THE INHiBITING EFFECT OF SODIUM FLUORIDE ON … INHiBITING EFFECT OF SODIUM FLUORIDE ON THE ACTION...

THE INHiBITING EFFECT OF SODIUM FLUORIDE ON THE ACTION OF LIPASE.

(Second Paper.)

BY A. S. LOEVENHART AND GEORGE I’EIRCE.

(From the Laboratory of Physiological Chemistry of the Johns Hopkins VniveYsity*)

(Received for publication, November 13, 1906.)

Kastle and Loevenhart’ first noted the remarkable inhibiting action of hydrofluoric acid and sodium fluoride on the ester- splitting property of liver and pancreas extracts. Hanriot hart previously stated that sodium fluoride does not inhibit the action of lipase on monobutyrin. Arthus3 has since confirmed the statement of Kastle and Loevenhart that sodium fluoride inhibits the action of lipase. He fouhd that the action of blood serum on monobutyrin is considerably retarded by this salt. In their experiments Kastle and Loevenhart studied the effect of sodium fluoride on the hydrolysis of ethyl acetate and ethyl butyrate by turbid liver and pancreas extracts. The experiments with ethyl butyrate and liver extract were of fifteen minutes’ duration; the fluoride and hydrofluoric acid acted in a dilution of I to 5000. The per cent. hydrolysis was decreased from 8.54 to 1.13 by the sodium fluoride and from 8.54 to 0.63 by the hydrofluoric acid. In connection with other work to be presented later, we were led to study more closely the inhibiting action of sodium fluoride on the hydrolysis of fats and lower esters with the result that our observations seemed sufficiently interesting to warrant separate publication.

Experimental Methods.-Unless otherwise stated the enzymie preparations used in this work were from the liver and pancreas of the pig. In a few instances corresponding preparations from the dog were employed. We have used: (I) turbid extracts of

1 Anzer. Chew. Journ., xxiv, p. 491, 1900.

*Arch. de physiol.. x. p. 797, 1898. s Jouvn. a% phytiol., iv, p. 5 6. 1202.

33:

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Effect of Sodium Fluoride on Lipase

these organs prepared by grinding IO grams of the fresh tissue with coarse white sand and extracting with water. This was then strained through cloth and the total volume made up to IOO cc. Toluene was always added as the preservative. This preparation we shall refer to as IO per cent. turbid extract. (2) Clear liver extracts prepared as follows: The turbid liver extract just described was placed in the thermostat at 400 C. for about eighteen hours, when coagulated masses appeared. Tt was then repeatedly filtered through an ordinary pleated filter, when a perfectly clear highly active filtrate is invariably obtained. The color of this filtrate varies from a straw color to bright red. We shall refer to this undiluted preparation as clear IO per cent. liver extract, and in experiments in which it was diluted the degree of dilution will be indicated by the per cent. The corresponding preparation from the pancreas has only very slight activity, the enzyme apparently remaining in the coagulum. We have obtained clear extracts of the pancreas by the method proposed by Kastle, Johnston, and Elvove i for obtaining clear extract of the liver. One gram of fresh pig pancreas was ground up with sand and extracted with 75 cc. of water. Then 2 C.C.

of & sodium hydroxide were added and the mixture warmed on the water bath to 380 C. when, 3.5 cc. of & butyric acid were added. This causes a coagulum to separate and on filtering a very faintly opalescent filtrate is obtained. While the filtrate has some activity it is very decidedly less active than the turbid extract. The experiments were conducted with ethyl acetate, ethyl butyrate, diacetin, triacetin, and olive oil. The first four were employed in such quantities that for complete saponification they would require approximately 40 cc. of & sodium hydroxide. That is to say they were employed in acid-equivalent but not in molecular-equivalent quantities. This was done because Kastle and Loevenharta found the hydrolysis to be largely independent of an excess of the ester, and furthermore it has been repeatedly shown that the acid produced inhibits further hydrolysis. For these reasons the esters were employed in quantities which would yield equivalent amounts of acid on complete hydrolysis. The

