solution and again subjected toheat-precipitation the solution of the proteid grows turbid at the regulartemperature, followed by a slow separation of another coagulum as thefluid is boiled. Consequently, on the basis of the results obtained byheat-coagulation in this case, one might well assume the above globulinto be a mixture of two distinct proteids, yet such is not the case if theresults of analysis and general reactions are to be trusted. The globulinis simply incompletely coagulable by heat. Such observations as these,however, necessarily make one somewhat cautious about relying tooimplicitly on the indications gained by a study of the temperatures atwhich heat-precipitation occurs.

The sodium chloride preparation of the ferment with the associatedproteids may be obtained as a dry, scaly residue by evaporating thedialyzed solution of the NaCl precipitate, at 400 C. By this evapora-tion of the neutral aqueous solution of the ferment, the reaction of thefluid is not altered, but whenever the temperature is raised sufficientlyhigh to lead to even incipient heat-precipitation the reaction of thefluid becomes distinctly alkaline, and at the same time the proteolyticpower of the ferment is destroyed. The dry residue obtained byevaporation at 400 C. is readily soluble in water and shows markedproteolytic action on blood-fibrin in neutral, acid and alkalinesolutions.

The amouint of ferment obtained by this method is not large.

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Thus, in one experiment, ten pineapples of moderate size were crushedand pressed, yielding 8600 c.c. of clear, filtered juice. This, afterneutralization, was saturated with sodium chloride, the precipitatewashed with saturated salt solution, dissolved in water and dialyzeduntil free from chloride. The filtered solution, on evaporation at400 C., left a residue weighing 2 34 grams; the yield of ferment from8-6 litres of fresh juice. It is somewhat suggestive that the amount offerment obtainable by this method is exactly equal to the amount ofproteid directly separable from pineapple juice by heat. Thus by heatalone, 1 litre of pineapple juice yielded 0-27 gram of dry proteid (driedat 1100 C.) while the same volume of juice precipitated by salt, etc.,likewise gave 0 27 gram of dry proteid (ferment) by evaporation of thedialyzed solution. In this connection it is to be noted that separationof the proteids from neutralized pineapple juice by heat is far morecomplete and characteristic than from aqueous solutions of the so-calledisolated ferment. As has been already stated, the purified precipitatesof the proteid or proteids associated with the ferment are veryincompletely, or only slightly precipitated by heat. This differencewe attribute, in part at least, to the modifying influence of thesalts and other substances present in the juice which are naturallyabsent to a greater or less extent, from solutions of the isolatedferment.

Saturation of neutralized pineapple juice with sodium chloridealone, either in neutral or acid solution, does not precipitate all of thecontained proteid matter. The filtrate, on addition of ammoniumsuilphate in substance, yields a second precipitate composed in greatpart of albuiminous matter. This is soluble in water, and on removal ofthe sulphate by dialysis usually yields a clear solution, which onevaporation at 40°C. gives a small, flaky residue. Occasionally, thesolution after dialysis shows a slight separation of proteid as seen in thesodium chloride preparation. When this is the case, the turbidity ispartially cleared up by heat and completely on addition of a little saltsolution, or by 0-2 per cent. hydrochloric acid and 0 5 per cent. sodiumcarbonate. This residue shows the presence of proteid matter by boththe biuiret and Millon's test, and when dissolved in water gives a slightprecipitation by heat, increased by the addition of a drop or two ofacetic acid. Treated with absolute alcohol, the proteids are precipitatedand retain their solubility in water even after three weeks of contact withthe strong alcohol. As a rule, this ammonium sulphate precipitate,when freed from the ammonium salt, shows some proteolytic action on

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blood-fibrin in a weak acid solution, but as a rule, never any curdlingaction on milk. From these reactions it is evident that such proteidsas are precipitated under the above conditions are simply traces of thesame proteids as are contained in the sodium chloride precipitate andwhich have escaped precipitation by the latter reagent. The amountof this secondary precipitate is exceedingly variable; in some instancesthe quantity is fairly large, in others quite small and wholly devoid ofproteolytic power. Taking the majority of the results, however, itwould seem that the great bulk of the proteolytic ferment with theassociated proteids is precipitated by sodium chloride alone, while themilk-curdlina ferment is almost always completely precipitated by thissalt. This statement assumes that the proteolytic and rennet-likeferments are two distinct bodies. This view, in the writer's opinion, isjustified by the various results obtained, although in the majority ofcases the two ferments are usually associated in the same precipitate.

MgSO4 Precipitation:-Neutralized pineapple juiice saturated withmagnesiunm sulphate yields a flocculent precipitate of proteid mattercontaining more or less of the proteolytic ferment. Dissolved in waterand dialyzed until the magnesium salt is entirely removed a fairlyclear solution is obtaiined, which on evaporation at 4000. leaves a flakyresidue of proteid matter. This shows proteolytic action, etc., and isapparently of much the same character as the ammoniuin sulphateprecipitate, although less in amount than the precipitate produced bythis salt. An aqueous solution of the dried residue (at 400C.) growsdistinctly turbid at 70W C., and in all other respects resembles theprecipitate produced by ammonium sulphate.

Any proteid matter not thrown down from the neutralized juice bymagnesium sulphate can be precipitated by addition of sodium sulphateto saturation, the sodio-magnesium sulphate thus formed precipitatingnearly all of the residual proteid matter. This precipitate has only aslight proteolytic action, but in its general chemical reactions does notdiffer markedly from the magnesium sulphate precipitate.

It is thus evident, as previously stated, that saturation of neutralizedpineapple juice with sodium chloride, magnesium sulphate, or ammo-nium sulphate results in a precipitation of the proteid matter presentin the fluid; with ammonium sulphate, the separation of the proteids iscomplete, with sodium chloride less so, while with magnesium sulphatea smaller portion only is precipitated. The sodium chloride precipitate,however, contains by far the largest proportion of ferment, or in otherwords, a smaller proportion of admixed, inactive proteid. This is

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shown by the following experiment which gives the relative proteolyticaction of the three precipitates, produced by saturation of neutralizedpineapple juice with the respective salts, after their purification bydialysis and evaporation of the resultant solutions to dryness at 40° C.

Proteolytic action was determined by ascertaining the amount ofcoagulated egg-albumin dissolved in a given time by a given weight ofthe ferment. Each digestive mixture contained 10 grams of the moistcoagulated egg-albumin, 0 05 gram of the ferment and 100 c.c. of waterthe mixtures being warmed at 40° C. for six hours. The 10 grams ofalbumin coagulum contained 2-184 grams of dry proteid (dried at1100C.).

Weight of undissolved AlbuminFerment albumin digested

NaCl preparation 1 5810 grams' 27-6 per cent.MgSO4 ,, 1-8761 ,, 14-1(NHAIISO4 ,, 19149 ,, 12-3

While these figures show well the relative proteolytic activity ofthe three preparations, they do not give an adequate idea of their trueproteolytic power. Coagulated egg-albumin is far more resistant to theaction of the ferment than either blood-fibrin, or the proteids of muscletissue. The two latter proteids are not only much more quicklyattacked by the ferment, but a far greater amount is dissolved andconverted into proteoses and peptone.

A sodium chloride preparation of the ferment, made as describedabove, is the strongest we have thus far been able to obtain. Appar-ently, however, all of these methods of separation produce somedeterioration of the ferment; i.e. the activity of the isolated ferment isnot equal to the proteolytic strength of the volume of pineapple juicefrom which the ferment is extracted. This loss of power we have notbeen able to locate; in fact, it may be more apparent than real, thegreater proteolytic strength of the natural juice being possibly due tothe beneficial or stimulating effect of the salts and other substancespresent in the fluid.

In view of the fact that pineapple juice contains proteids other thanthose with which the ferment is associated, it seemed possible that longcontinued warming of the fresh juice at 400 C. would result in, at least,a partial peptonization of the proteids, and consequently in a purer andstronger preparation of the ferment by either of the above methods of

I After drying at 1100C,

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precipitation. But, to our surprise, it was found that the naturalproteids of the juice are exceedingly resistant to the proteolytic actionof the ferment, so that even after several hours warming of the juice at400 C., heat-precipitation occurs at the regular temperatures and thereis no indication of an appreciable diminution of precipitable proteids byeither ammonium sulphate or sodium chloride.

We are well aware that none of the above preparations of theferment are to be considered as pure. In fact, they are undoubtedlyfar from being, so, but we have not been able, by any method ofprocedure as yet tried, to obtain any quantity of a preparation purer(judging from the relative proteolytic action) than the above describedsodium chloride preparation. We have therefore subjected this bodyto a partial analysis with a view to ascertaining something regarding itschemical composition.

For this purpose, a sufficient amount of the NaCl precipitate wasprepared and after purification by the method already described andevaporation of the resultant solution to dryness at 400 C., it was finallydried at 1100C. until of constant weight. Analysis by the usualmethods gave the following results:-

NaCl Preparation of Bromelin.

I. 03684 gram substance gave 01930 gram H20 =5-73 per cent. Hand 0-6208 gram CO,= 45 95 per cent. C.

11. 0-5571 gram substance gave 0X2889 gram HEO = 5 76 per cent. Hand 09370 gram CO= 45-86 per cent. a.

III. 0.3576 gram substance gave 28-0 c.c. N at 4.300. and 746-1 mm.pressure = 9 50 per cent. N.

IV. 0 4090 gram substance gave 317 c.c. N at 4.50 C. and 749-1 mm.pressure= 9 44 per cent. N.

V. 0-4523 gram substance gave 0-0429 gram ash = 9-48 per cent.

Percentage composition of the ash-free substance.Average

C 50-76 50-67 50-71H 6-33 6-36 - 6-34N - 10-49 10-43 10-46

} - 32-49

10000

The large percentage of ash in this preparation obviously detractssomewhat from the value of the above analysis, but it is plain from the

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results that the substance is especially characterized by a very lowcontent of nitrogen. The content of carbon is not far removed fromthat of many vegetable proteids, but the nitrogen is exceedingly lowand is strongly suggestive of the probable admixture of some non-nitrogenous body, if it is assumed that the ferment is proteid in itsnature, or is associated with a proteid substance. At the same time,pure trypsin as prepared by Kiihne contained only 12 5-13'4 per cent.of nitrogen, an amount much smaller than is usually found in a trueproteid body'. The ash in the above preparation was comiposed mainlyof calcium sulphate, with some calcium phosphate and a little ferricoxide. In view of the large percentage of ash, or mineral matter,associated with this preparation of the ferment, it becomes necessary toconsider its influence on the general reactions of the body. Thus, forexample, the apparent solubility of the ferment preparation in watermay be due to the adherent calcium salts, although obviously we cannotspeak with certainty on this point. However this may be, it is evidentthat the proteid body associated with the proteolvtic ferment holds oInto the above salts most tenaciously, for when once in contact with themno ordinary method of treatment, such as long-continuied dialysis orrepeated precipitation, is adequate to effect a complete or even partialseparation.

Taking all of the above observations at their apparent value, it isplain that the proteolytic ferment of pineapple juice is associated witha proteid body more or less completely precipitable from a neutralsolution by saturation with ammonium sulphate, sodium chloride andmagnesium sulphate. This body is soluble in water and consequentlyis not precipitated by dialysis. It is further not coagulated by longcontact with strong alcohol and its aqueous solution is very incomipletelyprecipitated by heat. It resembles somewhat Martin's B-phytalbumosein the juice of the papaw, but apparently is not as easily or ascompletely precipitated by heat as that body. Placing it in line withthe known forms of albuminonis bodies, it is not far removed fromprotoalbumose or heteroalbumose, differing, however, from the latter inthat it is soluble in water without the addition of sodium chloride. Atthe same time, in its behaviour towards nitric acid and heat and in someother ways it does not show the typical proteose reactions. In com-position, it is especially characterized by a low content of nitrogen.

Associated with this proteid in pineapple juice is a body moreI The Chemical Basis of the Animal Body, p. 55, 1893. By A. Sheridan Lea. Com-

pare also 0. Loew. Pfluger's Archiv fir Physiologie, Bd. xxvii. p. 204.

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PROTEOLYTIC A(TION OF BROMELIN.

closely related to true heteroproteose or to a genuine globulin; a bodyless soluble in water, especially when cold, requiring for its completesolution the presence of salt and consequently separating, in part atleast, on dialysis. This body is more completely precipitable by heatthan its neighbour and is apparently present in much smaller quantity.Its separation from the more soluble albumose is exceedingly incompleteand it is liable to show more or less proteolytic power. Possibly thislatter body is the particular proteid with which the rennet-like fermentis associated.

Proteolytic action of the ferment under varying conditions.As already stated, fresh pineapple juice is strongly proteolytic in a

neutral, acid and even alkaline reacting fluid. There are, however,many modifying circumstances that need to be considered in attemptinga demonstration of the above statement. Thus, the character of theproteid used is an important element in modifying the rate of proteolyticaction of the ferment. On blood-fibrin, either fresh or soaked for along time in alcohol, juice of the natural acidity acts more energeticallythan the neutralized fluid, while on coagulated egg-albumin and onthe proteids of muscle tissue the neutralized fluid shows the greatestproteolytic power. Blood-fibrin, which has been thoroughly hardenedby long boiling with water, is very resistant to the action of both acidand neutralized pineapple juice. Addition of dilute hydrochloric acid(02 per cent. HCI) in small amount to neutralized pineapple juice, orto juice of natural acidity, checks, but does not entirely inhibit proteo-lytic action. Further, sodium carbonate can be added to neutralizedpineapple juice in small amount (0O1 per cent.) without greatly checkingthe proteolytic power of the fluid on coagulated egg-albumin. Asmight be expected, however, the isolated ferment is far more sensitiveto acid and alkaline fluids than pineapple juice, since in the latter fluidthe large amount of salts and other substances present exert a protectiveinfluence which is wholly, or in great part, wanting in the case of thepurer ferment solutions. Further, the ferment itself, especially in itsaction on blood-fibrin, is directly influenced by the presence of neutralalkali salts, the latter contributing greatly to the proteolytic action ofthe ferment in a neutral fluid.

A. Action of the proteolytic ferment on blood-fibrin in the presenceof acids.

The blood-fibrin used in these experiments was a thoroughly washed

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preparation which had been soaked for a long time in strong alcohol.Comparative tests were made using like amounts of the isolated ferment(scale form) with equal weights of fibrin and equal volumes of fluidcontaining the designated percentages of acid. The mixtures werewarmed at 400C. for a stated time and the rate of digestive actionmeasured by simply observing the extent of disintegration and solutionof the fibrin, aided by application of the biuret test to the filtrates ofthe respective mixtures.

