STUDIES ON THE HEMORRHAGIC SWEET CLOVER DISEASE

IV. THE ISOLATION AND CRYSTALLIZATION OF TEE HEMORRHAGIC AGENT*

BY HAROLD A. CAMPBELL AND KARL PAUL LINK

(From the Department of Biochemistry, ‘CVisconsin Agricdtura~ Experiment Station, University of Wisconsin, Madison)

(Received for publication, September 19, 1949)

In a previous paper (1) we presented an extraction scheme for the preparation of hemorrhagic concentrates with a prothrombin- reducing activity’ approximately 200 times greater than the original spoiled hay. These concentrates are essentially free from neutral fats and waxes, certain pigments, sugars, glycosides, water-soluble polysaccharides, water-soluble acids, amines, al- kaloids, water-soluble proteins, and the water-soluble decomposi- tion products of chlorophyll. At this stage the concentrates are dark blue-green in color, indicating the presence of pigments arising from the degradation of chlorophyll.2 The concentrates also contain neutral materials along with acidic substances which arc water-insoluble, but soluble in 95 per cent ethanol, methanol, and ether, and which form water-soluble sodium salts.

The ratio of physiologically inert materials to the hemorrhagic agent is still too high to permit a practical separation of the latter

* Published with the approval of the Director of the Wisconsin Agri- cultural Experiment Station. Cooperative studies with the Division of Forage Crops and Diseases, United States Department of Agriculture, Washington, through Dr. E. A. Hollowell. Personnel and supply assistance since July 1, 1938, in part through the Natural Science Research Project No. 65-l-53-2349 of the Federal Works Progress Administration (Madison).

i Standardized susceptible rabbits were used as the test animals (2, 3). ) When sweet clover hays undergo spoilage, the temperature in the stacks

rises appreciably. Temperatures between 69-70” have been observed in this work (4). The degradation of the chlorophyll pigments to the por- phyrins is apparently quite complete, for in badly spoiled hays the original green color is completely destroyed.

21

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22 Hemorrhagic Sweet Clover Disease. IV

by any of the usual procedures or the more specialized ones like high vacuum distillation, chromatographic adsorption, etc. All attempts to find a chemical test for the hemorrhagic agent which could be correlated with physiological activity as determined by the prothrombin assay were unsuccessful (2, 3, 5).

No useful purpose would bc served by listing in detail at this time the numerous and diverse unsuccessful trials which finall? led to the fractionation scheme presented below (5). From Step 9 on (see Paper I (l)), the problem of eliminating inert classes of compounds was largely one of freeing the concentrates from certain water-insoluble acidic substances, non-saponifiable sub- stances, compounds with acidic properties containing tertiary nitrogen, acids with basic functional groups, and the higher hydroxy fatty acids soluble in alcohol, ether, and benzene, but insoluble in n-pcntane.

The complete removal of the chlorophyll decomposition products without loss of the hemorrhagic agent prcscntcd the most for- midable problem. After many trials the technique originally employed by Willstiitter in his classical researches in this field (6) was found to be the most practical. Willstatter used various concentrations of hydrochloric acid to separate acidic chlorophyll degradation products (phytochlorins and phytorhodins) having tertiary nitrogen groups. Thus these acids arc separated from one another by variations in their basin’ty rather than their relative acidity, as is customarily done in the separation of acids. When we applied this technique (to fractionate the crude ether extract from Step 9 (1)) using various concentrations of hydrochloric acid, a series of pigmented fractions was obtained.* The pig- mented fractions extracted by the acid from the ether solution were for all practical purposes physiologically inert, while the ether solutions, including the one treated with the concentrated (36 per cent) acid (with an olive-yellow color) retained the bulk of the original biological activity. These experiments demon- strated unequivocally that the hemorrhagic agent does not have a basic group. The presence of an acidic group has been indicated previously (see above).

After the chlorophyll degradation products were removed via Step 10, and the absence of a basic group in the hemorrhagic agent was established, further fractionation techniques were

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H. A. Campbell and K. P. Link 23

concerned largely with the task of removing traces of inert neutral and acidic entrainments. These entrainments, which passed through the extraction process as emulsions, were finally eliminated by extraction with ether, while the hemorrhagic agent was in the form of its water-soluble sodium salt (Step 13). After removal of the non-acidic entrainments the acids were liberated from their sodium salts with hydrochloric acid (Step 14) and shaken out with ether. The residue obtained after the ether was removed possessed an extremely high physiological activity. At this stage the acidic hemorrhagic agent is separated from the higher fatty acids by fractional crystallization from 95 per cent ethanol (Step 15).