* Amer. Chem. Joum., xxxi. p. 52x. x904. 1 Lot. cif.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A. S. Loevenhart and George I’eirce 399

quantities actually used were as follows: ethyl acetate-0.1747 gram, ethyl butyrate - 0.23 gram, diacetin - o. I 7 I 4 gram, triacctin-0.1497 gram. Three-tenths of a gram of neutral olive oil was used in each experiment unless otherwise stated. This amount would require 21.36 C.C. of & potassium hydroxide to saponify it. The titrations in the experiments with the lower esters were made with $‘,, aqueous sodium hydroxide. In some experiments litmus was used as the indicator, in others phcnol- phthalein, no difference between the two being noted in the experiments with clear extracts. When turbid extracts were employed, litmus was always used. In the experiments with olive oil 20 C.C. of neutralized 85 per cent. alcohol, made up from absolute alcohol, were added at the conclusion of the incubation and the titration carried out with & alcoholic potassium hydroxide, phenolphthalein being used as the indicator. All the experi ments were carried out in the thermostat at 380-400 C. Toluene was used as the antiseptic. Blanks of two kinds were carried through with each series : (I) Experiments with the active enzyme without the addition of ester; (2) experiments with the boiled extracts to which the ester was added. The initial acidity has always been deducted in the numbers given. The amount of acid formed in these blanks was insignificant except in one case which will be pointed out later.

The numbers given in the tables which follow state the amount of & alkali in cubic centimeters required to neutraliT& the acid produced by the action of the enzyme on the ester. All necessary deductions have been made. The duration of the experiments varied and will be given in connection with each series. As will be seen in the tables, duplicate experiments were carried through in many instances. The averages of identical experiments ase given and these were used in the calculation of the inhibition or acceleration. In the tables the concentration of the sodium fluoride in the total mixture is given.

EFFECT OF SODIUM FLUORIDE ON THE HYDROLYSIS OF OLIVE OIL

AND ETHYL BUTYRATE BY IO PER CENT. TURBID PANCREAS

EXTRACT.

Se&s I.-Duration of experiments, zo hours. Temperature, 3S” C. One cc. of a IO per cent. turbid pancreas extract and 4 C.C. of water,


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

400 Effect of Sodium Fluoride on Lipase

or 3 cc. of water and r CC. of sodium fluoride solution were used in each experiment.

The total volume in each case therefore was 5 C.C. _.~---̂ -.--~--. _.-.---------_---------

N~~i&$~- cmppption

____---_-. --

; ii.“0

43 1l50,000 1: 5,000 “ 6 I:500 6 1:250

- ..--- -.-. I.._-- --- _.... .- . . . ---..-.-.-.-- _... -~- __.._._ -

‘er cept. of Inhibttion. --

-

Y5 13.0 31.5 35.0

-..-

In Experiment I of the above series the pancreas extract showed a large production of acid on standing even when no olive oil was added. This has always been observed. It is un- doubtedly to be attributed to the hydrolysis of the fat of the pancreas itself, which is invariably present in the turbid extract. This large production of acid in the turbid pancreas extract is not observed in aqueous titrations and is therefore due to the higher fatty acids. As shown in Experiments 2 and 3, the acid production was considerably increased by the addition of olive oil. Furthermore, the acid production is decreased by the presence of sodium fluoride, I : 2 50 (Exp. 6), to a point below that observed in Experiment I in which neither olive oil nor fluoride was added. It will be observed that the duplicate experiments do not sholv a perfect agreement. This is probably due to the fact that in measuring these turbid extracts, in spite of thorough shaking before each filling of the pipette unequal amounts of the suspended mat.erial were obtained. The agrcemcnt is sufficiently close, however, for our purpose. Although the differences between some of the duplicates are nearly as great as the difference seen with different concentrations of sodium fluoride, yet the averages show distinctly that with increasing dilution the inhibiting action of the fluoride gradually decreases. When the sodium fluoride acted in a concentration of I: 50,ooo (Exp. 3) practically no inhibition (4.5 per cent.) was noted. It was very important for our purpose to make an accurate comparison of the inhibitory effect of sodium fluoride on the hydrolysis of olive oil and ethyl butyratc under conditions as nearly identical as