As a result of the many trials made it may be stated that, as arule, an aqueous solution of the NaCl preparation of the ferment hasonly a slight and in some cases no proteolytic action on blood-fibrin, ifthe solution be perfectly neutral and free from soluble alkali salts.The presence of very small amounts of hydrochloric acid, however,brings out at once the proteolytic power of the ferment, even 0-012 percent. HCI being sufficient for this purpose. The rate of digestiveaction increases with the further addition of hydrochloric acid up to0-025 per cent. Beyond this point it is not materially affected by thefurther addition of acid until the mixture contains 0 0o per cent. HCI,after which proteolytic action rapidly declines and in the presence of0-1 and 0-2 per cent.-HCI is practically nil.

With a MgSO4 or (NH4)2SO4 preparation of the ferment, wherethere is a somewhat larger proportion of inert proteid matter, maximumproteolytic action is reached in the presence of 0-05 per cent. HCI,beyond which point it quickly declines.

Towards organic acids, the isolated ferment is far less sensitive.Thus with acetic acid, both the NaCl and (NH,)2SO4 preparation ofthe ferment show little proteolytic action on blood-fibrin until 0-25 percent. of the acid is present. When this degree of acidity is reacheddigestive action is very marked and continues good even in the presenceof 10 per cent. of the acid.

With citric acid, proteolytic action in the case of the NaCl prepa-ration commences in the presence of 0-03 per cent. of the acid andremains strong in the presence of 0'12 per cent. Beyond this degree ofacidity, digestive action diminishes and the presence of 10 per cent.practically checks all proteolytic action. With the less pure (NH4)2SO4preparation, maximum digestive action is seen in the presence of0 5-P15 per cent. of citric acid.

With tartaric acid, slight proteolytic action is manifested in thepresence of 0 06 per cent. of the acid, but maximum digestive actionoccurs when the mixture contains from 0-15 to 10 per cent. of the acid.

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PR(TEOLYTIC ACTION OF BROMELIN.

With oxalic acid, strong digestive action in the case of the NaCipreparation is seen only in the presence of 012-025 per cent. of theacid. The presence of 1-0 per cent. of oxalic acid practically checks allpI.oteolytic action. With the (NH4)2SO preparation of the ferment,maximum digestive action occurs in the presence of 025-O05 per cent.of oxalic acid.

From these results it is seen that while bromelin in a neutralsolution, free from salts, has little or no digestive action on blood-fibrin,its proteolytic power is called forth by the presence of small amounts ofacid, the exact percentage of acidity required to bring out maximumproteolytic action being dependent upon the character of the acid.Thus with a strong mineral acid, as hydrochloric acid, digestive actionis most pronounced on blood-fibrin in the presence of 0-025-005 percent., while with the weaker organic acids, as tartaric acid, proteolyticaction is vigorous even in the presence of 1'0 per cent. All of theorganic acids tested afford a good medium for digestion, and differ froineach other only in the strength required for bringing out the highestactivity of the ferment.

B. Action of the proteolytic ferment on egg-albumin in the presenceof acids and alkali carbonate.

On coagulated egg-albumin, as already stated, the isolated fermentappears to be most active in a neutral fluid; a simple aqueous solutionof the ferment, free from salts, giving the best results with this form ofproteid matter. In the presence of small amounts of hydrochloric acid,(0012-0025 per cent. HCl), however, there may be more or lessproteolytic action, and, likewise, in the presence of small amounts ofsodium carbonate (0025 per cent. Na2CO3). Indeed, in several experi-ments, with different forms of the isolated ferment, proteolytic actionseemed more marked in the presence of traces of hydrochloric acid andof sodium carbonate than in a perfectly neutral solution, but themajority of our results show that a neutral solution of bromelin givesthe best proteolytic action on coagulated egg-albumin. Probably thecalcium salts which adhere so tenaciously to the ferment together witha possible lack of complete neutralization are the cause of these some-what variable results.

Organic acids, likewise inhibit the action of the ferment on coagu-lated egg-albumin. The ferment is active in the presence of varyingamounts of these acids, but not to the same extent as in a neutral

263

R. H. CHITTENDEN.

solution. Thus, in one experiment in the presence of 0-1 per cent.citric acid, only 12-5 per cent. of the coagulated albumin was dissolved.while the same amount of ferment dissolved in water alone, otherwiseunder exactly the same conditions, digested 30-9 per cent. of thecoagulated proteid.

With raw egg-albumin, somewhat different results were obtained.The albumin made use of in these experiments was prepared accordingto the method of Schiitz'; 300 c.c. of undiluted white of egg were freedfrom globulin by the addition of 42 c.c. of HCl Sp. Gr. 1-12, filteredafter standing some time and then carefully neutralized. The amountof albumin contained in a given volume of the so-prepared solution wasascertained by taking 10 c.c., diluting with water and heating thesolution until the albumin was completely separated. The coagtulumwas then collected on a weighed filter, washed with hot water andeventually dried at 1 10 C. until of constant weight.

With so-prepared fluid egg-albumin the following quanititativeresults were obtained: Each digestive mixture contained 10 c.c. of theprepared albumin solution, 0 05 gram of bromelin (NaCl preparation)and 90 c.c. of water, with an amount of acid or alkali carbonate to makethe indicated percentages. The mixtures were warmed at 400 C. for 16hours, a few drops of an alcoholic solution of thymol being added toeach solution. The 10 c.c. of albumin solution contained 0 8862 gramof albumin.

Reaction Undigested albumin2 Albumin digestedNeutral 0-7383 gram 16-6 per cent.0-012 per cenit. HCI 07330 ,, 17-30-025 ,, ,, 0-6920 ,, 21-90 100 ,, ,, 0-8309 ,, 6-20 050 ,, Na20C0 0-8783 ,, 0-8

From these results we draw the conclusion that bromelin acts beston liquid egg-albumin in the presence of very small amounits ofhydrochloric acid, although its digestive action is quite pronounced in aneutral solution.

Organic acids, however, do not have the same effect as hydrochloricacid. This is plainly shown by the following experiments: Eachdigestive mixture contained 15 c.c. of the prepared albumin solution,

1 Zeit8chriftfiir physiologische Chemie, Bd. ix. p. 581.2 Determined by heating the mixtures to boiling, carefully neutralizing and collecting

the coagulated albumin on weighed filter papers. The precipitates were then thoroughlywashed with hot water and finally dried at 1100C. until of constant weight.

264


0 05 gram of ferment and 90 c.c. of water with the necessary amounts ofacid to give the designated percentages. The mixtures were warmedat 400 C. for 20 hours. The 15 c.c. of albumin solution contained 1P0033grams of dry albumin.

Reaction Undigested albumin Albumin digested

Neutral 0-6471 gram 35 5 per cent.0-25 per cent. Tartaric acid 0-7687 ,, 23-3 ,,0 50 , ,,0-8361 ,, 16-6 ,0.10 ,, Oxalic acid 0-8123 ,, 190 ,,0.10 ,, Citric acid 0-8158 ,, 18-6 ,,0-20 ,, ,, 0-8419 ,, 1817

It is thus evident that of the organic acids tested, tartaric acid hasthe least inhibitory effect, but even in the presence of this acid proteo-lytic action does not approach that of the neutral solution.

Influence of neutral alkali-salts on the proteolytic action of theferment.

As previously stated, neutralized pineapple juice shows distinctproteolytic action on blood-fibrin, although the action is not as markedas in the presence of small amiounts of dilute acid. It was found,however, that a neutral solution of the isolated ferment frequentlydoes not exhibit any proteolytic action on blood-fibrin, or at the bestshows only slight and incomplete digestive power. In looking aboutfor a reasonable explanation of this apparent conffiction, it was foundthat the difference in digestive action between the neutralized juiceand a neutral solution of the ferment lies in the presence of neutralalkali salts in the one case and their absence in the other. Thus, anaqueouis solution of bromelin, showing practically no proteolytic actionon blood-fibrin, is quickly made active by the presence of a smallamount of sodium chloride. In the presence of this and other similaralkali salts the ferment quickly disintegrates and dissolves the proteid,forming proteoses and true peptone. As is well known, fresh blood-fibrin is somewhat soluble in dilute and stronger salt solutions, but thissolubility is something quite different from the solvent and digestiveaction of bromelin; a 10 per cent. solution of sodium chloride, forexample, will cause blood-fibrin to swell up and in time will dissolve afairly large proportion of the proteid matter, but this reaction in no-wise explains the ready disintegration and fairly rapid solution of fresh

PH. XV. 18

265

R. H. CHITTENDEN.

blood-fibrin in a neutral solution of bromelin containing, say 1 per cent.of sodium chloride.

Thus, fresh fibrin may be soaked for comparatively long periods oftime in various salt soluitions without any very appreciable change, butas soon as the ferment is added, disintegration of the proteid irnmedi-ately sets in. Even boiled fibrin is slowly disintegrated and dissolvedby the ferment in the presence of 10 per cent. sodium chloride. Thesame result, only more marked, is obtained when blood-fibrin which hasbeen soaked for a long time in alcohol is treated with a neutral solutionof bromelin in the presence of 1-3 per cent. of sodium chloride. Here,naturally, the proteids of the fibrin are sufficiently coagulated to resistthe ordinary solvent power of the neutral salt, but the latter aidsdecidedly the proteolytic power of the ferment, and the fibrin is slowlydisintegrated and dissolved.

The salts most active in promoting the digestion of blood-fibrin bya neutral solution of bromelin are sodium chloride, sodium sulphate andammonium sulphate, although ammonium chloride and magnesiumsulphate are, likewise, useful.

With sodium chloride, sodium sulphate and ammonium chloride, theactivity of the ferment is manifested in the presence of 1-10 per cent.of the salts. When present in larger amounts, the above salts retardthe proteolytic action of the ferment and in the presence of 20 per cent.of these salts the ferment is no more active than in a simple aqueoussolution. With ammonium sulphate and magnesium sulphate, the bestresults follow in the presence of 1-5 per cent. of the salts. Largeramounts check the action of the fermnent. Practically, to promote thedigestion of blood-fibrin by a neuitral solution of bromelin, it is best touse sodium chloride in quantities ranging from 1 to 3 per cent. Eventhen the rate of proteolytic action is not equal to that of a slightly acidsolution of the ferment.

The above salts have no noticeable effect on the digestion of blood-fibrin by an acid solution of bromelin, except when present in largeamounts, when they check the proteolytic action of the ferment.

On the digestion of coagulated egg-albumin by bromelin in a neutralsolution, the above salts are without any apparent effect, except tocheck digestion when present in too large quantity.

Influence of temperature on the proteolytic action of the ferment.

Experiments previously made with neutralized pineapple juiceindicated that the temperature best adapted for its solvent action on

266


coagulated egg-albumin lay between 50O-600 C., and that even at 7000.ferment action was very pronounced. With the isolated ferment, wefind that proteolytic action on all kinds of proteid matter, in bothneutral and acid solution, is most vigorous in the neighbourhood of6000.

The following series of experiments with a very weak preparation ofthe ferment shows results in conformity with this statement. Eachdigestive mixture contained 10 grams of coagulated egg-albumin 0-05gram of a somewhat inactive specimen of the ferment and 100 c.c. ofdistilled water. The mixtures were warmed at the respective tempera-tures for 5 hours, when the undissolved albumin was collected, washedand weighed. The 10 grams of coagulated albumin contained 2-1036grams of dry proteid.

Temperature Undissolved albumin Albumin digested400 C. 19394 grams 7 8 per cent.45 1P9004 ,, 9-6 ,,50 1P8636 ,, 114 ,,55 1*8522 ,, 119 ,,60 1P8391 ,, 12-5 ,,65 1P9044 ,, 9.4 ,,70 1-9293 ,, 8-3

Owing to the weakness of this specimen of the ferment, digestionwas not very pronounced at any of these temperatures, but it is to benoticed that the largest amount of albumin was dissolved at 600 C.,although even at 700 C. more proteid was digested than at 400 C.Evidently, a temperature approximating to 5 5-600 C. is best adapted forthe proteolytic action of the ferment.

Results of essentially the same order were obtained in the digestionof blood-fibrin, both in a slightly acid (HC1) solution of the ferment andin a neutral solution in the presence of sodium chloride.

2. Products formed in the Digestion of Blood-fibrin with Bromelin.

(From experiments made by Theodore C. Janeway, Ph.B.)

As already stated, bromelin digests blood-fibrin vigorously in thepresence of 0025 per cent. hydrochloric acid, and nearly as well ina neutral fluid provided sodiu-m chloride is present to the extent of 1 to5 per cent. The isolated ferment does not show much digestive actionon fibrin in the presence of sodium carbonate, neither does it digestboiled fibrin very rapidly. Under favourable circumstances the amount

18-2

267

2. H. CHITTENDEN.

of ferment required to digest a large amount of blood-fibrin is verysmall. Thus in one experiment, 300 grams of moist fibrin, pressed asdry as possible, were conmpletely dissolved by 0 25 gram of the ferment,in the presence of 2500 c.c. of 0-025 per cent. HCl, in 5 hours at 400C.

The digestive action of bromelin on blood-fibrin is marked by adistinct eating into and disintegration of the proteid, especially notice-able in a slightly acid solution, but likewise pronounced in a neutralfluid, thus showing a resemblance to the action of trypsin. Moreover,the products of digestive action, especially the final products, resemblethose formed by trypsin; leucin and tyrosin being particularly promi-nent at the end of a vigorous digestion, both in a neutral and acidfluid.

Acid4Bromelin Digestion.

The general characteristics of the products formed in an acid-bromelindigestion of blood-fibrin, together with the methods of separation, areindicated by the following statement outlining the results of a singledigestion experiment. 300 grams of moist, thoroughly pressed blood-fibrin, were warmed at 400 C. with 2500 c.c. of 0-025 per cent.hydrochloric acid and 0-25 gram bromelin (NaCl preparation) for 5hours. At the end of this time the fibrin was entirely dissolved save asmall flocculent residuie which was apparently composed of somealteration product. The acid fluid, after filtration from the above flockyresidue, was neutralized with dilute sodium carbonate, giving a smallneutralization precipitate. The neutralized fluid, on being boiled for ashort time, yielded quite a heavy precipitate or coagulum, which,however, was readily soluble in warm 0-2 per cent. hydrochloric acid andin warm 0 5 per cent. sodium carbonate, thus indicating that it was notan ordinary coagulated proteid. Freed from this heat-precipitate, theneutral fluid was concentrated to a small volume on the water-bath andprecipitated while hot with a large excess of warm 95 per cent. alcohol.From the alcoholic filtrate, leucin and tyrosin were separated in con-siderable quantity by evaporation of the alcoholic solution and afterpurification by recrystallization, etc., were thoroughly identified by theircrystalline form, and by their reactions with Piria's, Hoffmann's andScherer's tests.

