THE SULFUR OF INSULIN.

BY VINCENT DU VIGNEAUD.

(From the Department of Vital Economics, the University of Rochester, Rochester, New York.)

(Received for publication, July 1, 1927.)

Dudley (1) was probably the first to hint at the possibility that insulin contained sulfur, but he did not feel justified in drawing any conclusions as to the relation between activity and sulfur content. He marked the very strong organic sulfur reaction given by his purified product when boiled with lead acetate and sodium hydroxide. He was unable to obtain, however, a positive nitroprusside reaction on insulin itself or in a reduced solution of his “insulin hydrochloride.” He was therefore convinced that the sulfur in the intact substance was not reducible as it is, for example, in glutathione.

Piper, Allen, and Murlin (2) and Shonle and Waldo (3) also reported a negative nitroprusside test for the sulfhydryl group. These investigators made the interegting observation that after boiling with acid the lead- blackening sulfur was reduced three-fourths.

Blatherwick (4) likewise obtained a negative test on the intact insulin. Upon reduction, however, with sodium cyanide he was able to get a very distinct positive test, showing that although the sulfhydryl group, as such, is not r~ :sent inI the insulin material the sulfur is present in a re- ducible fc,m.

It .remained for Abel and Geiling (5) to demonstrate the high degree of lability of the sulfur and its relationship to potency. They found that merely boiling with so weak an alkali as 0.1 N sodium carbonate so affected the sulfur that upon acidification hydrogen sulfide was given off. These authors felt justified from their work to draw the conclusion that the labile sulfur was directly proportional to the degree of hypoglycemic activity and that “this unstable sulfur is an integral part of the insulin moleculeand that the alteration in its condition consequent upon heating with sodium carbonate bears to the destruction of the physiological activity of the hormone the relation of cause to effect.”

Abel (6) has also found that his crystalline insulin contains sulfur in a

labile form. In a recent publication (7) he and his collaborators have confirmed this finding although their method of obtaining the crystals is somewhat different.1

1 I have recently been able to isolate by the method devised by Abel ,ind his coworkers a small quantity of crystals identical with those de-

393

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394 Sulfur of Insulin

Brand and Sandberg (8) have brought forth some very interesting evi- dence concerning the lability of the sulfur of cystine derivatives. They have shown that although the sulfur of cystine itself is quite stable, the linking of other amino acids to it has such an effect that the sulfur of the resulting compound is labile. For instance, under the same con- ditions of boiling with 0.1 N sodium carbonate for 45 minutes, cystine gave off only 2.8 per cent of it,s total sulfur, whereas dialanyl-cystine gave off 18.6 per cent and dialanyl-cystine dianhydride, 91.8 per cent. Insulin they found under the same experimental conditions gave off 35.6 per cent of the total sulfur and glutathione, 31.2 per cent. They, therefore, feel that the finding of a sulfur lability different from that of cystine for insulin cannot be interpreted as “evidence that, (a) a sulfur-containing amino acid other than cystine is present, or that (6) the sulfur in such compounds is present in more than one form of combination.” Abel and Geiling’s observation on the high degree of lability of insulin-sulfur is therefore not opposed to the idea that cystine is a constituent of the insulin molecule.

Brand and Sandberg tried to isolate cystine from the hydrolytic products of a sample of insulin. They obtained 150 mg. of dark brown crystals from the neutralized acid hydrolysate of 1 gm. of insulin containing ap- proximately 21,100 units. The crystals gave a positive Millon’s reaction and a very strong reaction for lead-blackening sulfur. The further purification of the material, they stated, was very difficult. From about one-half of the material they obtained some slightly yellowish crystals, which looked microscopically like cystine platelets and showed a rotation of [a]“,” = - 212.5O.