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A. S. Loevenhart and George Peirce 401

possible. It was necessary in making this comparison to use an extract which has considerable action on both of these substances. As will be pointed out later liver extract has very little action on olive oil, whereas pancreas extract is quite active on both. Therefore, two series (II and III) of experiments were carried through simultaneously which were identical in all respects except that olive oil was used in one series, while ethyl butyrate was employed in the other. It was necessary to use the same extract for both series because the effect of the fluoride varies to a certain extent with different extracts.

Series Il.-Duration of experiments, 20 hours. Temperature, 3P C. Two C.C. of a turbid IO per cent. pancreas extract and 3 C.C. of water, or

2 C.C. of water and I C.C. of sodium fluoride solution, were used in each experiment. The total volume in each case, therefore, was 5 C.C.

No. of Ex. periment.

tx 115,000,000 1: 500,000 1: 50.000 1: 5,000 1:500

_-

9

_--

--- I _-~--

3.1 6.15 6.90

I‘ 6.30 I‘ 6.05 “ 5.20 ‘I 3.10

live Oil, hms.

Increase in Acidity.

c-3 Averagt - . .-- ____

.__.__..__ I _-_._. -

-- - - - - 13 -

: 3 - 50 -

We wish to emphasize that we do not attach much significance to small results as the method is hardly trustworthy for small differences. There is no doubt, however, that the fluoride, I: s,ooo,ooo (Exp. 3), accelerated the process. We have frequently seen such accelerations when very small amounts of the fluoride are used. Similar results have been obtained with the esters as will be pointed out. It has been observed by various investigators that in many instances a substance which in ordinary concentrations exerts a powerful retarding influence on a process, may in great dilution accelerate it. This has been observed with remarkable frequency and the case under consideration is no exception to the rule. This fact deserves recognition.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

402 Effect of. Sodium Fluoride on Lipase

Series III.--Conditions identical in all respects to Series II, except that ethyl butyrate was used instead of olive oil.

Concegrgtion of

2. 0.0 S.QS 9.28 9.08

43 : : ~~~0~~~00

1 i 50,600 txx 7:7s %

: 1: 5,000 2.73 2183 1:500 1.38 1.38

-.-

Increase in Acidity, -__-- (2) AVWilSX!.

---.-

-- Per cent. of Inhibition.

The comparison of Series II with Series III, in the following summary,shows that the action of turbid pancreas extract on ethyl butyrate is inhibited to a much greater extent ‘by the fluoride than is the action on olive oil.

Thus the concentration, I: ~,ooo,ooo, was approximately the vanishing point of the inhibition with the butyrate, while I: 5,0&o represented the same point with the olive oil. Viewed in this way therefore, the inhibiting effect of the fluoride on the hydrolysis of the butyrate is about one thousand times as great as on the hydrolysis of olive oil. In some experiments with a specially prepared pancreas powder we have been able to inhibit the action on ethyl butyrate to the extent of 85 per cent. by means of sodium fluoride J: 5,000, while the action on olive oil showed no inhibition whatever under the same conditions.

EFFECT OF SODIUM FLUORIDR ON TBE HYDROLYSIS OF ESTERS BY

CLEAR LIVER EXTRACT.

The action of clear liver extract on the lower esters proved to be even more sensitive to the fluoride than the action of the turbid extracts of the pancreas.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A. S. Loevenhart and George f’eirce 403

Series IV.-Duration of experiments, 17 hours. Temperature, 40~ C. One C.C. of clear IO per cent. extract of pig liver, 0.23 gm. of ethyl

butyrate, and 4 cc. of water, or 3 C.C. of water and I C.C. of sodium fluoride solution, were used in each experiment. ------_---__--.^-- -__---.._--. _ ..-.-..-

No. of Ex- periment.