I

FIG. 1. Hemorrhagic agent from spoiled sweet clover hay (crystallized from cyclohexanone; magnification 67 X).

From Step 15 on, the hemorrhagic agent is readily purified by recrystallizing it from a benzene-methanol mixture; after two recrystallizations from acetone the maximum melting point of 288-289” is realized.

In the pure state, the hemorrhagic agent has a low solubility in the common organic solvents, but solution is readily effected in basic media owing to its acidic nature (Step 9 (1)). The pure product (Fig. 1) is optically inactive, ash-free, and also free from nitrogen, sulfur, and phosphorus. The molecular diagnosis points to the formula C1~HnOe. A crystalline dimethyl ether CloHloOl(OCH&, m.p. 16%170”, was obtained by methylating with diazomethane.

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24 Hemorrhagic Sweet Clover Disease. IV

The substance gives a positive Folin test (7) and forms a colored, coupled product with diazo;..red sulfanilic acid. The FeCh test is negative when made in ?l&er or 95 per cent ethanol, but posi- tive in cyclohexanone. Attempts to classify the substance on the basis of its solubility and behavior in the usual characteriza- tion schemes failed (8, 9).

The acidic nature of the hemorrhagic agent is reflected in the isolation scheme and its ability to form salts, as well as its behavior in ether towards diazomet;) ; :e. The acidic properties fall be- tween the usual carboxylic acids and the phenols. If the amount

I a

0 I I I I I I

I 9

I

6

6

3

2 IO 15 20 25 30 35 40 45 50 55 60 65

ML. OF 0.01062N H2S04

FIG. 2. Electrometric titration curve of the hemorrhagic agent (A and B indicate the inflection points at pH 8.3 and 5.5).

of acid necessary to change the basic solution from pH 8.3 to 5.5 be taken (the points of inflection A and B on the electrometric titration curve, Fig. 2), a neutralization equivalent approximating 160 is obtained. When the substance, dissolved in neutral acetone containing an excess of sodium hydroxide, with phenolphthalein as the indicator, is titrated back, a neutralization equivalent of 168 is obtained.

The physiological activity (prothrombin reduction (2, 3)) of the pure substance is represented by Fig. 3. Experimentally produced hays and supplies of hay encountered in agricultural

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H. A. Campbell and I<. P. Link 25

practice,3 which had. killed cattle in 1938 and 1939 (5), yield identical products. The yield of the crystalline hemorrhagic agent from the Westfield hay and he experimentally produced hays is essentially the same, and the products have parallel bio- logical activity. These findings are significant, since the pro- thrombin assay (2, 3) and the entire fractionation scheme were based on work done with spoiled hays produced experimentally in 1934-36 (1, 5).

On the basis of our knowledge of the compound, and of prc- viously reported substances with approximately the same elc-

65 r

75

25 I2 24 36 40

1 IME AFTER FEEDING @OU?~

FIG. 3. Increase in the clotting time of the plasma (8 per cent) from Rabbit S-51 after feeding 1.8 mg. of the hemorrhagic agent.

mentary composition, the hemorrhagic agent represents a product which has hitherto not been found in nature.4 The chemical and

J Obtained in February, 1939, from a farm near Westfield, Wisconsin. In a herd of twenty cows, seven died and all the others were in a serious hemorrhagic condition when we took charge of the herd and the hay for experimental purposes. These cattle had been eating the hemorrhagic hay ad Zibilvm for about a month before the characteristic sweet clover disease symptoms became apparent.

4 The causal relationship of the total coumarin content of the common strains of sweet clover (Melilotus alba and ojicinalis) and the tendency of hay made from them to become hemorrhagic when improperly cured have

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26 Hemorrhagic Sweet Clover Disease. IV

physical properties exhibited by the pure substance are as ex- ceptional and anomalous as the disease which it is capable of inducing (12, 13).

In contrast to the known anticoagulants (naturally occurring and synthetic), the hemorrhagic agent from spoiled sweet clover hay appears to have no effect on the clotting powers of normal blood or plasma in vitro (14). Its unique physiological activity becomes apparent only when fed by mouth or after injection into the blood stream. Detailed studies on its mode of action and further work on its constitution and structure are in progress.