The alcohol precipitate, composed of proteoses and peptone, was1 The fibrin, from sheep's blood, was prepared by thorough washing with water, then

soaked in alcohol for some weeks, and again washed in water until the alcohol was entirelyremoved.

268

PROTEOLYTIC ACTI(N OF BROMELIN.

extracted repeatedly with hot alcohol for the more complete removal ofleucin and tyrosin, after which it was dissolved in water and theproteoses and peptone separated by saturating the solution withammonium sulphate. This was accomplished by adding the ammoniumsalt in substance to the solution acidified with acetic acid, and keepingthe mixture at the boiling point for some time in order to bring aboutas complete a separation of the proteoses as possible. The gummyprecipitate of proteoses was then dissolved in water, the solutionacidified with acetic acid and again saturated while boiling hot withammonium sulphate, after which the precipitate was repeatedly washedwith a saturated solution of the ammonium salt. In this manner it washoped to remove from the proteoses the greater portion of the adherentpeptone.

The two ammonium sulphate-saturated-filtrates, containing the pep-tone, were then concentrated somewhat and the excess of ammonium saltallowed to crystallize out. The filtrate was next dialyzed in runningwater, putrefaction being prevented by the addition of thymol, until thesulphate was entirely removed. The solution, which had obviously lostconsiderable peptone by diffusion, was then concentrated to a syrup andthe peptone precipitated by strong alcohol.

The ammonium sulphate precipitate of proteoses, after beingthoroughly washed with a saturated solution of ammonium sulphate,was next dissolved in a small volume of water and the individualproteoses separated by the usual methods. The solution was madeperfectly neutral, filtered from any insoluble heteroproteose or othermatter, and then saturated with sodium chloride. This produced amore or less gummy precipitate of protoproteose with some little hetero-proteose. The precipitate was filtered off, washed thoroughly with asaturated solution of sodium chloride, and then dissolved in water anddialyzed until the chloride was completely removed. The solution wasnext filtered from any separation of heteroproteose, concentrated to asmall volume, and the protoproteose precipitated with alcolhol.

On adding a little acetic acid to the original salt-saturated filtratefrom the above precipitate of protoproteose a heavy guimmy precipitateresulted, presumably a mixture of proto aind deuteroproteose. This wasfiltered off, washed thoroughly with a saturated solution 'of sodiumchloride containing a trace of acetic acid, then dissolved in water, thesolution neutralized, and dialyzed in running water until the salt wasentirely removed. The neutral fluid was then concentrated to a smallvolume and the mixed proteoses precipitated with alcohol.

269

R. H. CHITTENDEN.

The filtrate from the above acetic acid precipitate obviously containednothing but deuteroproteose. In order to separate this advantageouslyfrom the salt-saturated solution, the latter was dialyzed until the greaterportion of the salt was removed, after which the deuteroproteose wasprecipitated by saturating the solution with ammonium sulphate. Thegummy precipitate resulting was washed with saturated ammoniumsulphate-solution, then dissolved in water, the fluid carefully neutralizedand dialyzed in running water until the sulphate was entirely removed.The fluid was next evaporated to a syrup and the deuteroproteoseprecipitated by alcohol.

From this description it is plain that in the digestion of blood-fibrinwith bromelin, in the presence of a trace of hydrochloric acid, bodiesresembling the ordinary products of proteolytic action are formed.Variations in the relative proportion of fibrin and ferment and thevolume of acid fluid, together with variations in the length of time themixtures are warmed at 400 C., naturally lead to corresponding variationsin the relative proportions of the several digestive products. Quitenoticeable, however, in the acid-bromelin digestion of fibrin is thepeculiar heat-precipitate of a heteroproteose-like body, produced onboiling the neutralized digestive fluid. In fact, in the above digestionof 300 grams of moist fibrin, nearly 14 grams of this purified productwere obtained. It is also to be noticed that when this heat-precipitateis so pronounced there is usually very little neutralization precipitate;and, on the other hand, when the neutralization precipitate is heavy, theheat-precipitation is slight. Evidently, there is a close relationshipbetween these two bodies. Further, in some cases the peculiar characterof the neutralization precipitate is to be noted, as it may consist almostentirely of a heteroproteose-like body instead of acid-albumin. In otherwords, heteroproteose or a closely related body is frequently present insuch amount as to separate in considerable quantity at this point whenthe acid fluid is neutralized, eventually taking on a gummy form anddissolving not only in dilute acid and alkali carbonate, but also in saltsolution (10 per cent.).

In a second digestion experiment of this same order, 230 grams ofmoist fibrin which had been soaked for a long time in alcohol werewarmed at 400C. for 17 hours with 0 3 gram of bromelin and 2-5 litresof 0-025 per cent. HlI. Here, obviously the yield of the secondaryproteose and true peptone was considerably greater than in the precedingdigestion, but the same products were observed, only differing in theirproportion. As products, then, of the digestive action of bromelin on

270


blood-fibrin in a slightly acid fluid, we have: (1) an insoluble antial-bumid-like residue; (2) a neutralization precipitate; (3) a peculiarheat-precipitate of a heteroproteose-like body; (4) protoproteose;(5) deuteroproteose; (6) peptone; (7) leucin and tyrosin.

We have already outlined the general method pursued in theseparation of these bodies, and we will now consider more in detail themethods pursued together with the reactions and composition of theisolated substances.

A. Insoluble residue.

As already stated, in the digestion of blood-fibrin with a slightlyacid solution of bromelin there appears a flocculent residue insolublein 0025 per cent. HCI. This may be filtered off and on re-digestion,with a fresh solution of bromelin in 0 025 per cent. HCI, is not materiallydiminished in amount, provided, of course, the previous digestion hasbeen sufficiently vigorous to convert all of the digestible proteid intosoluble products. Otherwise, any adherent, unaltered fibrin or globulinswill be digested by this second treatment with fresh ferment, and thusapparently diminish the amount of non-digestible residue. Undersuitable conditions, then, this undissolved residue is incapable of beingconverted into soluble products by the further action of the ferment inan acid solution. On being filtered off and washed with water, it isfound soluble in 05 per cent. sodium carbonate, yielding on filtration asomewhat turbid fluid, from which the substance can be reprecipitatedby addition of dilute hydrochloric acid in slight excess. Treated againwith 05 per cent. sodium carbonate it passes into solution and remainsdissolved on exact neutralization of the fluid with weak hydrochloric acid.By dialysis of this solution until all traces of chloride are removed theabove proteid is separated into two portions; one soluble in water andthe other insoluble in the neutral fluid free from salt.

On washing the precipitated portion with alcohol and ether it maybe obtained as a grayish-white powder. So prepared it is insoluble inwater and salt solutions, but dissolves readily in 05 per cent. sodiumcarbonate, especially on warming the fluid. In 02 per cent. hydro-chloric acid it is only slightly soluble. Dissolved in an alkaline solutionit is reprecipitated by neutralization, but is not entirely, if at all,dissolved by an excess of the acid; evidently, then, it is not transformedinto alkali-albuminate when dissolved in the alkaline fluid. Suspendedin water and the fluid heated to boiling the body retains its solubility in0.5 per cent. sodium carbonate, hence it cannot be a globulin. It

271

R. IH. CHITTEArDEN.

shows the ordinary proteid reaction with Millon's reagent and givesa violet colour with potassium hydroxide and dilute solution of cupricsulphate.

From an alkaline solution it is precipitated by addition of nitric acid,but is not redissolved by an excess of the acid, and is only slightlysoluble on heating the acid fluid.

From these reactions it would appear that this portion of the abovenon-digestible resi(lue is an antialbumid-like body, corresponding to thebody formed by pepsin-hydrochloric acid under similar circumstances.

Dried at 1100C. until of constant weight it shows on analysis thefollowing results:

Anticdbumid-like body.I. 0 4000 gram substance gave 0-2492 gram H,O = 6-92 per cent. H

and 0-7478 gram CO,= 50 98 per cent. C.II. 0-3582 gram substance gave 0-2208 gram HO= 6-85 per cent. H

and 0-6717 gram CO,= 51-13 per cent. C.III. 0-3920 gram substance gave 518 c.c. N at 22.00 C. and 773 9 mm.

pressure= 16-01 per cent. N.IV. 0 3990 gram substance gave 0 0079 gram ash = 198 per cent.

Percentage compo8ition of the a8h-free mubstance.Average

C 52-00 52 16 - 52-08H 6-84 6 99 6-91N 1 6X33 16X33

S} - - 24-68

100-00

By evaporation of the neutral dialyzed solution, containing thesoluble portion of the original non-digestible residue, to a thick syrupand precipitation with alcohol the substance is separated in a somewhatgummy form readily convertible into a powder by successive treatmentwith alcohol and ether. So prepared it is soluble in water, and givesthe ordinary proteid reactions with Millon's and the Xanthoproteictest. With potassium hydroxide and dilute cupric sulphate it gives aviolet colour. From an aqueous solution it is not precipitated by nitricacid until a little salt solution is added, but with acetic acid andpotassium ferrocyanide it yields a light flocculent precipitate. It isevidently a proteose-like body, presumably precipitated with the anti-albumid owing to the formation of an acid compound, aided doubtless

272


by the salts present in the fibrin. In any event, it is quite different inits nature from the associated antialbumid and is present only in smallamount.

Dried at 1100C. until of constant weight it gave on analysis thefollowing results, which point to a relationship in composition with theneutralization precipitate about to be described.

Portion of thte non-digestible residue soluble in water.

I. 0-3701 gram substance gave 02123 gram H20 = 6 37 per cent. Hand 0-6689 gram CO, = 49-28 per cent. C.

II. 0-3241 gram substance gave 43-6 c.c. N at 21.60 C. and 760 0 mm.pressure = 16 02 per cent. N.

III. 0-4044 gram substance gave 0-0131 gram ash = 324 per cent.


C 50 93 50*93H 6-58 - 6-58N - 16-55 16 55

° - - 25-94

100*00

Comparison of the composition of these two bodies with that ofblood-fibrin shows that the above described soluble proteid differs quitewidely from the other two, especially in its content of carbon. Theantialbumid-like body, however, differs only slightly from the oliginalfibrin in composition, and fails to show the higher percentage of carbonusually found in an antialbumid.

Fibrin according Antialbumid-liketo Hammarsten body Soluble body

C 52-68 52-08 50*93H 6 83 6-91 6-58N 16-91 16-33 16-55S 1 10} 24-68 25-94O 22.48

It is thus evident that the insoluble matter found at the end of avigorous and complete digestion of blood-fibrin with an acid solutionof bromelin is composed of an antialbumid-like body, with which maybe associated a more soluble proteose, presumably thrown down as aninsoluble acid compound which from the presence of possible adherentsalts, or from some other cause, resists the further digestive action of theferment.

273

R. H. CHITTENDEN.

B. Neutralization precipitate.The amount and character of the so-called neutralization precipitate

formed in the acid fluid resulting from the digestion of blood-fibrinwith bromelin in the presence of 0-025 per cent. HCI, is dependentupon the extent and thoroughness of the digestion. Thus, in twodistinct experiments, where the digestions were very vigorous, theneutralization precipitates obtained on cautious addition of dilute sodiumcarbonate quickly took on a gummiy character, and after standing forsome hours settled ouit on the bottom of the beakers as a thin layer ofgum, having all the appearance of heteroproteose as it appears on thewalls of a parchment dialyzer tube after the salt has been removed bydialysis. This separation of a gummy neutralization precipitate occurseven when the solution is quite dilute and is not due apparently to theconcentration of the fluid. The gummy character of the precipitatestill persists, even after purification by solution in dilute sodiumcarbonate and reprecipitation by neutralization. So treated, the preci-pitate is exceedingly sensitive to the least excess of eitber acid or alkali,quickly dissolving when the neutral point is passed. This neutralizationprecipitate is likewise completely soluble in 10 per cent. solution ofsodium chloride, thus clearly showing that it is not a precipitate ofacid-albumin. Further, on dialyzing the salt solution, the proteid again-appears as a gummy precipitate resembling heteroproteose, as alreadystated. Dissolved in salt solution it is readily precipitated by even 0-2per cent. hydrochloric acid. It is likewise precipitated by nitric acid andthe precipitate so formed is almost entirely soluble in excess of the acid,especially on warming, and on cooling the solution the precipitatereappears, thus testifying to its proteose character. Cupric sulphateproduces a heavy precipitate, while potassium hydroxide with dilutecupric sulphate gives a violet colour. This body was not obtained, inthe form of a neutralization precipitate, in sufficient amount for analysis,but it is evident from its reactions that it is a form of heteroproteose,precipitated at this point because of insufficient sodium chloride to hoidit in solution after removal of the dilute hydrochloric acid.

More generally, the above neutralization precipitate shows thereactions of a dysproteose, i.e. a coagulated heteroproteose. Thus, in onedigestion of blood-fibrin with an acid solution (0-025 per cent. HCI) ofbromelin the bulky neutralization precipitate, after thorough washingwith water, alcohol and ether appeared as a white powder soluble onwarming in 0 5 per cent. sodium carbonate and less readily in 0 2 percent. hydrochloric acid. From these solutions it was reprecipitated by

274


neutralization, but on boiling the neutral fluid with the suspendedprecipitate for some time the latter remained soluble in 0 5 per cent.sodium carbonate, thus showing that it was not composed of ordinaryacid-albumin or globulin, coag,ulable by heat. Unlike the precedingneutralization precipitate, however, it was almost wholly insoluble in a10 per cent. solution of sodium chloride, and hence could not consist ofunaltered heteroproteose. Dissolved in 0 5 per cent. sodium carbonateit yielded a heavy precipitate with nitric acid which dissolved somewhaton the application of heat, reappearing as the fluid cooled.

Evidently then, in the digestion of blood-fibrin with an acid solutionof bromelin, heteroproteose is a characteristic primary product; but, afterprecipitation by neutralization of the slightly acid digestive fluid, it isprone to change into a more insoluble dysproteose-like body in whichform it is generally found. In this state it differs from an ordinaryneutralization precipitate in that it is not converted into coagulatedproteid by being heated at 1000 C. in a neutral fluid, as is shown byretention of its solubility in dilute sodium carbonate.

Occasionally, however, there may be a greater or less admixtuire ofacid-albumin or globulin present, as indicated by the lack of completesolubility in dilute alkali carbonate after subjection to the action ofboiling water.