Other workers have reported values for the cystine content of hydro- lyzed insulin by the Folin-Looney method and by the method of Van Slyke. By the former method Shonle and Waldo (3) reported a positive reaction for cystine, and by the latter found that the cystine nitrogen was 3.2 per cent of the total nitrogen. Scott (9) found only 0.5 per cent of the total nitrogen as cystine nitrogen, and Glaser and Halpern (10) also using the Van Slyke method found none. Doisy and Weber (ll), on the other hand, using the calorimetric procedure of Folin and Looney obtained a value of 13 per cent for cystine. Their product was a very pure one assaying about 49 units per mg. Blatherwick, with two products containing 25 units per mg., found by the Folin-Looney method 6.6 per cent in one and 7.1 per cent in the other. It is interesting to note that the more potent

scribed by t,hem. The crystals were washed with 30 cc. of ice-cold water and with 30 cc. of 95 per cent ethyl alcohol. 0.02 mg. per kilo lowered the blood sugar of a rabbit 77 mg. per 100 cc. of blood. The rabbit unitage per mg. is no doubt even higher but this happened to be the smallest amount used in the series of tests made. Slightly larger amounts caused very severe convulsions which were relieved by administration of glucose. The presence of labile sulfur in the crystals was also confirmed.

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preparations contain a very high percentage of cystine as determined by the Folin-Looney method.

Neither the Folin-Looney reaction nor the Van Slyke method is specific for cystine in its application to an unknown substance. The great dis- crepancy between the two methods, however, is most likely explainable by the fact that boiling with strong acid so affects cystine that only 40 per cent is precipitable by phosphotungstic acid in the Van Slyke method (12). Working with small amounts of material as with insulin it is readily under- standable why such low figures have been reported by this method.

That the lability of sulfur is not peculiar to insulin, but more or less a general property of proteins, is well recognized. In determining the labile sulfur of a number of proteins Blatherwick (4) found that “the labile sulfur in the insulin proteins is no more sensitive to alkalinity than the labile sulfur in keratin and zein.” With gelatin and casein he obtained only a trace of sodium carbonate labile sulfur.

These workers have been led to regard the labile sulfur of insulin prepara- tions as associated with the adsorbing proteins, and yet, they state that their biuret-free insulin contains 7 per cent labile sulfur. This is much higher than that of any of the products reported that contained protein. The observation that their biuret-free insulin contained such a large amount of labile sulfur would seem to be in favor of the view that insulin contains sulfur as an essential element. We have no evidence, however, that the disulfide linkage, per se, carries the physiological activity; there may well be other necessary structures present, such as the guanidine or imidazole groupings. In spite of this high sulfur content the potency of Blatherwick’s biuret-free compound was not any greater per mg. ; in fact, less than some of the biuret-positive samples. This might be explained in two ways. In the first place, in the process of obtaining the product, some other grouping might have been attacked, destroying some of the potency without affecting the labile sulfur. Secondly, the body of the assay animal might be able to destroy the biuret-free compound much more easily, with the result that its apparent activity is much lower than that actually present. The demonstrated greater susceptibility of the biuret-free insulin to inactivation makes this seem very likely.

Since evidence has accumulated that insulin is actually a sulfur-containing compound, it becomes imperative to discover the type of sulfur.linkage. This is important as a step towards the solution of the structure of the molecule and also for estab- lishing the minimum molecular weight from the empirical formula. It was therefore the purpose of this investigation to determine the type of sulfur linkage present and the source of the labile sulfur. It was also hoped that the study of this problem might yield a chemical method of assay.

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396 Sulfur of Insulin

EXPERIMENTAL.

The disulfide and the sulfhydryl types of sulfur are most com- mon and possibly the most important linkages in biological com- pounds. Enough evidence existed at the time this work was undertaken to eliminate the sulfhydryl group from consideration as being present in the intact molecule. Although other types were possible, it was natural to consider first the disulfide linkage. The question immediately arose as to whether or not unhydro- lyzed insulin would reduce the phosphotungstic acid reagent of Folin and Denis after preliminary reduction with sulfite, a reaction applied to the quantitative determination of cystine by Folin and Looney (13).