EXPERIMENTAL

Extraction Procedure (See Diagram)

Step IO-The blue-green ether solution (3 to 4 liters) realized in Step 9 (see Paper I (1)) is shaken in a separatory funnel with four successive 150 ml. portions of concentrated hydrochloric acid (sp. gr. 1.17 (6)). The highly colored acid layers are com- bined and shaken with fresh ether (150 ml. portions) to remove the entrained hemorrhagic fraction. The ether layers (colored a light yellow or olive-green) are combined. They contain the bulk of the hemorrhagic agent. The dark green hydrochloric acid layers which carry out the phyllo-, pyrro-, and rhodoporphyrins2 and the solids which separate from the ether layer usually contain entrainments of the hemorrhagic agent, but it is not practical to attempt to remove the small quantity lost.

Step II-The ether solution from Step 10, which is now es- sentially free from pigments of the chlorophyll class, is concen- trated to dryness. About three-fourths of the ether can bc removed by distillation at atmospheric pressure; the balance is removed under reduced pressure at 25-28” in an atmosphere of nitrogen or carbon dioxide. The residue is taken up in 10 ml. of benzene, in which the bulk of the hemorrhagic agent dissolves. Some physiological activity is lost in a dark gummy residue which is insoluble in the benzene.

already been pointed out in previous publications by us (10). However, it can be stated with certainty that the hemorrhagic agent described in this communication is not identical with any of the 60 or more coumarin deriva- tives previously isolated from various species of plants including the differ- ent members of the Melilotus family (11).

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H. A. Campbell and K. P. Link 27

Step 12-The benzene solution from Step 11 is diluted with 25 volumes of Skellysolve A. A brown solid separates which is removed by filtering through a layer of Filter-Cel on a Buchner funnel. The Skellysolve A solution is distilled under reduced pressure; the residue is taken up in benzene, and Skellysolve A is added again.

Steps in Concentration of Hemorrhagic Agent Ether solution (from Step 9 (1))

10. Extract 4 time; with HCl (36%)

1 1 HCl extract (discard) 11. Ether solution concentrated to dry-

ness; residue taken up in benzene

I 1 Insoluble material (discard) 12. Add Skellysolve A to benzene solu-

tion; concentrate filtrate to dry- ness; dissolve in benzene, again add Skellysolve A

1 Ppt. (discard)

I

1 13. Concentrate filtrate to dryness; take

rr;hen 0.5% NaOH, extract with

-1 Eth\r solution (discard) 14. Acidify alkaline ‘aqueous solution,

extract with ether

1 Aqueous solution (discard) 15. Concentrate etherlsolution add 9501

ethanol; dense crystals ar;? collectedO by filtration; m.p. 250-255”

1 1 Ethanol filtrate, stearic, oleic 16. Crystals are recrystallized from ben-

acids, etc. (discard) zene-methanol mixture; m.p. 270- 280”+; recrystallized from acetone (2 times), m.p. 288-289” (maximum physiological activity)

Step 13-The Skellysolve A solution obtained from Step 12 is finally concentrated to dryness under reduced pressure at 25-28”, yielding a residue that contains appreciable quantities of non- acidic substances. These are removed by shaking out a solution of the sodium salts with ethyl ether. The residue is taken up in about 200 ml. of 0.5 per cent sodium hydroxide. It is then shaken carefully with several portions of ethyl ether. If,a stable emulsion is formed, the solution is again made acid with dilute hydrochloric acid. By adding the sodium hydroxide to the acidic

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28 Hemorrhagic Sweet Clover Disease. IV

solution carefully the troublesome emulsions are avoided. The hemorrhagic fraction is in the aqueous solution that contains the sodium salts. The ethereal solution of the non-acidic material carries no physiological activity.

Step lQ-The alkaline solution from Step 13 is acidified with dilute hydrochloric acid. The liberated acidic material forms a cloudy suspension. This is separated from the aqueous solution by shaking the suspension with ethyl ether in a separatory funnel. The aqueous layer is discarded.

Step Is-For crystallization of the hemorrhagic agent the ether solution from Step 14 is concentrated to a volume of about 1 ml., to which 5 ml. of 95 percent ethanol are added. At this stage dense crystals form spontaneously at the bottom of the vessel. They are collected by filtration and are thus separated from the fatty acids (physiologically inactive) which also crystallize at this point when the solution is cooled to room temperatures.