Concefitraption of

- ~

Xncreasc in Acidity. Per cmt. of _-.-. -_---___- __-...__.- - Inhibition. (1) (2) Average ____-- - -

i 0.0 8.43 8.47 8.45

4”

; : ~~~~~~0

1 i so,doo 73’:: 1:os

f% 1:07

::z 3.5

1:OS x7” -._--..-.c --____---____

Thus the fluoride when acting in a concentration of I : 500,ooo

depresses the actions over 50 per cent. but at a dilution of I :s,ooo,- ooo very little inhibition was noted in this series. The effect of the fluoride I : 5,000,ooo varied indifferent series. In some cases it caused an inhibition of as high as II per cent., while in one instance it caused an acceleration of 6 per cent. The action of the fluoride varies considerably with different esters of the lower fatty acids. In this connection we have studied the behavior towards ethyl acetate, diacetin, and triacetin.

Series V.--Ethyl acelate. Experiments were performed in manner described in previous series. Duration, 18 hours at 40°C.

,_____...- --~~- ---.- --- ._.. --,- _--_____.__.

No. of Experiment.

Conce;;tara~.ion of

t

Increase in Acidity, -._---~

(1) l--G---lAGGi: Per cent. of Inhibition.

0.0 1: 50,000.000 ; : gpb”,“”

1 i 60,dOO 1: 6,000

4.43 43’:: 4.81 3.94 3.95 2.23 2:08 2.16

0.65 0.60 0.15 0.16 E 0.10 0.13 0:12

--

Series VI.--Diuc&in. Otherwise, same as above.

m Aver&. -- --__

2.2 --I-- 1.97 29 1.80 2:oo 1.40 1.31

-

Per cent. of Inhibition.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Series VII.-Triacetin. Conditions same. --_-~

The last three series of experiments show that these three esters of acetic acid show about the same degree of inhibition. It seems to make little difference whether the combined alcohol is ethyl alcohol or glycerin. On comparing them with ethyl butyrate (Series TV) it will be seen that the hydrolysis of the latter is inhibited to a much less extent than the esters of acetic acid. The inhibition with acetic esters is about ten times as great as with the butyrate ester calculated from the values found with sodium fluoride, I : 5,000,ooo.

In order to determine whether the inhibition is influenced by the proportion of fluoride to enzyme, a series was carried out in which the amount of enzyme vgried, while the concentration of the fluoride in all the tubes was the same, viz., I: IOO,OOO.

Series VIII.---Duration of experiments, 24 hours. Temperature, 40~ C. Ten per cent. clear extract of pig liver, ethyl butyrate, Na F in every experiment, I :IOO,OOO. The total volume was 5 C.C. in each experiment.

I No. of Ex-

I

Liver perimant. Extract.

-- Na F.

_-.----_

-I-

-I;

‘$

-I-

5

--. C.C. 0

(1)

11.97 5.87

10.41 3.26 8.48 1.86 5.97

:-Et oh4

a 37

b NaOH Required.

(2) I

Average. ----......

12.32 12.14 5.77 5.82

10.56 10.48 3.06 3.16 8.43 8.45 1.86 1.86 El 5.92

E 0:lS

Pet cent. of Retardation.

G - -

52

ii

5;

ii.2

ii.8

From this series of experiments we see that for a given strength of sodium fluoride the smaller the amount of enzyme acting the greater is the percentage inhibition. This proves that there


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


exists a quantitative relationship between the enzyme and the fluoride. ‘V\Te will return to the significance of this result later.

By using very dilute liver extract it is possible to show that, the fluoride, I : ~oo,ooo,ooo, exercises a distinct inhibiting action on the process, as shown in the following.

Series IX.---Duration of experiments, 20 hours. Temperature, 39’ C. One C.C. of IO per cent. clear liver extract was diluted to 16 cc. with water and I C.C. of this was used in each experiment together with ethyl butyrate, and 4 C.C. of water, or 3 C.C. of water and 1 CC of sodium fluoride.

___.-.-.--~~-.~-_

No. of Ex- Concerption of C.C. of ps NaOH Required. Per cent. periment. -. -_~-~-~ ---._---._ ------ of Ketard-

(1) (2) (3) Average. ation. -.~--~~-.-- -... ~-

!