While many other substances reduce the phosphotungstic acid, giving a blue color, the significant fact is that most of these sub- stances give the color directly with the reagent and do not require to be reduced first with sodium sulfite as does cystine. It can readily be seen, however, that this test cannot be considered speci- fic for cystine, but rather for the disulfide linkage. In the field of general organic chemistry there are, at least theoretically, innum- erable substances which upon reduction might in turn reduce phosphotungstic acid. In the more restricted range of biochemi- cal compounds only cystine has been shown to act in this manner. Folin and Looney (13) state that neither tryptophane nor tyro- sine, nor any other known amino acid except cystine gives the reaction in this procedure. Of sulfur compounds the disulflde linkage is the only type that will not react directly but which upon reduction with sodium sulfite in alkaline solution will give the blue color with the reagent. Such compounds as thiourea, thioaceta- mide, and thiobarbituric acid fail to respond to the above pro- cedure. There is no reason to believe that sulfur linkages other than disulfide would react in the manner described for cystine.

It was found, as others have found, that insulin preparations do not give a blue color directly with the phosphotungstic reagent but upon preliminary treatment with sulfite they do react. A disturbing factor arose in the fact that upon saturation of a solu- tion of insulin with sodium carbonate the insulin material was thrown out of solution. It was therefore decided to study the reaction on the hydrolyzed material. Later a method was found to apply the reaction to the unhydrolyzed insulin.

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V. du Vigneaud 397

Source and Pur$i.cation of Insulin Used.

The insulin used in the following studies was samples of puri- fied iletin U-440. 110 cc., containing 48,000 clinical units, by the manufacturer’s assay, were purified by Abel’s procedure (5). Sample PK represents Fraction III, in this procedure, the por- tion insoluble in ~/6 acetic acid after treatment with phenol. It was a dark brown powder weighing about 0.2 gm. Fraction IV, the portion soluble in ~/6 acetic acid after phenol treatment, was further purified by pyridine precipitations, by salting out, and by a modification of Abel’s brucine method. Upon the addition of a ~/6 solution of brucine to a solution of Fraction IV in ~16 acetic acid, a precipitate is formed. Addition of ~16 pyridine to the supernatant fluid causes a further precipitate to form. The bru- tine precipitate can be redissolved in ~16 acetic acid and repre- cipitated by the addition of brucine. Again a precipitate is formed upon the addition of pyridine to the supernatant fluid. This process of dissolving the brucine precipitate in acetic acid, reprecipitating with brucine, and then adding pyridine to the supernatant fluid was carried out until the addition of pyridine no longer gave a precipitate with the supernatant fluid. This frac- tion which was entirely precipitable by brucine was designated as PS. The fraction that had originally remained in solution upon the addition of brucine and which had been precipitated by pyri- dine, was redissolved in acetic acid and the brucine solution added again. A precipitate formed. It was separated by centrifugation and the insulin material remaining in solution precipitated by the addition of pyridine, The latter precipitate was again dissolved in acetic acid, brucine added, etc. This was repeated until the addition of brqcine failed to give a precipitate. This fraction which contained practically no material precipitable by the bru- tine solution was labeled PR. The pyridine precipitates from the PS series and the brucine precipitates from the PR series were collected together into one fraction and called PU. The three fractions were purified by pyridine-acetic acid precipitations until the products no longer gave a test for brucine by the nitric acid test. In spite of this complete separation of the twofractions, PS possessed a very high degree of potency. It contained about 30 clinical units per mg., almost as great as that of PR. The latter had a potency of about 40 clinical units per mg. and within the

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accuracy of animal testing it was not distinguishable in activity from PU. PS was light brown in color and required more acid for solution than either PR or PU. The latter products were almost white. The yields of the products were as follows:

PR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.317 gm. PU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.636 “ PS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.360 “

Hydrolysis of Insulin.