Step If?-The crude greenish yellow crystals (6.8 mg.) which melt over the range 250-255” are physiologically active (reduce the prothrombin level). By recrystallizing from a benzene- methanol mixture, the melting point is raised to the 270-280” range. After two recrystallizations from acetone the melting point remains constant at 288-289”.

After the first crystalline hemorrhagic material was obtained, as indicated above, subsequent operations yielded the same crystals in Step 14 at the interface between the water and the ether. Later it was found that the hemorrhagic agent could also be removed fromthe insoluble fractions of Steps 11 and 12 with the methanol-ether mixture, On seeding this solution crystalliza- tion occurred promptly.

Yields of Hemorrhagic Agent-6 mg. of the pure substance were realized from 3.0 kilos of the hemorrhagic hay in the first successful extraction. The next three separations yielded 16, 18, and 23 mg. respectively. Finally the yields rose to 60 mg. in those instances when all of the sixteen operations demanded by the fractionation scheme were executed successfully.

, Analysis and Properties of Hemorrhagic Agent

Melting Point-Capillary, 288-289”. Optical Rotation-[[ar]i5 = 0” in benzene; C = 0.17 in a 4 dm.

tube.

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H. A. Campbell and K. P. Link 29

Qualitative Analysis-Ignition test, negative, ash-free; Beil- stein’s test, negative, no halide; fusion with KN03-Na&Os, nega- tive, no phosphorus; Emich’s nitrogen test, negative, no nitrogen.

Quantitative Analysis-13.940 mg. lost approximately 8 y; the volatile matter was less than 0.1 per cent (Abderhalden drier, 1 mm. of Hg, 78”, 10 hours).

Carbon and Hydrogen Determinations--We are indebted to Dr. H. K. Alber, Biochemical Research Foundation, Philadelphia, for the determinations listed under Analyst B as well as for control qualitative tests which confirmed all of our findings. On a dried sample basis, the results, measured in per cent, were as follows:

I Analyst A I Analyst B

Carbon found. 67.70 Hydrogen found.. Oxygen (by difference). . .

/ 3.50 1 ;g 1 ;g 1 y; 28.80

The ultimate analysis and the molecular weight figures (see below) suggest that the empirical formula is in the range C~~HUO~.

Molecular Weight by Micro-Rust Method-The substance shows no appreciable solubility in camphor. Its solubility in borne01 is sufficiently high so that this medium can be used. The average value of 322 was obtained from several determinations. This value is undoubtedly low, since the hemorrhagic agent undergoes some decomposition in the borne01 melt.

Neutralization Equivalent-An excess of 0.148 N NaOH was added to the hemorrhagic agent dissolved in neutral acetone. After the solution had stood for 12 hours it was titrated back with standardized HCl with phenolphthalein as the indicator (pH 9.0); 6.20 mg. required 2.47 ml. of 0.148 N NaOH. This indicates a neutralization equivalent of 168.

Solubility-The pure crystals, m.p. 288-289”, are slightly soluble in benzene (10 mg. per ml. at 76”). Their solubility in acetone and ethyl ether is somewhat lower. In cyclohexanone approximately 8 mg. dissolve in 1 ml. at 25’. The substance shows no substantial solubility in the other common alcohols, ethers, ketones, glycols, and hydrocarbons. Solution is readily effected in pyridine (salt formation) and also in quinoline. The hemorrhagic agent partitions between ethyl ether and dilute bicarbonate solution. Dilute NazCOa or NaOH dissolves about

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30 Hemorrhagic Sweet Clover Disease. IV

1 mg. per ml. In alkaline solution above 12 per cent concentra- tion, the sodium salt is precipitated (salted-out).

Tests for Functional Groups-A positive reaction is obtained with the Folin phenol reagent (7). The ferric chloride test is negative when made in water or 95 per cent ethanol, but positive in cyclohexanone. No reducing action on warm alkaline solutions of silver and cupric salts is observable. On coupling with di- azotized sulfanilic acid, a colored product is obtained. All other qualitative tests gave negative results (8, 9).

Electrometric Titration of Hemorrhagic Agent (Fig. 29-30 mg. were dissolved in 5 ml. of 0.1065 N NaOH. The acidic parent substance was then liberated by titrating with 0.0108 N HzS04. The titration curve was followed by pH measurements with a glass electrode.