..-- __-___ --.-.-- -~---_

i 0.0 5.20 5.13 5.05 5.13 1: 1,000,000,000 5.08 5.10 5.11 5.10 OS,

3 1: 100.000,000 4.95 4.66 5.02 4.88 4.9 - -..---_ _ .-.-~~-_- -.-~ ~.-~~~--.-- - _..__________ _._ ~.~__ ._~~ . .._.__

Another series of experiments was performed with even more dilute liver extract.

Series X.-Duration of experiments, I 7 hours. Temperature, 4r0 C. One cc. of IO per cent. clear liver extract was diluted to rco c.c. with water and I C.C. of this was used in each experiment together with ethyl butyrate and 4 C.C. of water, or 3 c.c. of water and I cc. of sodium fluoride.

No. of Ex- periment.

C.C. of ZS NaOH Required. Per cent. of

Acceleration.

Thus with this very dilute extract the fluoride, I: ~,ooo,ooo,ooo,

caused a remarkable acceleration in the process. We have already pointed out that the fluoride when acting in very dilute solution also accelerates the action on olive oil, but the amount required to show the acceleration with olive oil would be sufhcient to very greatly inhibit the action on the lower esters.

It was frequently noted after titrating a series that those tubes which contained the fluoride developed acid much faster than the


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Effect of Sodium Fluoride on Lipase

corresponding tubes in which no fluoride was present. Thus TUJXS 3 and 4 of Series VIII were allowed after titration to stand one hour at room temperature when they were again titrated.

No. 3 (no fluoride) required 0.55 C.C. of & NaOfI No. 4 (fluoride, l:lOO,OOO) required 3.10 C.C. of $s NaOlI.

This fact proves that the fluoride does not destroy the enzyme but merely inhibits its action. * It has been shown repeatedly that the enzyme is destroyed by acids, and since the tubes containing no fluoride developed such a large amount of acid during the incubation, the enzyme in these tubes was largely destroyed, The fluoride by inhibiting the action of the enzyme decreased the amount of acid to which the enzyme was exposed during the incubation and thus indirectly conserved it. This is probably the principal reason why the fluoride tubes show a greater acid production than the water tubes after the titration. We found, however, that there is at least one other important factor which must be taken into consideration here. It is this: The inhibiting action of the ‘fluoride increases as acid develops. This was shown by the following experiments:

(1) 1 c.c. of clear 10 per cent. extract of pig’s Iiver. 4 C.C. of water. Ethyl butyrate. Litmus.

(2) Exactly the same as No. 1.

(3) ! cc. of the same liver extract. 3 cc. of water. 1 C.C. of sodium fluoride (1 :lO,OOO). 0.26 C.C. of ethyl butyrate. Litmus.

(4) Exactly the same as No. 3.

Tubes I and 3 were kept constantly very near the neutral point by adding & NaOH as rapidly as they became acid. Tubes L and 4 were

--- * Note added during proofreading.- We have found ‘that the flu&ride

may be completely removed from the extract by dialysis leaving the enzyme uninjured. The activity after dialysis was as great as that of the extract which hsd not been treated with the fiuoride.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


allowed to stand at room temperature. All of the experiments lasted 36 minutes. The results were as follows:

Remarks. Per cent. of Inhibition.

(1) required 5.02 c.c. & NaOH. Kzpt CL! ne+ral p+nt. - 2;; :: ‘( 47.8 “ 0.35 (‘ *‘