Two distinct preparations of this neutralization precipitate, dried at11000. until of constant weight, were analyzed with the following results:

Neutralization Precipitate, 1.I. 0-4103 gram substance gave 0-2605 gram H20 = 7 05 per cent. H

and 0-7766 gram CO. = 51-37 per cent. C.II. 04134 gram substance gave 0-2550 gram HO = 6-85 per cent. H

and 07755 gram CO, = 51-16 per cent. C.III. 0-3568 gram substance gave 48-7 c.c. N at 19.40 C. and 7617 mm.

pressure = 16-27 per cent. N.IV. 0-4213 gram stubstance gave 57-4 c.c. N at 19.60 C. and 764-3 mm.

pressure = 16-26 per cent. N.V. 05442 gram substance gave 0-0015 gram ash = 0-27 per cent.


C 51-50 51-30 51-40H 7'06 6-86 6'96N 16-30 16-29 16-29S} - 25035

100-00

27a

B. H. CHITTENDEN.

Neutralization Precipitate, 2.

I. 0 3005 gram substance gave 01813 gram H,O = 6-70 per cent. Hand 0-5689 gram CO= 51-62 per cent. C.

II. 02725 gram substance gave 01625 gram H20 = 6'63 per cent. Hand 0-5112 gram CO. = 51U15 per cent. C.

III. 0 4797 gram suibstance gave 64-55 c.c. N at 19.2500. and 759-7 mm.pressure = 16-13 per cent. N.

IV. 0-5385 gram substance gave 72-36 c.c. N at 22.50C. and 767-0 mm.pressure = 16-21 per cent. N.

V. 0f6324 gram substance gave 0 0037 gram ash = 0-58 per cent.


C 51-92 51 45 51'68H 6-73 6-67 6-70N - 16-22 16-30 16-26

s} - 25'36

100*00From these two analyses, which show fairly close agreement, it is

seen that the above neutralization precipitate contains a somewhatlower percentage of carbon than the original blood-fibrin. The nitrogen,too, is correspondingly lower.

C. Heat-precipitate.As stated in a preceding paragraph, the neutralized digestive fluid

freed from both the non-digestible residue of antialbumid and from theneutralization precipitate, gives on heating a heavy precipitate orcoagulum. When first observed, this heat-precipitate was thought tobe a separation of coagulated globulin such as is frequently seen in theinitial stage of digestion when fresh unboiled fibrin is treated with anactive trypsin solution, or when raw fibrin is warmed with a weakpepsin-acid mixture. But it is to be remembered that the blood-fibrinused in the above bromelin digestions had been soaked for some time inalcohol, presumably sufficiently long to bring about a more or lesscomplete coagulation of the proteid. However this may be, the aboveheat-precipitate is not composed of an ordinary coagulated globulin,since it is completely soluble in both 02 per cent. hydrochloric acid andin 0 5 per cent. sodium carbonate, especially when heated.

Dissolved in dilute acid or alkali carbonate it is reprecipitated by

276


neutralization, and is then exceedingly sensitive to the least excess ofeither acid or alkali, quickly dissolving as the fluid passes beyond theneutral point. Treated with nitric acid, a solution of this substancegives a heavy precipitate which is somewhat soluble on heating andreappears as the fluid cools. With cupric sulphate and potassiumhydroxide it gives a reddish violet colour.

The amount of this peculiar suibstance is exceedingly variable, beingapparently dependent upon the extent of the preceding neutralizationprecipitate. If the latter is large in amount, the heat-precipitate isusually small in quantity. On the other hand, if the neutralizationprecipitate is small in amount then the heat-precipitate is more volu-minous. In other words, it is our opinion that the bulk of theneutralization precipitate and the above heat-precipitate are essentiallythe same. Bromelin, contrasted with the animal proteolytic ferments,is comparatively weak, and in its initial action gives rise to a largeamount of a primary proteose not far removed from the mother proteid.This proteose is a body closely related to, if not identical with, theheteroproteose so frequenitly studied in previous digestion experimentswith ferments of animal origin. Being present in large amount inthese bromelin digestions it is partially precipitated by neutralization,and is thus detected either as heteroproteose or as the more insolubledysproteose in the neutralization precipitate. The amount throwndown by so-called neutralization will depend in part upon the exactnessof the neutralization and in part upon the amount of salt present in thefluid, which in turn is dependent upon the amount of acid to beneutralized. Under any circumstances, precipitation of this hetero-proteose by neutralization is not complete; consequently when theneutralized fluid is heated to boiling more or less of the remainingsubstance is precipitated as coagulated heteroproteose, easily distin-guished from coagulated globulin by its ready solubility in bothdilute acid and alkali carbonate. Obviously, there is not a great deal ofdifference between such a heteroproteose as is here described and theglobulin resulting from a weak trypsin digestion of raw fibrin. In ouropinion, however, the difference in solubility of the above heat-pre-cipitate in dilute acid and alkali is sufficient to indicate a slight change,preliminary to a further transformation into more pronounced proteoses.In fact, the body might be described as a heteroproteose-like globulin,or as a globulin-like heteroproteose.

A sample of this heat-precipitate washed thoroughly with hot water,alcohol and ether and finally dried at 11O C. until of constant weight

277

R. H. CHIITTENDEN.

gave on analysis the following results, which indicate a composition notfar removed from that of the mother proteid.

Heat-precipitate, after neutralization.I. 0 4349 gram substance gave 0-2730 gram HI20 = 6'97 per cent. H

and 0-8318 gram CO = 52-15 per cent. C.II. 0-3551 gram substance gave 0-2181 gram H20= 6-82 per cent. H

and 0-6770 gram CO = 51V99 per cent. C.III. 05408 gram substance gave 75-3 c.c. N at 22.30 0. and 762 0 mm.

pressure = 16 62 per cent. N.IV. 0-6009 gram substance gave 83-6 c.c. N at 23.30 0. and 763-1 mm.

pressure - 16-63 per cent. N.V. 05165 gram substance gave 0 0010 gram 4sh=0 19 per cent.

VI. 0-6817 gram substance gave 0-0012 gram ash = 017 per cent.

Percentage compositton of the ash-free 8ubstance.Average

C 52-24 52-08 - 52 16H 6-99 6'83 - 6-91N - 16 65 16-66 16'65O - - 24-28

100X00

D. Soluble proteoses and peptone.On evaporatinog the filtrate from the above described neutralization

and heat-precipitates, a syrupy fluid remains which, as already stated,contains soluble proteoses and peptone together with leucin and tyrosin.The essential steps in the process of separating these bodies havealready been outlined, but they may be again stated here more indetail in order to avoid any misunderstanding as to the inethods used.The syrupy residue while still warm was treated with a large excess ofhot 95 per cent. alcohol, whereby the proteoses and peptone wereprecipitated. The alcoholic solution containing the leucin and tyrosinwith some peptone was evaporated to the point of crystallization, andthe leucin and tyrosin separating were purified by repeated crystalliza-tions from alcohol and water. The alcohol precipitate of proteoses andpeptone was dissolved in a small amount of water, concentrated to athick syrup and again precipitated with hot alcohol. The more or lessgummy precipitate wvas then repeatedly boiled with fresh 95 per cent.alcohol for the more complete removal of any adherent leucin andtyrosin.

278


The peptone and proteoses were next separated by the ammoniumsulphate method; i.e. the alcohol precipitate was dissolved in water, thefluid acidified slightly with acetic acid and saturated while hot withammonium sulphate, the mixture being kept at the boiling point forsome time to effect as complete a separation as possible. The resultantgummy precipitate of proteoses was then redissolved in water and againseparated by treatment with ammonium sulphate, and the precipitatewashed repeatedly with hot saturated ammonium sulphate solution.The filtrates from the above proteose precipitates, containing all of thepeptone present, were treated according to the method to be describedlater, under the head of peptone.

For the separationi of the individual proteoses, the gummy pre-cipitate produced by saturation with ammonium sulphate was dissolvedin a small amount of water, the solution made exactly neutrall to testpapers by addition of a little sodium carbonate, and the clear neutralfluid saturated with sodium chloride. There resulted a heavy flocculentprecipitate composed mostly of protoproteose with some little hetero-proteose. This was filtered off and washed thoroughly with a saturatedsolution of salt.

E. Protoproteose.

The above sodium chloride precipitate was dissolved in water, thendialyzed' in running water until the solution was entirely free fromchloride. When this was accomplished there was usually a slightprecipitate of gummy heteroproteose adhering to the walls of theparchment dialyzer tube. This was removed by filtration and theneutral, salt-free solution concentrated to a syrup and precipitated withhot 95 per cent. alcohol. The protoproteose thus precipitated wasboiled repeatedly with dilute alcohol (80-90 per cent.), then treatedwith absolute alcohol and ether. So prepared, it is a white powderreadily soluble in water, yielding a slightly turbid fluid; the turbiditybeing doubtless due to a trace of adherent heteroproteose which, as iswell known, almost invariably resists complete removal from the relatedproteoses.

1 On neutralization of the acid fluid at this point, a precipitate may form whichappears to be heteroproteose, or its alteration product dysproteose. The appearance ofthis precipitate is apparently dependent upon the completeness of the previous separationof the body, either by neutralization of the acid digestive mixture or by the heating of theneutralized fluid. This point will be further discussed under the head of heteroproteose.

2 In dialyzing these solutions, putrefaction was prevented by the occasional addition ofan alcoholic solution of thymol.

279

R. H. CHITTEATDEN.

An aqueous solution of this protoproteose gives the following re-actions, all of which testify to its proteose character.

A drop or two of concentrated nitric acid produces a white pre-cipitate, which dissolves on warming and reappears on cooling the fluid.An excess of concentrated nitric acid dissolves the first formed precipi-tate to a clear yellow solution. Addition of 20 per cent. salt solutionfollowed by a drop or two of dilute nitric acid gives a heavy white pre-cipitate, readily dissolved by heat and reappearing as the solution cools.

Cupric sulphate produces a heavy precipitate insoluble in excessand only slightly soluble on application of heat.

Mercuric chloride gives a precipitate insoluble in excess, and practi-cally insoluble on heating the mixture.

Neutral and basic lead acetate both produce heavy precipitates,soluble on application of heat and reappearing on cooling, and sligbtlysoluble in excess of the lead salts in the cold.

Acetic aci(d alone gives no precipitate, but potassium ferrocyanideadded to the acid fluid produces a flocculent precipitate soluble onheating and reappearing as the solution cools.

Tannic acid gives a heavy white precipitate somewhat soluble onheating, the soluble matter again appearing as the fluiid cools.

Picric acid likewise yields a precipitate, almost completely solubleon heating and reappearing as the mixture cools.

Phospho-tungstic acid gives a heavy white precipitate somewhatsoluble on the application of heat.

Platinic chloride added in excess yields a yellowish coloured pre-cipitate.

Potassio-mercuric iodide gives a precipitate somewhat soluble onapplication of heat.

Phospho-molybdic acid yields a precipitate partially soluble onheating and reappearing as the fluid cools. The precipitate is insolublein an excess of the precipitant in the cold.

With potassium hydroxide and cupric sulphate, a reddish violetcolour is obtained, while with Millon's reagent and heat the ordinaryred precipitate results.

Boiling with potassium hydroxide and lead acetate gives only aslight darkening of the fluid for sulphur.

As is characteristic of protoproteoses in general, an aqueous solutionof the substance is only partially reprecipitated by saturation withsodium chloride; addition of a few drops of acetic acid, however, to thesalt-saturated solution throws down the remainder of the body.

-280


From these reactions it is-plain that protoproteose as formed in thebromelin digestion of blood-fibrin is essentially akin to the ordinaryform of this substance as found in pepsin-acid digestions of the sameproteid, the only noticeable difference in reactions being that the pro-teose formed by pepsin yields precipitates with the metallic salts moresoluble in excess of the precipitants.

Dried at 1100C. until of constant weight, the substance gave onanalysis the following results, which coincide with what is usuially foundin the case of primary proteoses, viz.: a somewhat lower content ofcarbon than is present in the mother proteid.

Protoproteose, NaCI precipitate.I. 0'3981 gram substance gave 0-2348 gram H20 = 6-55 per cent. H

and 07419 gram CO2= 50-82 per cent. C.II. 0-3294 gram substance gave 0-1988 gram H2O= 6-70 per cent. H

and 0-6149 gram CO,,= 50 90 per cent. C.1II. 03576 gram substance gave 49 5 c.c. N at 19-5 (C. and 762-9 mm.

pressure = 16A49 per cent. N.IV. 0-3512 gram substance gave 49-1 c.c. N. at 19.00C. and 760-5 mm.

pressure = 16-59 per cent. N.V. 0'5496 gram substance gave 0 0110 gram ash = 2-00 per cent.


C 51-85 51P93 51-89H- 6-68 6-83 6-75N - - 16-82 16-92 16-87°} - -- 24-49

100*00

F. Heteroproteose and Dysproteose.

As is well known, heteroproteose is a well characterized primaryproduct of digestion, insoluble in pure water, but readily soluble in saltsolutions and exceedingly prone to change into an insoluble modificationknown as dysproteose. As already stated, in a weak bromelin digestionthis body is apparently formed in considerable quantity, and owing tothe above peculiarities is exceedingly liable to appear at various placesin the examination of a solution containing the products of bromelindigestion. Thus, as already pointed out, the neutralization precipitateand the above described heat-precipitate are both composed, in part at

281

19PH. XV.

B. H. CHITTENDEN.

least, of this proteose and its alteration product. We have also found,as might be expected, that a certain amount of this body is, likewise,liable to be present in the precipitate of proteoses produced by saturationof the neutralized and concentrated digestive mixture with ammoniumsulphate. To be sure, the amount present in the above precipitate isdependent in great part upon the completeness of the preceding separa-tions, but from the very nature of the conditions it is plain that somriemust escape precipitation by both neutralization and boiling of theneutral fluid.

When the ammonium sulphate precipitate of proteoses is dissolvedin water there is sufficient adherent acetic acid present to render thesolution distinctly acid and the entire precipitate usually dissolvescompletely. But when the fluid is neutralized with sodium carbonatepreparatory to precipitation of the primary proteoses by saturation withsodium chloride, a precipitate usually forms readily soluble in eitherexcess of acid or alkali. This consists of a portion of the heteroproteoseapparently altered either into dysproteose or into a related body. Theprecipitate is evidently not acid-albumin, since the body composing ithas already been exposed to neutralization and to a long-continuedevaporation at 1000 C. in a neutral fluid. Further, when now heated forsome time, suspended in water, it is not transformed into coagulatedproteid, since it preserves its solubility in warm dilute acid and alkali.It cannot consist, however, of unaltered heteroproteose, as it is almostentirely insoluble in salt solution. Evidently, it is a transformationproduct of heteroproteose, presumably so changed by long exposure tothe action of boiling saturated ammonium sulphate-solution.