In our first hydrolyses of insulin 15 to 20 per cent sulfuric acid was used. Upon addition of sulfuric acid a precipitate began to flock out at a concentration of about 5 per cent. On heating, it first seemed to clear somewhat and then reprecipitate as if it had coagulated. It was very slow to dissolve. Although the hydroly- sates gave a negative biuret after 28 hours of heating, a precipitate formed on saturation with sodium carbonate. The hydrolysis had proceeded far enough to break down the linkages responsible for the biuret test, yet the material was not broken down to such an extent that it was not precipitated by sodium carbonate.

20 per cent hydrochloric. acid was found to be much superior to sulfuric acid for the hydrolysis of insulin. With the addition of hydrochloric acid to the insulin solution a precipitate formed at a concentration of about 3 per cent. As the concentration was

‘increased the precipitate seemed to grow less and at 20 per cent the solution had cleared considerably but not entirely. Upon heating, the solution cleared to a great extent and with cooling a precipitate formed again. It was found that after 2 hours heating in a boiling water bath the insulin solution became biuret-free. In the fol- lowing experiments 4 hours were allowed to insure ample time for hydrolysis.

Phosphotungstic Reaction on Hydrolyzed and Unliydrolyzed Insulin and a Possible Method of Assay.

A study was made to correlate if possible the intensities of the phosphotungstic reaction on hydrolyzed and unhydrolyzed insulin preparations with the potencies of the products. The Hunter (14) modification of the Folin-Looney technique was employed. It was hoped that such a study might reveal a chemical assay for

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insulin or at least show whether or not a parallelism existed bk- tween the strength of the reactions and the potency.

To aliquots of the neutralized hydrolysate or solutions of insulin containing about 2 mg. of the substance, 1.4 cc. of 0.1 N sodium hydroxide, 0.1 cc. of 20 per cent lithium sulfate, enough water to make a volume of 3.5 cc., and then 2 cc. of 20 per cent sodium sul- fite were added. After standing 5 minutes, 0.5 cc. of the phos- photungstic reagent of Folin and Trimble were added with shaking. They were then allowed to stand 10 minutes for color develop- ment, diluted to 20 cc., and compared in a calorimeter with stand- ards containing 0.2 mg. of cystine treated in the same manner.

The percentages of cystine found are compared in Table I

TABLE I.

Phosphotungstic Reaction and Potency.

Per cent cystine equivalents.

Intmlin preparation.

U-273 5.3 7.0 15 u-440 5.9 18 PK 6.0 6.3 20 PS 9.0 9.2 30 PR 9.8 11.2 40 PU 12.2 13.2 40 CA 3.0 2 D.4 2.0 I

Unhydrolyzed. Hydrolyzed. Clinical units per mg.

with the potencies of the products. The values shown are aver- ages of a number of determinations. The rabbit testing on the highly purified preparations is extremely unsatisfactory. The column indicating unitage shows, however, the general order of the potency of the products. The color reaction with the hy- drolyzed material is stronger than that with the unhydrolyzed, but seems to run quite parallel to it. The study brings out dis- tinctly an increase in the strength of the reactions with increasing potency. It is very encouraging as a possible method of assay. It is admittedly unsuited for very impure samples containing large amounts of protein impurities. So far this method has not been tried on insulin inactivated by ultra-violet light. Insulin inac- tivated by alkali however gives only a very weak reaction if any.

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400 Sulfur of Insulin

The fact that insulin inactivated by special means still responds chemically in an in vitro method does not necessarily rule that particular method out. If this inactivation, however, could occur in the ordinary procedures used in dealing with insulin then it would be, of course, a serious objection.

A somewhat analagous case might be presented for the chemical method of epinephrine in which a blue color is produced with a phosphomolybdic reagent. It is possible to destroy or at least greatly attenuate the physiological activity of epinephrine with- out affecting the phenolic groups responsible for the in vitro reaction. Nevertheless, the method is a very valuable one.