Ac,‘d alloted t2 accumulate. “ 79.4

Thus the tubes which were kept as nearly neutral as possible showed much less percentage inhibition than those in which the acid was allowed to accumulate. Hence the inhibiting action of the fluoride becomes greater as acid develops. The probable explanation of this is that hydrofluoric acid is a more powerful inhibitor than sodium fluoride and as the solution becomes more acid more hydrofluoric acid is produced. The fact that an inhibition of 47.6 per cent. was noted even when the mixture was kept neutral proves, however, that sodium fluoride is a powerful inhibitor in itself entirely independent of the production of hydrofluoric acid. Hydrofluoric acid is quite weak and even the organic acids can decompose sodium fluoride with the production of hydrofluoric acid. The difference observed in the inhibiting effect of the fluoride on different esters may be in part attributed to variations in the amount of hydrofluoric acid produced by the acids resulting from the hydrolysis. We are sure, however, that t.his does not explain the very great difference noted between the esters of the lower and higher fatty acids because even when the mixtures were kept neutral in the above experiments, the fluoride I: 50,000 caused an inhibition of 47.6 per cent. whereas sodium fluoride I:~O,OOO only very slightly inhibits the action on olive oil.

Three facts prove conclusively that the inhibition caused by the fluoride is in no way connected with the precipitation of calcium, viz: (1) The amount of fluoride required is so small that at such dilution calcium fluoride is soluble: (2) Ammonium oxalate does not inhibit the reaction to any extent: (3) The addition of calcium chloride does not decrease the inhibiting action of the fluoride.

In order to be sure that the inhibiting action possessed by the iiuoride is not shared by other salts, a series was carried


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


through with the other halides of sodium and potassium and with several other substances.

Series XI.-Duration of experiments, 21 hours. Temperature, 38O C. One C.C. of a xo per cent. clear extract of dog liver, ethyl butyrate, and 4 cc. of water, or 3 C.C. of water and I C.C. of the solution (I : aso), were

used in each experiment. The substance therefore acted in a con-

Substance.

centration of I : 1250.

Water NaCl

iE KC1 KBr

-- Per cent. of Retardation.

Per cent. of Acceleration.

-

-ii - - - - - - - - -

27

ii

-i

Thus we have been unable to find any substance which exercises an effect on the reaction which is comparable to that of the fluoride. Several interesting facts are brought out in the table, however. A comparison of the halides of sodium and potassium shows that in both cases the bromide inhibits the reaction less than the chloride or iodide. In the case of halides and the nitrate practically no difference is seen between the sodium and potassium salts. The chlorides of calcium, barium, man- gane’se; and cadmium all inhibit the process. The results obtained with ammonium sulphocyannte and thiourea are interesting, The former exercises a marked inhibiting action, while the latter is practically without effect. Kastle and Loevenhartr

g Amer. Chem. /own., xxix, p. 397, ‘go3


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A. S. Loevenhart and George Peirce. 409

fdund exactly the same thing in their work on liver catalase. Here also the sulphocyanate exercised a marked inhibiting effect, while its isomer, thiourca, somewhat accelerated the decomposition.

Thus the action of sodium fluoride1 is perfectly characteristic as far as we have been able to determine and the question presents itself, What is the mode of action of the fluoride3 Does it act on the enzyme or on the ester? It is impossible, in the present state of knowledge of enzyme action, to give all the possible ways in which an enzymic process may be affected by a substance. We may say, however, in a general way that there are at least five general modes of action by which a foreign substance (& e. a substance which is not essential to the process or a product of the reaction) may accelerate or retard an enzymic process. (I) It may react with the enzyme. The inhibiting and destruc- tive’action of acids, in general, on animal lipase is an instance of this kind. (2) It may react with the substance upon which the enzyme acts, the zymolyte? An instance of this kind i$ the retarding action of formatdehyde on the coagulation of milk by rennin. Bliss and Novy3 found that rennin is not affected by formaldehyde but that this substance acts on the caseinogen changing it to a body which is not readily coagulated by rennin. (3) It may react with one of the products resulting from the process and remove it from the field of action. An excellent example of this is the accelerating effect of calcium salts on the action of rennin. The calcium salts precipitate t.he paracasein as rapidly as it is formed. (4) The solubiiity of the enzyme or the zymolyte may be altered. (5) The possibility exists that the substance may react with an intermediate compound formed by

I Hydrofluoric acid and in all probability all soluble fluorides would act similarly.

1 It is necessary to have some general term to designate the substance upon which any enzyme acts. The Germans have introduced the term “substrat” and this has recently been used by English and Americafi writers in the forms substrate or substratum. logical justification for this term.