Precipitated in this manner by neutralization of the slightly acidsolution of the mixed proteoses, it may be obtained after thoroughwashing with water, alcohol and ether as a perfectly dry powder,insoluble in water and sodium chloride solutions, but readily dissolvedby dilute acid and alkali carbonate. Dissolved in either of these fluidsit yields a heavy precipitate with strong nitric acid, which consistentlywith its proteose nature redissolves completely as the mixture is warmedand reappears as the solution cools.

It would thus appear that heteroproteose as formed in an acid-bromelin digestion of blood-fibrin is especially characterized by the easewith which it undergoes transformation into a body insoluble in sodiumchloride solutions. How great a transformation this involves of coursecannot be stated; presumably, however, the change resembles thatwhich syntonin and myosin undergo on long contact with water for

282


examnple, or the gradual change in solubility towards dilute sodiumchloride which vegetable myosin shows on long contact with saturatedsalt solution.

Dried at 1100C. until of constant weight it shows on analysis thefollowing composition:

Insoluble Heteroproteose.

I. 0-2791 gram substance gave 0-1712 gram H1O = 6-81 per cent. Hand 0-5179 gram CO,= 50-60 per cent. C.

II. 0-3105 gram substance gave 0-1878 gram H,O=6672 per cent. Hand 0 5737 gram COs,= 50-41 per cent. C.

III. 0-3362 gram substance gave 46-4 c.c. N at 23.50 C. and 771-0 mm.pressure= 16-65 per cent. N.

IV. 0 3101 gram substance gave 0 0028 gram ash = 0'90 per cent.

Percentage composition of the ash-free substance.

AverageC 5106 5086 50'96H 6-87 6-78 6-82N - - 1680 16-80

S} - 25-42

100-00

The analysis shows this body to contain a considerably lowerpercentage of carbon than the original fibrin, and even lower than theoriginal neutralization precipitate. From the methods of separationand the attendant results it is evident that the body just described isfar more probably an alteration product of a puire heteroproteose thanthe previously described neutralization and heat-precipitates. At thesame time, such reactions as can be made use of serve to show, asalready recorded, that both of these latter preparations are certainly notordinary neutralization precipitates or heat coagula, but approach farmore closely to our ordinary conception of a hetero body. Betweenheteroproteose and an ordinary globulin there are not many sharp pointsof distinction, but such as can be applied plainly indicate the hetero ordysproteose character of all three of these preparations. Possibly theyrepresent successive or different stages in the transformation of theglobulins of blood-fibrin into hetero or protoproteose. Certainly theremust be some good reason for the noticeable variation in composition ofthese three distinct preparations or products. Evidently, however, the

19-2

283

R. H. CHITTENDEN.

body just described as dysproteose must, prior to its final isolation, haveexisted in the digestive mixture and in the first proteose precipitate asa genuine heteroproteose.

G. NaCI + GH3 COOH Precipitate of Proteoses.

As already stated, protoproteose is not completely precipitated fromneutral solutions by saturation with sodium chloride; there invariablyremains in the fluid a certain residue of this body precipitable by saltonly in the presence of acetic acid. When, however, deuteroproteose ispresent, the acetic acid precipitate produced in a saturated salt solutioncontains in addition to protoproteose some deuteroproteose. But thismethod of treatment, while it yields a mixed precipitate, is necessaryfor the complete removal of the proto body, in order to pave the wayfor the separation of pure deuteroproteose.

Accordingly, the salt-saturated filtrate from the first NaCl precipitateof protoproteose was treated with a little acetic acid, by which a moreor less gummy precipitate of proteoses resulted. This precipitate,though recognized as a mixture of proto and deuteroproteose, waspurified and studied. The ordinary method of purification consisted indissolving the precipitate in a small volume of water, neutralizing theadherent acetic acid with sodium carbonate, and then dialyzing theneutral fluid until the diffusible salts were entirely removed. The fluidwas then concentrated to a syrup and the proteoses precipitated withalcohol. These were washed thoroughly with alcohol and ether, andwere finally obtained as a dry powder readily soluble in water. Thereactions of this mixed precipitate were such as would naturally beexpected from a solution containing the two proteoses. All of thereactions of protoproteose previously described, were obtained, slightlymodified, however, by the presence of deuteroproteose. For example,solubility in water was more pronounced and many of the precipitatesobtained with the various metallic salts and with some of the acidswere more soluble on the application of heat than was seen with pureprotoproteose. Further, the reaction with potassium hydroxide andcupric sulphate gave a far more pronounced red colour than proto-proteose alone will give.

A portion dried at 1100C. until of constant weight yielded onanalysis the following results, which indicate that the above acetic acidprecipitate is composed mainly of protoproteose with only a smallamount of deuteroproteose.

284


Acetic acid precipitate; Mixture of Protoproteose and Deuteroproteose.

1. 0-5623 gram substance gave 0-3290 gram H20 = 6'50 per cent. Hand 10475 gram CO2 = 50-80 per cent. C.

IL. 04145 gram substance gave 0-2435 gram H20 = 6-57 per cent. Hand 0-7728 gram CO,= 50-84 per cent. C.

III. 0 5337 gram substance gave 75-1 c.c. N at 21.30 C. and 768-5 mm.pressure = 16-92 per cent. N.

IV. 0-5160 gram substance gave 72-6 c.c. N at 20.80 C. and 765-5 mm.pressure= 16-84 per cent. N.

V. 0-8365 gram substance gave 0-0141 gram ash = 1-68 per cent.

Percentage composition of the ash-free substance.

51-64 51-706X61 6-68

1

Average51-67

- - 6-647-20 17-12 17'16

24-53

100-00

H. Deuteroproteose.

The filtrate from the above sodium chloride and acetic acid precipi-tate contains only deuteroproteose, now completely freed from theprimnary proteoses. The deutero body, however, cannot be directlyseparated from the salt-saturated solution to advantage. The bestmethod of treatment is to neutralize the fluid exactly with sodiumcarbonate and then dialyze in running water until the greater portion ofthe sodium chloride is removed. The solution is then evaporated to a

small volume and saturated, while hot, with ammonium sulphate, bywhich the deuteroproteose is separated as a gummy precipitate. It isthen washed repeatedly with a saturated solution of ammonium sulphate,after which it is dissolved in water and dialyzed until entirely free fromsulphate. On concentration of the dialyzed fluid to a syrup, andprecipitation with alcohol, the proteose is finally obtained as a gummy

mass which by continued treatment with alcohol and ether can bepartially dehydrated and eventually converted by careful drying into a

yellowish white powder.So prepared it is very soluble in water, yielding a clear yellow

solution, which gives no precipitate whatever with nitric acid, unlessthe fluid is saturated with salt, when a slight turbidity is producedwhich disappears on heating and reappears as the solution cools.

CHNSOJ

285

R. H. CHITTENDEN.

Acetic acid and potassium ferrocyanide, likewise, give no precipitate,and both neutral and basic lead acetate produce only a slight turbidity.

Picric acid also fails to produce a precipitate.On the other band, platinic chloride, cupric sulphate, potassio-

mercuric chloride and tannic acid all produce precipitates readily dis-solved by heat and reappearing as the fluids cool.

Potassium hydroxide and dilute cupric sulphate give an exceedinglyred biuret reaction.

Phospho-tungstic and phospho-molybdic acids both produce precipi-tates, the former, in an acid solution, being somewhat soluble on theapplication of heat.

Millon's reagent and heat gives the ordinary red precipitate,characteristic of most proteid bodies.

In reactions, therefore, deuteroproteose as formed by bromelindigestion differs markedly from protoproteose, and agrees more or lessclosely with the reactions usually ascribed to deuteroproteoses in general.Further, analysis of the product dried at 1100 C. shows the usual fallingoff in the percentage of carbon characteristic of most deuteroproteoseshitherto analyzed.

Deuteroproteose.I. 0-4282 gram substance gave 0-2394 gram 120= 6-20 per cent. H (?)

and 0-7604 gram Co0= 48-42 per cent. 0.II. 0X4418 gram substance gave 0X2585 gram H20 = 6X50 per cent. H

and 0O7889 gram CO2= 48-69 per cent. C.III. 0-4271 gram substance gave 58X6 c.c. N at 18.30 C. and 755-1 mm.

pressure= 16 23 per cent. N.IV. 04696 gram substance gave 63-95 c.c. N at 20.30 0. and 759 3 mnm.

pressure= 16 -20 per cent. N.V. 0X4060 gram substance gave 0 0097 gram ash = 2X38 per cent.


C 49160 49-87 - 49.73H 6-65 6-65N 16'62 16-57 16-59S} - 27-03

100*00I. Peptone.

In all digestions of blood-fibrin with an acid solution of bromelinthere is a large production of peptone. Indeed, judging from such

286


results as have been obtained in the several digestions carried out, thereis relatively a much larger quantity of true peptone formed by theaction of bromelin in an acid solution than by pepsin-hydrochloric acid.In other words, the proteoses first formed are more quickly convertedinto true peptone, thus indicating in another way the relationship inaction between bromelin and trypsin. As already noted, this productcan be separated from the proteoses by saturation of the concentrateddigestive fluid with ammonium sulphate, after the manner alreadydescribed. The ammonium sulphate-saturated filtrate, containing thepeptone, is then freed from a portion of the ammonium salt by repeatedconcentration and crystallization, after which the solution is dialyzeduntil the sulphate is entirely removed. This process obviously entails aconsiderable loss of peptone by diffusion, but still sufficient remains tofurnish a reasonable amount of product. From the salt-free solution,the peptone is obtained by evaporation of the fluid to a syrup andprecipitation with strong alcohol. It is further purified by repeatedboiling with fresh quantities of alcohol and extraction with ether. Likeall preparations of pure peptone, free from proteose, bromelin-peptoneis extremely hygroscopic and difficult to dry, and when once dried showsa strong avidity for water, quickly becoming gummy in the presence ofmoisture and on addition of water quickly dissolving to a clear yellowsolution.

In reactions, the peptone is naturally characterized by lack ofprecipitation with many reagents which constitute good precipitants forthe proteoses.

Thus, nitric acid gives no precipitate whatever, even on the additionof strong salt solution. The solution, however, shows a yellow colour.

Acetic acid and potassium ferrocyanide, likewise cupric sulphate, failto produce any precipitate.

On the other hand, phospho-molybdic acid, phospho-tungstic acid inacid solution, tannic acid, picric acid, and potassio-mercuric iodide inacid solution all give heavy precipitates, some more or less gummy incharacter.

Neutral and basic lead acetate, mercuric chloride and platinicchloride all produce a slight turbidity.

Potassium hydroxide and dilute cupric sulphate give a deep pink, orred biuret reaction.

Boiling with potassium hydroxide and lead acetate gives no appre-ciable reaction for sulphur.

With Millon's reagent, no precipitate is obtained even on addition

287

R. H. CHITTENDEN.

of an excess of the fluid, and on boiling the mixture only a slight redcolouration appears which quickly fades into a dirty yellow. Thisreaction is somewhat significant, and has been previously commentedon in connection with the antipeptone1 formed by the action of trypsinon blood-fibrin. In fact, the above bromelin-peptone corresponds in itsreactions fairly well with those of fibrin-antipeptone formed by trypsin.This, taken in connection with the fact that large quantities of tyrosinare formed in an acid-bromelin digestion seems to point to the breakingdown of the proteid molecule into leuicin and particularly into tyrosinunder the influence of bromelin, the peptone left at the end of avigorous digestion being essentially antipeptone.

All of the peptone preparations separated from an acid-bromelindigestion were found to contain such a large percentage of ash thatanalysis of the products has little value. In the neutral bromelin-digestions, however, shortly to be described, peptone was separated witha much smaller content of mineral matter and its composition accuratelyascertained.

Neutral Bromelin Digestion.

On raw blood-fibrin, and on fibrin which has been soaked for sometime in alcohol, a neutral solution of bromelin exerts pronounced proteo-lytic action, especially in the presence of a small amount of sodiumchloride. The products which result are essentially of the same orderas those formed in an acid-reacting medium; minor differences, however,appear, some of which may be noted.

In one experiment, 400 grams of an alcoholic preparation of blood-fibrin were warmed at 400 C. with 3 litres of a 1 per cent. solution ofsodium chloride and 0 4 gram of bromelin for about 36 hours, putrefac-tion being prevented by addition of a little thymol. Disintegration andsolution of the fibrin took place quite rapidly, and at the end of 36hours only a small flocculent residue remained. This proved to be anantialbumid-like body similar to that formed in an acid digestion. Thesolution was found to have acquired an alkaline reaction during thedigestion, and on careful neutralization with dilute hydrochloric acid aslight neutralization precipitate was obtained which certainly was notalkali-albuminate, but reacted exactly like the peculiar heteroproteosebody precipitated by neutralization from an acid digestion, being more

1 Kuhne and Chittenden. " Ueber die Peptone." Zeitschrift fur Biologie. Bd.xxII. p. 444.

288


or less soluble in 10 per cent. salt-sokition and non-coagulable by heatwhen suspended in a neutral fluid. On boiling the neutralized digestivefluid, a very heavy heat-precipitate resulted which was far larger inamount thani that usually obtained in an acid digestion. Indeed, theappearance of this primary body in such quantity in a neutral digestionis emblematic of what has been previously stated, viz., that the fermentacts far more energetically on blood-fibrin in a weak acid solution thanin a neutral fluid. For while fibrin may be dissolved by a neutralbromelin solution with fair degree of rapidity, conversion of the pritnaryproducts into secondary proteoses and peptone goes on more slowly thanin a slightly acid medium. Certainly, the appearance of this body in aneutral digestion of fibrin is even more characteristic than in an aciddigestion.

This heat-precipitate, like the one previously described, is not acoagulated proteid, since it is readily soluble in 0-2 per cent hydro-chloric acid and in 0 5 per cent. sodium carbonate, but is composed ofcoagulated heteroproteose, i.e. it is a form of dysproteose. In the formof heteroproteose it is readily convertible by further ferment action intomore soluble proteoses and peptone, but in a neutral digestion of suchstrength as the one just described, where the fibrin may be rapidlydissolved, the usual primary proteoses seem to be in great part repre-sented by this peculiar hetero-like body. Both deuteroproteose andpeptone, however, were formed in considerable quantity, but very littlepure protoproteose unmixed with deuteroproteose was obtained.

Blood-fibrin, such as was used in this digestion, was only slightlydissolved by a 1 per cent. sodium chloride solution alone, hence the saltpresent in the digestive mixture could not have had any direct action inthe formation of this globulin-like proteose.