In the same way an in vitro method of assay for insulin, such as the one suggested here, may be of great value in following the insulin through certain purifications and manipulations, and when free enough from associated proteins be an actual aid in the assay- ing of the amount of insulin present.

Direct Reaction (Labile Sulfur).

In some of the preliminary work on the phosphotungstic reac- tion with the sodium carbonate method,. it was noticed that heat- ing with the sodium carbonate increased the strength of the reac- tion. This fact was evidently due to the labile sulfur and seemed to be worth while investigating as a means of assaying insulin.

It was demonstrated that by heating with 0.1 N sodium car- bonate the sulfur is split off in such a form as to reduce the phos- photungstic reagent directly. With insulin heated with carbonate it was unnecessary to reduce first with sulfite. This suggests that the sulfur is either split off as a sulfhydryl group or as sulfide ion, other evidence pointing more strongly towards the latter. With samples of cystine, heated in the same manner for 45 minutes with 0.1 N sodium carbonate, no direct reaction was obtained, demon- strating that the sulfur of cystine is comparatively stable.

The preliminary studies of this direct reaction did not give much promise for a chemical method of analysis, but nevertheless brought out a very interesting and important fact concerning the lability of the sulfur in hydrolyzed insulin preparations. Insulin hydrolyzed with acid behaved like t,he original insulin towards the phosphotungstic reagent. Only after reduction with sodium

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V. du Vigneaud 401

sulfite did it respond to this test. In marked contrast, however, with the original insulin the sulfur of the hydrolysate was found to be comparatively stable. Heating with 0.1 N sodium carbonate failed to produce the direct reaction just as it did in the case of cystine,

In order to eliminate the possibility of the influence of salt pres- ent in the hydrolysate resulting from the neutralization of the acid, a few mg. of insulin were added to the sample of hydrolysate. Heating with alkali immediately produced the direct reaction, showing that if the sulfur of the hydrolysate had been labile the presence of much sodium chloride would not have prevented its demonstration.

This clearly indicates that in the insulin molecule there is a sul- fur-containing moiety which can be broken off by acid hydrolysis. The sulfur of this fragment is comparatively stable toward alkali, while that in the intact molecule is labile. This observation fur- ther implies that if the active principle actually contains sulfur it cannot be a simple chemical entity adsorbed on the protein but must be a complex substance, the sulfur of which is labile. When this complex is hydrolyzed by acid, the sulfur of the sulfur-con- taining portion becomes stable, due to the absence of the influence of the other groups with which it had been attached. This is very similar to the behavior of the sulfur of cystine when the latter is linked to other amino acids and when it is in the free state, as shown by Brand and Sandberg.

Source of the Labile Suljur.

If this sulfur had as its source the disulfide linkage responsible for the phosphotungstic reaction, we would expect, after treat- ment with alkali and elimination of the sulfur formed as hydrogen sulfide, that the phosphotungstic reaction would be decreased. On the other hand, if this reaction retained its intensity after such treatment, the sulfur split off must have been present in the mole- cule in some other form than the disuhide.

Insulin preparations were heated in a current of nitrogen with 0.1 N sodium carbonate for 45 minutes in a boiling water bath. Acid was then added to liberate the hydrogen sulfide which was determined by an iodometric method (15). The material left in

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the test-tube after the- hydrogen sulfide had been driven off was then evaporated and made up to a volume of 3 cc. containing 20 per cent hydrochloric acid. It was next hydrolyzed under a con- denser for 4 hours in a boiling water bath. The evaporated hy- drolysate wasdissolved in water and the disulfide sulfur determined by the Hunter modification.