There is certainly nd We propose the term zymolyte because

it is in keeping with general chemical nomenclature and signifies definitely what is meant. It corresponds to “electrolyte,” etc.

d Jown. of Exper. Med., iv, p. 47, 1899.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

410 Effect of Sodium ‘Fluoride on Lipasc

the action of the enzyme on t.he zymolyte in consequence of which its stability may be increased or decreased.

Kastle and Loevenhastl found that hydrocyanic acid accelerates the decomposition of hydrogen peroxide by copper sulphate, and Loevenhartz showed that hydrocyanic acid accelerates the oxidation of formic acid by hydrogen peroxide in the presence of copper sulphate. The probable explanation of both of these facts is that the hydrocyanic acid facilitates in some way the iormation and decomposition of intermediate products formed in the reaction.

In order to be sure that the fluoride does not react in some way with the ester in consequence of which the stability of the latter might be increased, a series of experiments was performed to determine the effect of fluoride on the hydrolysis of the esters by acids and alkalies. It was found that sodium fluoride does not inhibit the alkaline hydrolysis of ethyl butyrate, diacetin, and triacetin to any noteworthy extent if at all, and in the hydrolysis of these esters by hydrochloric acid it acts merely as the sodium salt of any weak acid should. That. is to say, it retards the hydrolysis only by using up a part of the hydrochloric acid and replacing it with the much weaker hydrofluoric acid. Hence we are justified in stating that the fluoride probably does not inhibit the action of the enzyme by reacting with the ester.

It is obvious that the fluoride does not inhibit the process by reacting with either of the products or by altering the solu- bility of the enzyme or ester. Therefore but two of the five possibilities remain. Either it must react with ‘the enzyme or with some intermediate product. We have pointed out that it certainly does not destroy the enzyme. Furthermore, if it causes the inhibition by reacting with the enzyme, we would expect it to retard the hydrolysis of all the esters to a proportionate degree. But we have found large variations when different esters are used. These facts certainly speak against the view that the fluoride reacts with the enzyme.

We have found, however, that the effect of the fluoride increases

1 Amer.Chem. Journ., xxix, p. 397, 1903. a Ber. d. deutsch. them. Gesellsch., xxxix, p. 130, 1906.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A. S. Loevenhart and George Yeirce 411

as the amount of enzyme acting decreases. While we cannot be sure, we favor the view that it reacts with an intermediate product formed by the action of the enzyme on the ester in consequence of which the intermediate product is rendered more stable. This view seems to us to be more in harmony with all the facts which we have brought out.

In general chemicat behavior the fluorides differ widely from the other halides. Corresponding to this chemical dissimilarity the pharmacological action of sodium fluoride differs entirely from that of the other halides. It is highly poisonous to nearly all forms of life, the fatal subcutaneous or intravenous dose being put at 0.15 gram per kilo for dogs.1 Its pharmacological action is doubtless very complicated. Some of its effects are probably due to the removal of calcium, but this alone apparently cannot fully explain its action. The remarkable inhibiting action of sodium fluoride on thcenzymic hydrolysis of esters is the more striking when it is remembered that it is ordinarily considered a protoplasmic poison like chloroform, toluene, etc., which does not interfere with enzymic processes.2 Pavy 3 sounded a warning against this erroneous view and showed that sodium chloride in I per cent. solution inhibits the action of diastase on starch about 20 per cent. Its inhibiting action on the hydrolysis of esters, however, is incomparably greater than on any other enzymic process as far as we are aware, and the possibility naturally suggest itself that its toxic action may be due in part to the inhibition of this class of processes in the organism. It suggests the possibility that some cleavage of this type is of vital importance in the economy.

SUMMA'RY.