A portion of the above heat-precipitate, dried at 1100C. until ofconstant weight, gave on analysis the following results, which agreefairly well with those obtained from analysis of the like body formed inan acid digestion.

Heat-precipitate.

I. 04389 gram suibstance gave 02783 gram H2O = 7*04 per cent. Hand 08362 gram CO2= 51'96 per cent. C.

II. 02674 gram substance gave 01647 gram H20 = 6-84 per cent. Hand 05115 gram C2O= 52 16 per cent. C.

III. 0*4010 gram substance gave 55-2 c.c. N at 22.20 C. and 768-5 mm.pressure = 16 34 per cent. N.

289

R. H. CHITTENDEN.

IV. 04173 gram substance gave 56-9 c.c. N at 21.20 C. and 770-2 mm.pressure - 16 22 per cent. N.

V. 0-6562 gram substance gave 0 0004 gram ash = 0-06 per cent.


C 51F99 52-19 52-09H 7 04 6-84 6-94N - 16-35 16-23 16-29

- - - - 2468

100-00

Soluble Proteoses and Peptone.From the neutralized and concentrated digestive fluid, freed from

the heat-precipitate just described, proteoses, peptone, leucin andtyrosin were separated by the methods -detailed under the head of-the acid digestion. On dissolving the mixed proteose precipitate((NH,),SO4 saturation) in water, neutralizing and saturating with-sodium chloride, only a very small precipitate of protoproteose wasobtained. On adding acetic acid, however, to the salt-saturated fluid,quite a bulky precipitate resulted, composed of a mixture of proto-proteose and deuteroproteose, but in which, judging from the reactions,there was a predominance of protoproteose, although for some reason itwas not precipitable by salt alone. Purified after the method made useof in the acid digestion, the substance gave the usual reactions of theproto body, as modified by the presence of deuteroproteose. Especiallypronounced was the ready precipitation of an aqueous solution of thebody with nitric acid and the equally ready disappearance of theprecipitate on warming the fluid, followed by its reappearance as thesolution cooled.

Dried at 1100 0. until of constant weight, the following results wereobtained on analysis, which show a much lower content of nitrogenthan was found in the corresponding mixed precipitate from the aciddigestion.

Acetic acid prec pitate; Mixture of Protoproteose aud Deuteroproteose.

I. 0-6109 gram substance gave 0-3465 gram 1120 = 6-30 per cent. Hand 1 0848 grams CO= 48 42 per cent. C.

II. 0-3529 gram substance gave 0-2028 gram H20= 6-38 per cent. Hand 0 6287 gram CO2= 48-58 per cent. C.

290

PROTEOLYTIC ACTION OF BROMELIN. 291

IIT. 0-4014 gram substance gave 51-1 c.c. N at 22.30 C. and 765-0 mm.pressure = 15 11 per cent. N.

IV. 0 3777 gram substance gave 48-5 c.c. N at 22.30 C. and 763-3 mmpressure = 15 19 per cent. N.

V. 0 5859 gram substance gave 0X0327 gram ash= 5 43 per cent.


C 51-20 51'36 51-28H 6-55 6-74 - - 664N 15-97 16-06 16-01

OI - - 26-07

100-00

Deuteroproteose was separated from this digestion in the samemanner as from the preceding acid digestion, and although smaller inamount, showed essentially the same reactions as those previouslydescribed. Thus, it was not at all precipitated by addition of nitricacid, even to a salt-saturated solution of the proteose, and it likewisefailed to give any precipitate with acetic acid and potassium ferrocy-an'ide.

On analysis, the following results were obtained, the preparationhaving been dried at 1100C. until of constant weight.

Deuteroproteose.

I. 0-3032 gram substance gave 0-1743 gram H1O = 6-38 per cent. Hand 0-5320 gram 02= 47-84 per cent. C.

II. 0 3503 gram substance gave 0-2014 gram H20 = 6-38 per cent. Hand 0-6133 gram C02= 47-74 per cent. C.

III. 0-3569 gram substance gave 43-8 c.c. N at 22.60 C. and 761-5 mmpressure = 14-39 per cent. N.

IV. 0-3587 gram substance gave 0-0210 gram ash = 5'85 per cent.

Percentage composition of the ashfree substance.Average

C 50-81 50 70 - 50 75H 6-77 617 - 6-77N - 15-28 15-28S - _ 27-20

100*00

This analysis shows a wide divergence in composition from that of

2. H. CHITTENDEN.

the corresponding body formed in an acid digestion, the carbon being1 per cent. higher and the niitrogen more than 1 per cent. lower thanwas found in the previously described deuteroproteose. For thispronounced difference in composition we have no positive explanation;the reactions of the two proteoses show no marked differences, andthere is no reason for expecting any variation in the character of aproteose formed under conditions which differ so slightly. A differencein the conditions under which the proteolytic ferment performs its workmight well lead to differences in the rate of action and consequently inthe proportion of the several proteoses formed, but there would seem tobe no special reason why the individual products should show variation,providing, of course, the methods of separation are adequate for theirpurpose. Concerning the latter point we have no reasonable doubt,although it may be that deuteroproteose as formed in a bromelindigestion is less completely precipitated by saturation with ammoniumsulphate1 than ordinary deuteroproteose, but this possibility has littlebearing on the divergence in composition above noted. More plausibleis the view that in the weaker neutral bromelin digestion, less of thehemideuteroproteose is broken down into hemipeptone, leucin andtryosin than in an acid digestion and that consequently the compositionof the resultant product is modified accordingly. This assumes, how-ever, a difference in composition between hemi- and antideuteroproteose,of which we have no positive proof

The peptone formed in a neutral bromelin digestion is essentiallythe same as that formed in an acid digestion. From the neutraldigestion it was separated and purified by the methods previouslydescribed, and when so separated it showed all of the peculiaritiescommon to true peptone. The amount formed in this neutral digestionwas not as large as the amount formed in an acid-bromelin digestion,although the former mixture was kept at 4000. for a far longer period.In conformity with this lesser production of peptone, a much smalleryield of leucin and tyrosin was obtained, thus showing, as alreadystated, that the weaker action of a neutral bromelin solution is mani-fested more strikingly by the amount of secondary proteose and peptoneformed than by the rate at which the fibrin is dissolved.

The only striking point of difference between the peptone formed ina neutral digestion and that formed in an acid bromelin solution wasthe behaviour of the body towards Millon's reagent. As already

1 Compare Kiihne. Zeitschrift fiur Biologie. Bd. xxix. p. 1.

292


stated, the peptone separated from the latter digestive mixture gavelittle or no red reaction with the reagent, while the peptone separatedfrom the neutral digestion gave a good and characteristic red reactionwhen heated with a solution of mercuric nitrate. This difference inreaction is probably due to the lesser proteolytic power of the neutralbromelin solution, the hemipeptone formed being consequently onlypartially broken down into leucin and tyrosin. As a result, the pep-tone separated from such a digestion would naturally be composedof a mixture of hemipeptone and antipeptone, while in the morevigorous acid digestion the hemipeptone may have been completelytransformed into leucin and tyrosin, thus leading to the separation of afairly pure antipeptone.

This is obviously a mere conjecture which, however, if true wouldsatisfactorily explain this difference in the behaviour of the two pep-tones towards Millon's reagent.

The peptone separated from the above neutral digestion, after beingdried at 1100C. until of constant weight, gave on analysis the followingresults.

Peptone.

I. 03154 gram substance gave 0-1733 gram H20 = 6-10 per cent. Hand 0-5113 gram CO, = 44-20 per cent. C.

II. 04136 gram substance gave 48-7 c.c. N at 21.70 C. and 774-4 mm.pressure = 14'12 per cent. N.

III. 0-3882 gram substance gave 47-1 c.c. N at 21.50 C. and 762'2 mm.pressure = 14-28 per cent. N.

IV. 0'3766 gram substance gave 0'0345 gram ash= 9-16 per cent.

Percentage composition of the a8h-free substance.Average

C 48'65 48-65H 6-71 - 6-71N 15-54 15-72 15G63

O} _ - 29 01100*00

The above analysis shows the peptone to possess the usual lowcontent of carbon, characteristic of most of the true peptones (free fromproteose) thus far analyzed, but the percentage of nitrogen, in propor-tion to the nitrogen of the blood-fibrin, is exceptionally low. Thispeculiarity, however, is common to several of the digestive products

293

R. H. CHITTENDEN.

formed from blood-fibrin by the action of a neutral solution of bromelin.This might be interpreted perhaps as signifying that in bromelindigestion we have not only hydration of the proteid molecule, buthydration accompanied by cleavage of a nitrogen-containing radical.

The majority, if not all, of the proteoses and peptones formed byanimal digestive ferments hitherto analyzed have shown a content ofnitrogen at least equal to that of the mother proteid. Neumeister'rhowever, has described two products, atmidalbumnin and atniidalbumose,formed from blood-fibrin by the action of superheated steam, in whichthe content of nitrogen is 14'43 and 13-58 per cent. respectively. Thesame investigator claims that similar bodies ire formed in the digestionof blood-fibrin and other proteids with the juice of Carica Papaya.However this may be, the above deuteroproteose and peptone arepeculiar in their low content of nitrogen.

The composition of the various products analyzed, both those formedin a neutral and in an acid digestion, may be conveniently compared inthe following table:

Acid Digestion.

Blood-fibrin (Hammarsten)Antialbumid-like bodySoluble proteose in undigested t

residue JNeutralizationl precipitate, 1Neutralization precipitate, 2Heat precipitateProtoproteose, NaCl p.p.Insoluble heteroproteoseProto and Deuteroproteose, lmixed p.p. J

Deuteroproteose

C52-6852-08

H N6-83 16-916-91 16-33

6-58 16-55

S&O23-5824-68

25-94

Ash

1 98

3X24

51P40 6-96 16-29 25-35 0-2751P68 6-70 16-26 25-36 0-5852-16 6'91 16-65 24-28 0185189 6-75 16-87 24'49 20050'96 6-82 16-80 25-42 0 90

5167 6-64 17-16 24-53 1P6849 73 6-65 16'59 27-03 2-38

Neutral Digestion.

Blood-fibrin (Hammarsten)Heat precipitateProto and Deuteroproteose, l

mixed p.p. IDeuiteroproteosePeptone

52-68 6-83 16-9152-09 6-94 16'2951P28 6-64 16-01

50175 6177 15-2848&65 6171 15'63

Zeitschriftfur Biologie. Bd. xxvi. p. 71.

23-5824-68

26-07

27'2029-01

0'065.435-859-16

294

PRIOTEOLYTIC ACTION OF BROMELIN.

3. Products formed in the Digestion of Coagulated Egg-albuminwith Bromelin.

(From experiments made by W. H. P. Bronsoni, Ph.B.)

As indicated by the various statements already miade, coagulatedegg-albumin is considerably more resistant to the action of bromelinthan blood-fibrin. At the same time it is slowly dissolved, especially ina neutral fluid, and converted into the ordinary products of proteolyticdigestion. It is also to be noted that, as in the action of trypsin, whenalbumin is once dissolved the ordinary primary products are quicklytransformed into true peptone. In other words, the primary corrosionor solution of the coagulated proteid proceeds slowly, but when this isonce accomplished the secondary products are more rapidly formed, sothat an examination of the clear digestive fluid always reveals apredominance of true peptone with a proportionately smaller amount ofsoluble albumoses.

The albumin used in these experiments was prepared by taking thewhites of a large number of eggs, precipitating the globulin by theaddition of hydrochloric acid (4 2 c.c. of 20 per cent. HCl to every300 c.c. of albumin), and after some hours standing, with frequentstirring, removing the abundan t precipitate by filtration through paper.The perfectly cleax fluid was then coagulated in fine flocks by pouringit slowly into a large volume of boiling water slightly acidified withacetic acid. The resultant finely divided precipitate was then collectedon a cloth filter, and washed thoroughly with boiling water until allsoluble salts were entirely removed.

The ferment used in the several digestions was in two cases asodium chloride preparation, and in one case an ammonium sulphatepreparation.

Preliminary digestions with the above prepared coaguilated egg-albumin gave resuilts corresponding essentially with those alreadynoted, showing that the ferment is most active on this form of proteidmatter in a neutral solution, free from salt. Thus in one such series ofexperiments where like amounts of bromelin and albumin were em-ployed, the total volume of fluid being 25 c.c. and the mixtures warmedat 4000. for five hours, the most vigorous digestion, as indicated by thecharacter of the biuret test applied to the filtrates, was obtained in aneutral fluid; the presence of even 0-006 per cent. of hydrochloric acid

1 Deceased, June 1892.

295a

R. H. CHITTENTDEN.

tending to diminish slightly the proteolytic action of the ferment.Further, the presence of salt, either in a neutral or slightly acidsolution, offers no noticeable advantage. Indeed, it may be safely saidthat in the digestion of coagulated egg-albumin with bromelin, theferment sliows the most pronounced proteolytic action in a neutralmedium, free from salts; the presence of both salts and small amountsof acid interfering with the digestive action in proportion to theamount present, while larger quantities check the action of the fermentaltogether.

In the first digestion on a large scale, the coagulated albumin fromthree dozen of eggs was warmed at 400C. with two litres of distilledwater and 0'8 gram of a sodium chloride preparation of the ferment for40 hours, the mixture being thymolized to prevent putrefaction. Ex-.amination of samples of the clear digestive fluid revealed the presenceof a large amount of peptone, leucin and tyrosin, thus showing thatdigestive action had been quite vigorous. There still remained, how-ever, considerable finely divided undissolved matter, which on beingtested was found to consist in part of an antialbumid-like body, solublein 0 5 per cent. sodium carbonate. With this was naturally associateda certain amount of unaltered albumin. The entire portion of un-dissolved matter was filtered off, washed with water and the residueagain warmed at 40° C. for 24 hours with thymolized water containinga few decigrams of ferment. At the end of this time, the insolublematter was again separated by filtration and thoroughly washed withwarm water. The proportion of matter soluble in 0 5 per cent. sodiumcarbonate was now fouind to be greatly increased, more of the originalcoagulated albumin having been transformed into soluble proteoses andpeptone on the one hand, and into insoluble antialbumid on the other.This antialbumid-like body was readily soluble in dilute alkali car-bonate, but wholly insoluble in dilute (0-2 per cent.) hydrochloric acid,and in water. In order to separate this body from the unalteredalbumin which was still present, the entire residue after thoroughwashing with water was warmed at 500C. for an hour with a litre of0 5 per cent. sodium carbonate. The resultant solution was then filteredfrom the coagulated albumin and, after dilution, carefully neutralizedwitlh 0 2 per cent. hydrochloric acid. By this method of treatment aheavy flocculent precipitate was obtained, wbich was collected on afilter and washed with water until entirely free from soluble salts. Itwas next washed with weak alcohol, absolute alcohol and finally withether.