After the labile sulfur had been driven off, the disulfide content was most strikingly reduced. Sample PU which had given a value of 13.2 per cent cystine equivalents on the hydrolyzed mate- rial, contained only the equivalent of 2.2 per cent cystine after the above treatment. U-273, which had an original value of 7 per cent, dropped to 1.6 per cent. The amount of hydrogen sulfide driven off was about 32 per cent of the total sulfur. The latter was found to be about 2.8 per cent by the sodium carbonate-sodium peroxide fusion method as used by Blatherwick (16). In this method the sulfur is determined as BaS04 nephelometrically which is not particularly satisfactory. The value of 2.8 per cent sulfur if calculated as cystine would be equal to about 10.4 per cent cys- tine. This does not account for all the phosphotungstic reaction if the latter is calculated as cystine. As we shall show later, there is reason to believe that there is present in the material some sub- stance other than cystine that gives the phosphotungstic reaction under conditions to which cystine reacts.

Sullivan Reaction.

As we have stated the phosphotungstic reaction cannot be considered specific for cystine, for other disulfides can give the reaction. Sullivan (17) has recently found a specific reaction for cysteine. At least he has so far been unable to find any other compound (and he has tested many) that gives this reaction. Cysteine, after reduction by sodium cyanide, gives the reaction.

The cystine content of the insulin hydrolysate was determined quantitatively by this method, a solution of cystine being used as a standard. The unhydrolyzed insulin gave a negative Sulli- van reaction.

One of the experiments will be given. 30.1 mg. of PU were dissolved in 1 cc. of 0.1 N HCl and 2 CL’. of 30 per cent HCl added carefully. The mixture was heated for 43 hours in a boiling water

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bath. After evaporating to dryness, it was dissolved in 6 cc. of water.

Of this solution 2.4 cc. were introduced into a test-tube and after being neutralized were diluted to 5 cc. with enough HCl to make the solution 0.1 N, and 1 cc. of 5 per cent NaCN was added and the mixture allowed to stand 10 minutes. To this, 1 cc. of 0.5 per cent aqueous solution of the P-naphthoquinone sulfonate was added, followed by 5 cc. of a 10 per cent solution of sodium sulfite in 0.5 N NaOH. A red-brown color developed on standing 30 minutes which changed to a cherry-red color upon the addition of 1 cc. of a 2 per cent solution of sodium hyposulfite, NazSz04, in 0.5 N NaOH. Five standards of cystine were run at the same time containing from 0.4 to 2.0 mg. at 0.4 mg. intervals. The standards were in 5 cc. volumes of 0.1 N hydrochloric acid. The color produced in the insulin solution was very close to that pro- duced in the tube containing 0.8 mg. of, cystine. With the latter set at 10 the unknown read 9, giving a cystine value of 0.888 mg. in the unknown, which represents a cystine content of 7.37 per cent.

At the same time a solution of insulin was run which had been treated with 0.1 N sodium carbonate and then hydrolyzed with hydrochloric acid. Although containing the same equivalent amount of insulin the color production was almost nil. It was most striking, this difference in color of the two tubes; one was a bright cherry-red while the other was only a very pale orange. There could be no doubt that the splitting out of the labile sulfur had destroyed the compound which gave the Sullivan reaction. The source of this labile sulfur must have been this compound which, as far as we know now, is cystine, or a substance having. the SH and NH2 groups in close proximity, with the SH groups in the oxidized form; in other words, the disulfide linkage.

The phosphotungstic reaction was also run on an aliquot of the hydrolysate of the 30.1 mg. sample. The average value obtained was equivalent to 13.2 per cent cystine.

The values for cystine equivalents obtained by the two methods do not agree, the more specilic reaction indicating a much lower value. There is evidently present a compound which does not give the specific reaction for cystine but which gives the phospho- tungstic reaction afOer reduction with sulfite. This may be either

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a disulfide compound different from cystine or, possibly, a dipep- tide of cystine still unhydrolyzed. A compound such as the latter would not give the Sullivan reaction but would still respond to the phosphotungstic reaction.

It was conceivable that the boiling of cystine with 20 per cent HCl had so .affected the molecule that although still giving the disuEde reaction, it would fail to give the more specific test. However, 10 mg. of cystine boiled for 4 hours with 20 per cent HCl gave the theoretical value with both of the methods.

CONCLUSIONS.