I. Sodium fluoride inhibits in a remarkable way the hydrolysis of esters by liver and pancreas extracts.

2. The hydrolysis of ethyl butyrate by pancreas extract is from one hundred to one thousand times as sensitive to the

1 Tappeiner, Arch. f. exper. Path. H. Pharm., xxv, p. 203, 1889; xxvii, p. x08, 1890.

2 Arthus and Huber, Arch. de physiol., Se Ser. iv, p. 651, 1892. 3 Journ. of Physiol., xxii, p. 391, 1898.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

4=2 Effect of Sodium.Fluoride on Lipase

inhibiting action of the fluoride as is the hydrolysis of olive oil, by the same preparation and under the same conditions.

3. The action of clear liver extract on the lower esters is much more sensitivs to the fluoride than is the action of turbid pancreas extract. Sodium fluoride, Y: ~OO,OOO, inhibited the action of z per cent. clear liver extract to the extent of 78 per cent., while that of 2 per cent. turbid pancreas extract suffered an inhibition of only 5 per cent.

4. The hydrolysis of ethyl acetate is inhibited to a greater extent by the fluoride than that of ethyl butyrate. The fluoride inhibited the hydrolysis of ethyl acetate, diacetin, and triacetin to about the same extent. Hence the acid from which the ester is derived seems to be the principal factor in determining the extent of the fluoride inhibition. The inhibition seems to be largely independent of the alcohol from which the ester is derived.

5. Very small amounts of sodium fluoride (varying with different esters and extracts} accelerate the hydrolysis of the fats and lower esters by the extracts. Thus the fluoride, I: I,OOO,-

ooo,ooo, caused an acceleration of sg per cent. in the action of 0.02 per cent. liver extract on ethyl butyrate.

6. The inhibiting action of the fluoride increases as the tubes become more acid. This is probably due to the production of hydrofluoric acid.

7. Sodium fluoride does not influence materially, if at all, the hydrolysis of esters by alkalies and it only affects the hydrolysis of esters by acids as the sodium salt of any weak acid should. From this we conclude that the fluoride probably does not inhibit the enzymic hydrolysis of the esters by reacting with the latter.

8. The inhibiting action of the fluoride cannot be attributed to the precipitation of calcium.

g. The other halides of sodium and potassium as well as many other substances investigated do not possess an action comparable in extent to that of the fluoride. Hydrofluoric acid and in all probability all soluble fluorides affect these processes similarly to sodium fluoride,

JO. The greater the amount of enzyme acting the less will be the inhibiting effect of a given concentration of sodium fluoride.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


This proves that there exists a quantitative relation between the enzyme and the fluoride.

II. Sodium fluoride does not destroy the enzyme. 12. Of the two modes of action to be considered, (I) that the

fluoride reacts with the enzyme, (2) that it reacts with an intermediate product formed in the action of the enzyme on the ester and resulting in an increase in the stability of the intermediate compounds, we favor the latter view, because the enzyme is not destroyed by the fluoride and furthermore because the inhibition varies to such an extent with different esters.

13. We suggest that the substance upon which an enzyme acts be called the “ zymolyte, ” since this term is more in keeping with general chemical nomenclature and is preferable in every way to the term substrate which has recently been introduced.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A. S. Loevenhart and George PeirceLIPASE: Second Paper

FLUORIDE ON THE ACTION OF THE INHIBITING EFFECT OF SODIUM

1907, 2:397-413.J. Biol. Chem.

http://www.jbc.org/content/2/5/397.citation

Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

alerts to choose from all of JBC's e-mailClick here

#ref-list-1

http://www.jbc.org/content/2/5/397.citation.full.htmlaccessed free atThis article cites 0 references, 0 of which can be


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/2/5/397.citation

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;2/5/397&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/2/5/397.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=2/5/397&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/2/5/397.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/2/5/397.citation.full.html#ref-list-1

http://www.jbc.org/content/2/5/397.citation.full.html#ref-list-1

http://www.jbc.org/

THE INHiBITING EFFECT OF SODIUM FLUORIDE ON … INHiBITING EFFECT OF SODIUM FLUORIDE ON THE ACTION...

Documents

Transcript of THE INHiBITING EFFECT OF SODIUM FLUORIDE ON … INHiBITING EFFECT OF SODIUM FLUORIDE ON THE ACTION...