296


Antialbumid.

So prepared, this body gives the usual reactions characteristic ofwhat has been described in the past as an antialbumid. Like theantialbumid resulting from gastric digestion, this body when firstformed is entirely insoluble in 0-2 per cent. hydrochloric acid, but afterit has once been dissolved in dilute sodium carbonate and reprecipitatedby neutralization, it is then exceedingly soluble in both dilute acid andalkali. It is not, however, capable of being further digested, at leastnot readily so, by the action of bromelin, and hence differs from anordinary alkali-albuiminate. It gives the usual proteid reactions charac-teristic of this body, and contains a small amount of suilphur, asindicated by the reaction obtained with potassium hydroxide and leadacetate.

Dried at 1100 C. until of constant weight, one sample gave thefollowing results on analysis.

Antialbumid, 1.

I. 0-3864 gram substance gave 0-2376 gram H20 = 6-84 per cent. Hand 0-7437 gram CO2 = 52-48 per cent. C.

II. 0-4066 gram substauce gave 02555 gram H20= 6-98 per cent. Hand 0-7839 gram CO2 = 52-57 per cent. C.

III. 0-3046 gram substance gave 0-1916 gram H.0 = 6*98 per cent. Hand 0 5909 gram CO2= 52 90 per cent. 0.

IV. 0-4476 gram substance gave 57 9 c.c. N at 13.50 C. and 756-5 mm.pressure= la43 per cent. N.

V. 0 3405 gram substance gave 0-0015 gram ash = 0 44 per cent.


C 52-71 52-79 53-13 52-87H 6-87 7.01 7.01 - 6-96N - 1549 15 S;9S - - - - 24-68

100-00

Another preparation of this substance formed in a second digestion,and isolated in the same manner as the preceding, was also analyzedwith the following results. The analysis was made on a sample driedat 1100 C. until of constant weight.

297

20PH. XV.

9. H. CHITTENDEN.

Antialbumid, 2.I. 0-5567 gram substance gave 0 3540 gram H20 = 7-06 per cent. H

and 1 0917 grams CO. = 53-47 per cent. C.II. 0-4317 gram substance gave 0-2676 gram H.0= 6-88 per cent. H

and 0 8419 gram CO, = 53-18 per cent. 0.III. 0 3495 gram substance gave 44'6 c.c. N at 14.10 0. and 761-8 mm.

pressure= 15-27 per cent. N.IV. 0 4467 gram substance gave 57 4 c.c. N at 13.10 0. and 749 5 mm.

pressure = 15 -21 per cent. N.V. 0 3456 gram substance gave 0 0017 gram ash = 0 49 per cent.

VI. 0-3320 gram substance gave 0 0016 gram ash= 0-48 per cent.


C 53-73 53.45 - 53.59H 7.09 6-91 7 00N 15-34 15-28 15-31

3} - 24-10

100-00

From these data it is evident that the antialbumid-like body formedfrom coagulated egg-albumin by bromelin digestion does not differwidely in composition from the mother proteid. There is, however, asomewhat higher percentage of carbon, accompanied by a correspondingdecrease in the content of nitrogen, but the difference is not great.

Soluble products of digestion.In all of the digestions made with coagulated egg-albumin the

proportions of albumin, ferment and water used were essentially thesame as those already given, the mixtures being warmed at 40°-450 C.for 40-60 hours. The digestive action being somewhat slow, thislength of time was necessary in order to insure a sufficient amount ofsoluble products for study. On filtering the solution from the residueof undigested albumin and antialbumid it was usually found slightlyacid in reaction, although the thymol added had apparently preventedany appearance of putrefaction. With the neutral fibrin digestions, onthe other hand, it will be remembered an alkaline reaction was usuallydeveloped. Examination of the clear filtered fluid obtained in thesedigestions always revealed by acetic acid and potassium ferrocyanide,nitric acid, ammonium sulphate and the biuret test the presence oflarge quantities of albumose and peptone.

298


The methods of separation were eventually the same as thosedetailed under the head of fibrin digestion.

The clear fluid was first carefully neutralized with dilute sodiumcarbonate, without yielding any neutralization precipitate, then concen-trated to a small volume whereby a slight precipitate usually resultedcomposed of coagulated proteid. Filtered from.this precipitate, thefluid was concentrated to a syrup and then treated while hot with anexcess of 95 per cent. alcohol, whereby the albumoses and peptone wereprecipitated. The resultant gummy mass was then repeatedly heatedand boiled with alcohol to more completely remove leucin and tyrosin.Further, the precipitate was again dissolved in water and reprecipitatedwith hot alcohol, after which it was extracted with boiling alcohol untilthe latter failed to take up any more leucin and tyrosin. By concen-trating the alcoholic filtrate and extracts, an abundance of crystals ofboth leucin and tyrosin were obtained, the latter responding to bothHoffmnann's and Piria's tests.

The gummy precipitate of albumoses and peptone was dissolved inwater and the albumoses separated from the peptone by saturation ofthe solutiotn with ammonium sulphate, in the presence of a little aceticacid; the separation of the albumoses being made more complete byheating the salt-saturated fluid to boilirng and keeping it in thatcondition for some time. The albumose precipitate so obtained wasusually very oily, as if composed wholly of deuteroalbumose. It wasredissolved in water and again precipitated by saturation with theammonium salt under the same conditions as just described, and finallywashed repeatedly with hot saturated ammonium sulphate-solution.Redissolved in water, the solution usually gave only a slight precipitateby saturation of the neutralized fluid with sodium chloride and only asmall one on addition of acetic acid to the salt-saturated fluid, thusshowing the absence of any large amount of primary proteoses. Thiswas the usual result in the several digestions examined, and indicates,as already stated, a tendency for the first formed proteoses to be quicklyconverted into deuteroproteose and true peptone. Indeed, the solubleproducts in these digestions are especially represented by peptone, non-precipitable by ammonium sulphate. In no case was protoalbumose orheteroalbumose obtained in sufficient amount for analysis.

Protoalbumose.

Such amount of this product as was separated, was obtained bysatutrating the neutral solution of the above (N H4),SO4 albumose

20-2

299

00. H. CR1ITTENDEN.

precipitate with sodium chloride. The slight precipitate resulting waswashed with saturated salt solution, then dissolved in water and dialyzedin running water until entirely free from chloride. At the end of thedialysis, only a trace of heteroalbumose was, as a rule, found adherent tothe parchment wall of the dialyzer tube. The solution of the purifiedprotoalbumose was then concentrated to a syrup, and precipitated withalcohol. In all of our digestions the yield was small, insufficient foranalysis. The reactions, however, were such as are characteristic of anordinary protoproteose. From a neutral solution it was precipitated byaddition of salt in substance, added to saturation. With nitric acid aprecipitate was produced soluble on heating and in an excess of theacid. Acetic acid -and potassiuim ferrocyaiiide likewise gave a precipitatesoluble on heating, and reappearing as the solution cooled. Cupricsulphate gave a heavy precipitate, as did also mercuric chloride, leadacetate, platinic chloride, picric acid and tannic acid. The two latterprecipitates were dissolved somewhat on warming, but reappeared oncooling. Phosphotungstic acid gave a heavy permanent precipitate,likewise phosphomnolybdic acid. Potassio-mercuric iodide yielded aprecipitate which dissolved on the application of heat. Withi cupricsulphate and potassium hydroxide, a reddish violet colour was obtained,while Millon's reagent gave a heavy precipitate clhanging to theordinary red colour on boiling.

With salt and carefLul addition of nitric acid, a precipitate wasobtained readily soluble on application of heat and reappearing as thesolution cooled.

Deuteroalbumose.

In the salt-saturated filtrate from the protoalbumose precipitate,addition of a few drops of acetic acid produced a second more or lessflocculent precipitate, presumably composed of a mixture of the remain-ingf protoalbumose with some deuteroalbumose. This was so slight,however, it was rejected and riot examined further. In this last filtrate,only deuteroalbumose could be present. Preliminary to its separation,the solution was neutralized and then dialyzed for a few days until theexcess of sodium chloride was removed, after which the fluid wasconcentrated and then saturated with ammonium sulphate. The re-sultant almost oily precipitate of pure deuteroalbumose was washedsomewhat with a saturated solution of the ammonium salt, then dis-solved in water and dialyzed in running water until the sulphate wasentirely removed, after which the clear filtered fluid was concentrated

300

PROT'EOLYTIC ACTION OF BROMELIN.

and finally precipitated with strong alcohol. It was heated for sometime with fresh quantities of alcohol, then washed with ether and dried.The yield of pure deuteroalbumose in these digestions was not large,still the amount was far greater than of protoalbumose.

One sample, dried at 1100 C. until of constant weight, gave onanalysis the following results:

Deuteroalbumose.

I. 0-4205 gram suibstance gave 0-2445 gram H10 = 6-46 per cent. Hand 0 7433 gram CO = 48-21 per cent. C.

II. 0 3407 gram substance gave 0-1997 gram H,O= 6'51 per cent. Hand 0-6013 gram 0o, = 48 12 per cent. C.

IMI. 0-3632 gram substance gave 37 9 c.c. N at 13.80 C. and 749 9 mm.pressure = 12 31 per cent. N.

IV. 0-2754 grami substance gave 0-0133 gram ash= 4-83 per cent.

1'ercentage composition of the ash-free substance.Average

C 50-67 5058 50-63H 6-78 6-83 6-80N - - 12-93 12-93

O} - 29-64

100-00

Very noticeable is the exceedingly low content of initrogen. In fact,the percentage of this element is surprisingly low, and were it not forthe fact that the peptones analyzed likewise show a correspondingly lowpercentage of nitrogen, the above result would seem very improbable.As it is, we can offer no satisfactory explanation other than the mereconjecture that possibly it may signify a cleavage of the proteid moleculewith a splitting off of a nitrogen moiety.

In reactions, the pure deuteroalbumose differs markedly from theprimary albumoses. Thus, it is very soluble in water and is not pre-cipitated from either a neutral or acid solution by saturation withsodium chloride. Neither is it precipitated by nitric acid, acetic acidand potassium ferrocyanide, cupric sulphate, lead acetate, mercuricchloride, or platinic chloride. With picric acid, on the other hand, asalso with tannic acid, phosphotungstic acid, phosphomolybdic acid, andpotassio-mercuric iodide precipitates are formed more or less soluble onthe application of heat. With the biuiret reaction a bright red colour is

301

R. H. CHITTENDEN.

obtained, and with Millon's reagent a precipitate is formed, turningred on the application of heat.

Peptone.

As already remarked, peptone appears to be the characteristicproduct of bromelin digestion with coagulated proteids. The filtratefrom the ammonium sulphate precipitate of albumoses, in these diges-tions of coagulated egg-albumin, was always found rich in peptone; i.e.application of the biuret test to the digestive fluid, after completeremoval of the albumoses by saturation of the solution with ammoniumsulphate, invariably gave an intense red colour, indicative of the abun-dance of peptone.

The method of separation was essentially as follows: The saturatedammonium sulphate solution from the albumose precipitate, alreadydescribed, was concentrated somewhat and then allowed to stand in a coolplace until a considerable quantity of the ammonium salt had crystallizedout. The filtrate was then treated with dilute alcohol which gaverise to another separation of animonium sulphate. The fluid was thenconcentrated until the alcohol was entirely removed and another crys-tallization of sulphate comnmenced. The filtrate from this latter separa-tion was then carefuilly neutralized and dialyzed in running water, withthe usual addition of thymol, until the ammonium salt was entirelyremoved, as indicated by a negative reaction with barium chloride.The salt-free solution was then concentrated to a syrup and precipitatedwhile hot with strong alcohol. Obviously, the above method entailed alarge loss of peptone by diffusion, but the quantity formed was such thata reasonably large product was always obtained. The peptone so pre-pared was purified somewhat by repeated solution in water andreprecipitation with alcohol. It was finally boiled for some time withfresh quantities of 95 per cent. alcohol and then repeatedly extractedwith absolute alcohol and ether, by which it was partially freed frommoisture. Although, like all peptone preparations, very gummy, it wasobtained in the form of a powder by careful drying on a water-bath,after which it was thoroughly dried at 105-110° C.

So prepared, the peptone has the appearance of a light brownishyellow powder, readily soluble in water to a yellowish solution, and alsosoluble in a saturated solution of sodium chloride and ammoniumsulphate. It has a strong characteristic odour, like meat slightly burnt,both when dry and in solution. Like all true peptones, it is character-ized by lack of precipitation with the ordinary precipitants for albuinin-

302


ous bodies, giving no precipitate with acetic acid and potassiumferrocyanide, nor with metallic salts, such as cupric sulphate andmercuric chloride. With tannic acid and phosphomolybdic acid aprecipitate is formed which is more or less soluble on the application ofheat, reappearing as the solution cools. Phosphotungstic acid gives aheavy permanent precipitate, especially in the presence of dilute acid.With Millon's reagent only a slight precipitate is obtained, as in thecase of the corresponding fibrin-peptone previously described, but thesolution shows a distinct red colour when boiled. With nitric acid, noprecipitate is produced, but addition of ammonia to the strongly acidfluid brings out the ordinary orange yellow colour.

Trichloracetic acid fails to give any precipitate, as does also chromicacid and ferric chloride.

Two distinct preparations of peptone were analyzed, both samplesbeing dried at 1100C. until of constant weight. Following are theresults obtained:

Peptone, 1.1. 0 3393 gram substance gave 01947 gram H.0 = 6-37 per cent. H

and 0-5772 gram CO2= 46-39 per cent. C.II. 0 3719 gram substance gave 0'2137 gram H.0 = 6-38 per cent. H

and 0 6301 gram CO,,= 46-20 per cent. 0.III. 0Q4157 gram substance gave 49 5 c.c. N at 15.90 C. and 759-3 mm.

pressure = 14 12 per cent. N.JV. 0-4138 gram substance gave 49 3 c.c. N at 15.80 C. and 758-9 mm.

pressure = 14-12 per cent. N.V. 0A4966 gram substance gave 0-0170 gram ash = 3-42 per cent.

Percentage composition of the a8lifree substance.Average

C 48-01 47-82 - - 47-91H 6-59 6'60 - 6-60N - 14-62 14'62 14-62

-} 30-87

100-00Peptone, 2.