Crystal Habit-When analytically pure, the hemorrhagic agent crystallizes from benzene, acetone, and cyclohexanone as hexagonal prisms (see Fig. 1).

Preparation of Dimethyl Ether-The hemorrhagic agent can be methylated with diazomethane and yields a non-acidic dimethyl ether. Attempts to methylate it with dimethyl sulfate failed.

To 40 ml. of dry ether, 34.0 mg. of the hemorrhagic agent were added, whereupon the resulting suspension was cooled to -4’. Diazomethane (1.4 gm. in ethyl ether) was distilled into the chilled solution. The ether suspension which contained diazo- methane in excess was allowed to stand at 25” for 36 hours.

The dimethyl ether separated as a finely divided floe. After the excess diazomethane was removed under reduced pressure, the ether suspension was treated with 0.5 per cent NaOH to remove the acidic unchanged starting material. The non-acidic frac- tion (the methylated product) was collected on a filter and washed thoroughly with ether. As obtained the methylated product is analytically pure. By recrystallization from a large volume of methanol, crystals are obtained, which melt at 168-170’. The dimethyl ether does not show the color tests exhibited by the parent substance.

Analysis-Carbon found, 69.18, 69.16; hydrogen found, 4.40, 4.37; equivalent to C21HleO6, mol. wt. 364. Mol. wt. (micro-Rast) with borneol, 340 (average). Methoxyl content, calculated for CISH~~O~(OCHFJZ, 17.03; found, 17.15, 17.20.

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H. A. Campbell and K. P. Link 31

Physiological Activity (Prothrombin Reduction)

Bioassay of Crystalline Hemorrhagic Agent (2, S)-1.8 mg. fed to Rabbit S-51 (with a relative clotting index of 0.50 at the time of standardizations) gave an index of 0.50 after 24 hours and a relative clotting index of 0.10 after 40 hours.

Control dosage experiments made with rabbits whose relative clotting index values are between 0.50 and 0.30 at the time of standardization indicate that the reduction in the prothrombin level or activity realized with 50 gm. of the hemorrhagic hay sample (one dose, Paper II) is approximately equivalent to the reduction caused by 1.5 mg. of the pure crystals.

The increase in the clotting time effected by 1.8 mg. with Rab- bit S-51 is indicated in Fig. 3. This graph is representative of the response realized when rabbits with approximately the same sensitivity (relative clotting index = 0.50 at the time of standard- ization) are fed 60 gm. of the hemorrhagic hay sample instead of 50 gm. as in the standardization procedure (2).

Bioassay of Dimethyl Ether-5 mg. were fed to standardized susceptible rabbits with a relative clotting index of 0.50 to 0.30 at the time of standardization. No reduction in prothrombin level or activity resulted from this dosage. When the dimethyl ether was treated with alcoholic KOH at 25”, an acidic fraction was obtained which exhibited some physiological activity. This treatment most likely resulted in a partial demethylation of the ether. Since the ether does not reduce the prothrombin level, it appears that in the parent substance the free acidic hydroxyl groups are involved in the physiological activity.

E$ciency of Isolation Scheme and Percentage of Hemorrhagic Agent in Spoiled Sweet Clover Hays

With the bioassay data presented above as the basis for the calculation, the percentage of the hemorrhagic agent in the spoiled hays used during the past 6 years can be expressed. The experimentally produced spoiled hays from Melilotus alba with the highest physiological activity, as well as the Westfield, Wis- consin hay which killed cattle, contain approximately 0.003 per cent of the hemorrhagic agent on the dry substance basis. Since

6 The relative clotting index is the ratio of the concentration of the nor- mal plasma in the concentration range of 12.5 to 8.34 per cent to the con- centration of the pathic plasma which gives the same clotting time (2).

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32 Hemorrhagic Sweet Clover Disease. TV

the prothrombin-reducing action of 50 gm. of the hay is about the same as 1.5 mg. of the pure hemorrhagic agent, 3 kilos of the spoiled hays (the quantity employed in each extraction) contain approximately 90 mg. With the highest yields of the pure prod- uct realized consistently as the basis for thecalculation (60 mg. per extraction), the over-all recovery is 66 per cent. This yield is from many standpoints extraordinary when all the contingencies are considered.

SUMMARY

1. The hemorrhagic agent in spoiled sweet clover hay (Meli- lotus alba) has been isolated in a pure state, m.p. 288-289”, from experimentally produced spoiled hays as well as from hays that killed cattle in agricultural practice.