If the active principle itself actually contains sulfur, as we have good reason to hold, then we believe that the evidence presented in this study indicates that this sulfur is present as the disulfide link- age and that insulin is most likely a derivative of cystine.

By means of the phosphotungstic reaction we found that as the insulin became more putied the cystine content increased. The proportionality was so striking that it has given us hope that this might prove to be a suitable means of assay for pur%ed preparations.

When the sulfur was split out, the disulfide linkage was destroyed and the test for cystine greatly reduced in intensity, indicating this as the source of the labile sulfur.

Since it has been shown that insulin itself does not give the spe- cific Sullivan reaction for cystine, and since we have demonstrated the presence of cystine in the hydrolyzed insulin preparations, it must be concluded that the cystine is present as some derivative. Further, the sulfur of the hydrolysate is comparatively stable like free cystine, whereas the sulfur of the original unhydrolyzed insulin is very labile. This change of lability upon hydrolysis is identi- cal dvith what would be expected of an amino acid derivative of cystine. Glutathione, for instance, has a sulfur lability of the order of insulin. Upon acid hydrolysis free cystine would be formed and the sulfur of the hydrolysate would therefore be stable.

The behavior of the sulfur in insulin is quite parallel to the behav- ior of the sulfur in amino acid derivatives of cystine and suggests that the cystine in insulin is linked to the rest of the molecule by

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a peptide linkage. In this connection the high arginine, histidine, and tyrosine contents of purified preparations might be recalled.

In Abel’s analyses the calculated empirical formula of G5H75017N11S is based upon the presence of 1 sulfur atom in the molecule. From our work on the presence of the disulfide linkage the minimum value would have to be twice that given, or GoH~,o034N&.

The writer wishes to express his appreciation to Professor J. R. Murlin for his interest and helpful suggestions during the course of this investigation.

BIBLIOGRAPHY.

1. Dudley, H. W., Biochem. J., 1923, xvii, 376. 2. Piper, H. A., Allen, R. S., and Murlin, J. R., J. Biol. Chem., 1923-24,

lviii, 321. 3. Shonle, H. A., and Waldo, J. H., J. Biol. Chem., 1923-24, lviii, 731. 4. Blatherwick, N. R., Bischoff, F., Maxwell, L. C., Berger, J., and

Sahyun, M., J. Biol. Chem., 1927, lxxii, 57. 5. Abel, J. J., and Geiling, E. M. K., J. Pharmacol. and Exp. Therap.,

1925, xxv, 425. 6. Abel, J. J., Proc. Nat. Acad. SC., 1926, xii, 132. 7. Abel, J. J., Geiling, E. M. K., Rouiller, C. A., Bell, F. K., and Winter-

Steiner, O., J.Pharmacol. and Exp. Therap., 1927, xxxi, 65. 8. Brand, E., and Sandberg, M., J. Biol. Chem., 1926, lxx, 381. 9. Scott, D. A., J. Biol. Chem., 1925, Ixv, 601.

10. Glaser, E., and Halpern, G., Biochem. Z., 1925, clxi, 121. 11. Doisy, E. A., and Weber, C. J., J. Biol. Chem., 1924, lix, p. xxxiv. 12. Plimmer, R. H. A., Biochem. J., 1927, xxi, 247. 13. Folin, O., and Looney, J. M., J. Biol. Chem., 1922, li, 421. 14. Hunter, G., and Eagles, B. A., J. Biol. Chem., 1927, lxxii, 177. 15. Tre.adwell, F. P., Analytical chemistry, translated by Hall, W. T.,

New York, 4th edition, 1915, ii, 687. 16. Maxwell, L. C., Bisohoff, F., and Blatherwick, N. R., J. Biol. Chem.,

1927, ixxii, 51. 17. Sullivan, M. X., Pub. Ifealth Rep., U. X. P. H. S., 1926, xli, 1030.

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Vincent du VigneaudTHE SULFUR OF INSULIN

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