I. 0-3673 gram substance gave 0-2056 gram H 0 = 621 per cent. Hand 0-6043 gram CO, = 44-87 per cent. C.

II. 0-3644 gram substance gave 0-2058 gram H20 _ 6-27 per cent. Hand 06024 gram CO2= 45-08 per cent. C.

303

R. H. CIIITTENDEN.

III. 0-3982 gram substance gave 45-5 c.c. N at 16.90 0. and 766 9 mm.pressure= 13 -63 per cent. N.

IV. 0'3843 gram substance gave 43-8 c.c. N at 16.70 C. and 767-8 mm.pressure= 13 65 per cent. N.

V. 0-5051 gram sulbstance gave 0'0316 gram ash = 6 25 per cent.

Percentage composition of the ah-free 8Ubstance.Average

C 47-86 48-07 47-96H 6'62 6-67 - 6-65N -4-453 14-55 14-54

°} - - 30-85

100-00

These two results show very close agreement and are alike cha-racterized not only by a low content of carbon but also by a compara-tively small percentage of nitrogen.

The results obtained in the study of these digestions of coagulatedegg-albumin by bromelin testify to the true proteolytic power of theferment and indicate that the latter does not differ markedly in itsaction from the related animal ferment trypsin. Indeed, as with thelatter ferment, we find as characteristic products of its digestive actionon coagulated proteids, antialbumid, primary and secondary albumoses,true peptone, leucin and tyrosin. Fulther, bromelin like trypsin, al-though a weaker ferment, is especially a peptone-forming ferment, suchportion of the proteid as is dissolved by the action of the ferment beingquickly transformed into the final products characteristic of trypsindigestion.

For convenience in comparison, the analytical data, showing thecomnposition of the several products analyzed, are arranged together inthe following table:

C H N & 0 AshCoagulated egg-albumin' 52-21 6-96 15'80 25-03 0 37Antialbumid, 1 52-87 6-96 15-49 24-68 0 44

,, 2 53.59 7 00 15-31 24-10 0-48Deuteroalbumose 50-63 6-80 12-93 29-64 4-83Peptone, 1 47-91 6-60 14-62 30-87 3 42

,, 2 47-96 6-65 14-54 30-85 6-25

1 Chittenden and Bolten. Studies, Lab. Physiol. Chem. Yale Univ., Vol. Ii.p. 130.

304


Here again, as in the foregoing fibrin digestion, we are impressedwith the exceptionally low percentage of nitrogen in the deuteroalbu-mose and peptone. In fact, this is so contrary to our geTneral experiencewith digestion products formed by animal ferments, that we are forcedto consider it as something peculiar to the vegetable ferment. As iswell known', secondary proteoses and peptone formed by the action ofpepsin and trypsin usually show a much lower content of carbon thanthe proteid undergoing digestion, as in the above bronmelin products,but the percentage of nitrogen is ordinarily increased in proportion tothe decrease of carbon. Hence, on the strength of the above data wemight conjecture that the vegetable ferment bromelin, and possiblyother like vegetable ferments, are peculiar in giving rise to secondaryproteoses and peptones with a much lower percentage of nitrogen thanis contained in the rnother proteid, thus implying a cleavage of anitrogen-containing radical as a part of the proteolysis. This, if true,would constitute a good ground of distinction between the animal andvegetable proteolytic ferments.

4. Products formed in the Digestion of Myosin with Bromelin.

(From experiments made by Vertner Kenerson, B.A.)

Fresh muscle tissue is readily digested by bromelin, but myosin,when separated from the tissue by the usual methods of preparation, issomewhat more resistant to the proteolytic action of the ferment. It isdigestible, however, and yields the ordinary produicts of proteolyticaction. The myosin used in the experiments about to be described wasprepared from fresh lean beef by extracting the finely divided tissuewith large quantities of thymolized water until all soluble matter wasremoved, and then dissolving out the myosin with a 10 per cent.solution of sodium chloride. From this solution, after filtration throughcloth and finally through paper, the myosin was separated as a jelly-likemass by long continued dialysis, the latter process being continueduntil all traces of salt were removed. So prepared, the myosin wasalmost completely soluible in dilute hydrochloric acid, and was at oncesubjected to the action of the ferment without being dried.

Preliminary experiments tried with various solutions of the fermentshowed that the proteid was acted upon by the ferment in very weak

1 Kluhne and Chittenden. Zeitschrift fur Biologie. Bd. xx. p. 11; Bd. xxii.p. 409 and p. 423; Bd. xxv. p. 358, etc.

305

R. H. CHITTENDEN.

acid and alkaline solutions as well as in a neutral fluid, but proteolyticaction appeared most vigorous in the presence of very small amounts ofhydrochloric acid.

Consequently, such digestions as were made were carried on in thepresence of a very weak solution of hydrochloric acid. Thus, in oneexperiment, 1350 grams of moist myosin were warmed at 40°C. with3-5 litres of 0-025 per cent. hydrochloric acid, in which the myosinalmost entirely dissolved. To this solution 1 gram of bromelin wasadded and the mixture retained at the above temperature for 70 hours,a little thymol being added to prevent any putrefactive changes. Afew hours after the addition of the ferment, the solution became verythick and semi-gelatinous from the separation of what appeared to beacid-albumin. As the digestion proceeded, however, this separationgradually disappeared, but there still remained a certain amount ofmaterial apparently incapable of being further altered by the action ofbromelin in an acid solution. At least, repeated exposure of theinsoluble matter to the action of a fresh ferment solution failed tomaterially diminish the amount. This body may be considered simplyas a form of antialbumid, insoluble in dilute acid but soluble in weakalkaline fluids. The quantity obtained was too small for analysis, andthe reactions showed nothing at all peculiar, or especially characteristicaside from what has been already mentioned.

The clear filtered digestive fluid may, or may not give a neutraliza-tion precipitate on addition of dilute sodium carbonate. This is simplydependent upon the strength of the ferment solution, the rate ofdigestion, etc. In one experiment, where digestive action was some-what slow, a moderate amount of this neutralization precipitate wasobtained, having all the properties of an ordinary acid-albumin;i.e. readily soluble in both dilute acid and alkali, but insolublein water and salt solutions of various strength. This sample wasthoroughly washed with water, alcohol and ether, and when driedat 11000. until of constant weight gave on analysis the followingresults:

Neutralization precipitate.

I. 03619 gram substance gave 0-2232 gram H.0 = 6-85 per cent. Hand 0 6925 gram CO= 52'18 per cent. C.

II. 0 3720 gram substance gave 48-6 c.c. N at 15-00 C. and 764-0 mm.pressure = 1 5 63 per cent. N.

III. 0Q3963 gram substance gave 0-0036 gram ash = 0-98 per cent.

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Percentage composition of the ash.free substance.Average

C 52-69 52-69H 6-91 691N - 15-78 15-78s } 2462

100-00

The one peculiarity about the composition of this body is itscomparatively low content of nitrogen. Myosin contains 16-86 per cent.of this element, whereas the above neutralization precipitate containsonly 15 78 per cent. of nitrogen. The carbon content of the two bodies,on the other hand, is identical, a fact which might tend to throwdiscredit on the nitrogen result were it not that many of the productsformed by the proteolytic action of bromelin are apparently character-ized by a correspondingly low content of nitrogen.

On boiling the neutralized digestive fluid, freed from the abovedescribed neutralization precipitate, a heavy precipitate is liable toform which increases in amount as the fluid is concentrated. Thismight naturally be considered as simply coagulated proteid, but itdiffers from the latter body by being readily soluble both in 0-5 percent. sodium carbonate and in 0 2 per cent. hydrochloric acid, and isclosely akin to the peculiar beat-precipitate already described under thehead of fibrin digestion. It is apparently a heteroproteose-like body,coagulated or precipitated by heat. As already stated, it is readilysoluble in 0 2 per cent. hydrochloric acid and in 0 5 per cent. sodiumcarbonate, but is insoluble in .5-10 per cent. solution of sodium chloride.Dissolved in either dilute acid or alkali it is reprecipitated by neutra-lization. With cupric sulphate and potassium hydroxide, it gives a pinkcolour and yields the ordinary proteid reaction with Millon's reagent.

A portion dried at 1100 C. until of constant weight gave on analysisthe following results:

Heat-precipitate.I. 0-4159 gram substance gave 0-2622 gram H,O= 7*00 per cent. H

and 0)7932 gram CO = 52'00 per cent. 0.IL 0-5021 gram substance gave 0-3122 gram H,O= 6-90 per cent. H

and 0 9549 gram 0O, = 51 86 per cent. 0.Ill. 0 3887 gram substance gave 52 9 c.c. N at 15.50 C. and 76641 mm.

pressure= 16-30 per cent. N.

307

3. H. CHITTENDEN.

IV. 0-3515 gram substance gave 47-7 c.c. N at 15.00 C. and 767-2 mm.pressure = 16 31 per cent. N.

V. 04001 gram substance gave 0 0040 gram ash = 0 99 per cent.

Percentage composition of the asi-free substance.Average

C 52X51 52X37 - 52-44H 7-06 6-96 7-01N - 16-46 16-47 16-46S} -- 24-09

100-00On concentration of the filtrate from the above heat-precipitate, a

syrupy residue was obtained which was thoroughly extracted withboiling alcohol. By evaporation of this alcoholic solution, crystals ofleucin and tyrosin were obtained which responded to the appropriatetests for these bodies, thus showing that in the digestion of myosin withbromelin, amido acids are formed as in the digestion of other varietiesof proteid matter.

The alcoholic precipitate, presumably composed of proteoses andpeptone, was dissolved in water and reprecipitated by alcohol, after whichit was repeatedly boiled with alcohol for the more comnplete removal ofleucin and tyrosin. The precipitate was then dissolved in water and thepeptone and proteoses separated by saturation of the acidified fluidwith ammonium sulphate after the method already fullv described.The ammonium sulphate-saturated filtrate containing the peptonepresent was then freed from the ammonium salt by crystallization,addition of alcohol and dialysis, the final solution, entirely free fromsulphate, being then concentrated to a syrup and precipitated withalcohoL The gummy precipitate of peptone so obtained was verysoluble in water and gave no precipitate with cupric sulphate, nitricacid, or acetic acid and potassium ferrocyanide. It was, however,precipitated by phosphotungstic acid and phosphomolybdic acid, andgave a bright red colour with the biuret test.

A portion dried at 1100C. until of constant weight gave on analysisthe following results:

Ayosin-Peptone.I. 0 4407 gram substance gave 0-2589 gram H20 = 6-52 per cent. H

0-7486 gram Co, = 46 32 per cent. C.II. 0-2955 gram substance gave 0-1705 gram HSO = 6-41 per cent. H

and 0-5021 gram CO2= 46-33 per cent. C.

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IIf. 0'3833 gram substance gave 47-7 c.c. N at 16.10 C. and 765-5 mm.pressure= 14-86 per cent. N.

IV. 0-3265 gram substance gave 00189 gram ash = 578 per cent.


C 49-16 49-17 49X17H 6X91 6X80 6X85N 15X77 15-77

S} - 28-21

100-00

In common with most peptones hitherto analyzed myosin-peptone,as formed by bromelin, is seen to contain a much lower percentage ofcarbon than the mother proteid from which it originates, agreeing veryclosely in this respect with the myosin-peptone formed by trypsin'digestion. The nitrogen content of the bromelin product, however, isnearly one per cent. lower than that of the trypsin product.

The proteose precipitate ((NH4),SO4 saturation) after being entirelyfreed from peptone was dissolved in a small amount of water, the fluidcarefully neutralized and then saturated with sodium chloride. A smallprecipitate of proto- and heteromyosinose resulted. By dialysis, thesetwo bodies were separated, only enough of each being obtained for afew test-tube reactions. These, however, agreed essentially with thereactions previously described in our study of the gastric digestion2 ofmyosi.

The residue of these two primary myosinoses was removed from theabove salt-saturated fluid by addition of a little acetic acid, and fromthis filtrate pure deuteromyosinose was obtained, after partial removalof the salt by dialysis, on saturation of the concentrated fluid withammonium sulphate. When entirely freed from the sulphate by dia-lysis, and precipitated with alcohol it was obtained as a more or lessgummy mass which was eventually transformed into a yellowish-brownpowder bv long-continued treatment with absolute alcohol and ether.

So prepared, the myosinose was readily soluble in water yielding afaintly alkaline-reacting fluid, the clearness of which was not altered bycarefuil addition of very dilute acid. In reactions nothing especiallypeculiar was noted, the body behaving in essentially the same manneras deuteroalbumose.

1 Chittenden and Goodwin. " Myosin-peptone." This Journal, VoL xII. p. 34.2 Kiihne and Chittenden. Zeitschriftfilr Biologie. Bd. xxv. p. 358.

309

R. H. CHITTENDEN.

A portion dried at 1100 C. until of constant weight gave on analysisthe following results:

Deuteromyosinose.

I. 0-4177 gram substance gave 0 2427 gram H2O = 6 45 per cent. Hand 017266 gram CO, = 47-43 per cent. C.

II. 0 3073 gram substance gave 34 0 c.c. N at 15.90 C. and 767-2 mm.pressure= 13-25 per cent. N.

III. 0-3665 gram substance gave 0-0174 gram ash = 4-74 per cent.

Percentage composition o; the ash-free substance.Average

C 49-79 - 49.79H 6-76 6-76N 13-91 13-91

}} 29-54

100-00

Here again is to be noted the low content of carbon, so characteristicof nearly all deuteroproteoses, and in addition a strikingly low per-centage of nitrogen, which latter may perhaps be explained in the samemanner as the correspondingly low content of nitrogen in deuteroalbu-mose and albumin-peptone.

The following table shows the composition of the several productsanalyzed, and for comparison the composition of the myosinoses andmyosin-peptone formed by the digestive action of pepsin-hydrochloricacid and of trypsin.

C H N S &O AshMyosin 52-79 7-12 16-86 23-23 0-66Protomyosinose, Pepsin HCO' 52-43 7-17 16-92 23-48 1 14Deuteromyosinose, ,, ,, 50 97 7-42 17-00 24-61 1-74Myosin-peptone, Trypsin' 49-26 6-87 16-62 27-25

Bromelin DigestionNeutralization precipitate 52-69 6-91 15178 24-62 0-98Heat-precipitate 52-44 7-01 16-46 24-09 0.99Deuteromyosinose 49 79 6176 13-91 29-54 4-74Myosin-peptone 49-17 6-85 15177 28-21 5178

1 Kiihne and Chittenden. Zeitschriftfiir Biologie. Bd. xxv. p. 358.2 Chittenden and Goodwin. This Journal, Vol. xii. p. 34.

310

CHITTENDEN, Ph.D., Professor of Physiological Chemistry.

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