2. The substance has the empirical formula C19H1206. It is optically inactive. There are two acidic hydroxyls in themole- cule. The acidity of the pure substance falls between that of the phenols and the carboxylic acids. A crystalline dimethyl ether C19H1O04(OCH& with a melting point of 168-170” (physi- ologically inactive) has been prepared by methylation with di- azomethane.

3. In the pure state the substance has a low solubility in the ordinary organic solvents. It is insoluble in acid media. Basic solvents and dilute alkali effect solution readily (salt formation).

4. The substance could not be characterized or identified on the basis of its behavior towards the usual identification reagents. Its occurrence in nature has not previously been reported.

5. 1.5 mg. of the crystalline hemorrhagic agent cause approxi- mately the same reduction in the prothrombin level or activity of the plasma of standardized susceptible rabbits in 40 hours as 50 gm. of the standard spoiled hay sample.

6. Spoiled sweet clover hays produced experimentally from Melilotus alba and those realized in agricultural practice contain approximately 0.003 per cent of the hemorrhagic agent on the dry substance basis. The over-all yield of the substance in a fractionation scheme involving sixteen steps approximates 66 per cent of the quantity present.

During the 6 years that have elapsed since the first experiments on the isolation of the hemorrhagic agent were begun, various

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H. A. Campbell and K. P. Link 33

members of my laboratory not included as authors have contrib- uted much to this work in a very willing manner. Our thanks are due to Dr. E. W. Schoeffel for conducting the initial extrac- tion tests (1934) involving organic solvents. Mr. Lothar Joos assisted in the preparation of crude concentrates from July, 1938, to April, 1939. Mr. Ralph Overman has helped with the bioassays since July, 1938. The molecular weight determinations of the hemorrhagic agent and the photomicrograph were made by Mr. Mark Stahmann. Mr. William Sullivan made the electro- metric titration curves. Mr. C. F. Huebner and Mr. Mark Stahmann repeated the entire isolation scheme independently after the first crystalline material was obtained and the former also developed the technique for the preparation of the crystalline dimethyl ether of the hemorrhagic agent. Finally, Mr. R. J. Dimler and Mr. Mark Stahmann assisted in the preparation of the manuscripts for publication. (K. P. L.)

BIBLIOGRAPHY

1. Campbell, H. A., Roberts, W. L., Smith, W. K., and Link, K. P., J. Biol. Chem., 136,47 (1940).

2. Campbell, H. A., Smith, W. K., Roberts, W. L., and Link, K. P., J. Biol. Chem., 138, 1 (1941).

3. Campbell, H. A., Smith, W. K., Overman, R. S., and Link, K. P., J. Agric. Research, in press (1941).

4. Roberts, W. L., Summaries of doctoral dissertations, 1936-37, Madison, 2, 204-206 (1938).

5. Campbell, H. A., Summaries of doctoral dissertations, 193940, Madi- son, 6, 155 (1940).

6. Willstatter, R., and Stoll, A., Untersuchungen tiber Chlorophyll, Berlin (1913).

7. Folin, O., and Denis, W., J. Biol. Chem., 12,239 (1912). 8. Staudinger, H., Anleitung zur organischen qualitativen Analyse,

Berlin, 2nd edition (1929). 9. Meyer, H., Analyse und Konstitutionsermittlung organischer Ver-

bindungen, Berlin, 5th edition (1931). 10. Roberts, W. L., and Link, K. P., J. Biol. Chem., 119, 269 (1937); In&

and Eng. Chem., Anal. Ed., 9, 438 (1937). 11. Spiith, E., Ber. them. Ges., 70, 83 (1937); Monatsh. Chem., 69, 75 (1936). 12. Roderick, L. M., and Schalk, A, F., North Dakota Agric. Exp. Stat.,

Bull. 260 (1931). 13. Roderick, L. M., Am. J. Physiol., 96,413 (1931). 14. Luck, J. M., and Hall, V, E., Annual review of physiology, Stanford

University, 1,297 (1939).

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Harold A. Campbell and Karl Paul LinkOF THE HEMORRHAGIC AGENT

ISOLATION AND CRYSTALLIZATIONSWEET CLOVER DISEASE: IV. THE STUDIES ON THE HEMORRHAGIC

1941, 138:21-33.J. Biol. Chem. 

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