THE STABILITY OF CYSTINE IN ACID SOLUTION*

BY KAMENOSUKE SHINOHARAT AND MARTIN KILPATRICK

(From the John Harrison Laboratory of the University of Pennsylvania, Philadelphia)

(Received for publication, December 22, 1933)

It has been established by one of the authors (1) that cystine in acid solutions is oxidized by iodine as well as other oxidanm, mostly to cysteic acid. As shown in a preliminary report (2) the oxida- tion is sufficiently slow to be suitable for kinetic study. Since then an extensive study of the kinetics has been made, the results of which will be reported elsewhere. During this study it was found that cystine in acid solution gradually changes on standing, so that the initial consumption of iodine increases. With the purpose of ascertaining the primary cause of this peculiarity, a series of investigations was undertaken, in which it was found that cyst’ine undergoes hydrolysis on standing to produce cysteine. The study is so far semiquantitative, and yet this property of cystine, and disulfides in general, is so important for the under- standing of its chemical nature and biological significance that a report will be made here.

EXPERIMENTAL

In the study of the reaction between cystine and iodine, por- tions of a stock solution of I-cystine in hydrochloric acid, preserved in a glass-stoppered flask at the temperature of the laboratory, were removed from time to time. The portion removed was added to the reaction mixture containing the iodine, and the amount of

* The paper is the second part of the thesis presented in April, 1933, by Kamenosuke Shinohara to the Faculty of the Graduate School of the Uni- versity of Pennsylvania, in partial fulfilment of the requirement for the degree of Doctor of Philosophy.

t Robert McNeil Fellow at the Research Institute of the Lankenau Hospital, Philadelphia. The experimental work was carried out at the Institute.

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242 Stability of Cystine in Acid Solution

iodine consumed was determined at suitable intervals by titra- tion with sodium thiosulfate solution. It soon became evident that the older the stock solution of cystine, other things being equal, the greater was the initial consumption of iodine. The large initial consumption of iodine indicates the formation, on st’anding, of some reducing substance which reacts rapidly with iodine. To determine whether the reducing substance results from the partial oxidation of cystine by the air enclosed in the flask, the following experiments were carried out.

Eflect on Iodine Consumption of Aging of Cystine Solutions in Nitrogen Atmosphere

A 0.005 M I-cystinel solution in 0.025 M hydrochloric acid was

kept in a flask through which nitrogen, purified from oxygen with alkaline pyrogallol solution, was bubbled at a rate of roughly 100 cc. per hour. The bubbling was stopped during the night, but the pressure inside the apparatus was kept higher than that of the atmosphere, thus preventing possible entrance of air. The nitro- gen, after passing through t.he cystine solution, was introduced into lead acetate solution. The whole apparatus was exposed to the indirect light of the laboratory, and the room temperature was 25” f 3”. Portions of the solution were pipetted out when needed through a glass tube which was otherwise kept tightly covered. The technique used for determining the rate of iodine consumption was as follows:

The desired volume of the I-cyst,ine solution was added to the react’ion mixture, which contained iodine, potassium iodide, and sodium acetate and acetic acid, and which was at 25’ f 0.01”. The reaction mixture was then made up to a definite volume by adding wat,er, and the flask was shaken for 1 minute. Portions of the mixture were taken out at suitable intervals and run into sufficient standard sodium thiosulfate solution to react with the greater part of the iodine. The remaining iodine was then titrated with thiosulfate. A blank experiment in which dl-alanine was used in place of I-cystine was run parallel to the main experiment. The consumption of iodine by cystine was calculated from the

1 A 1 per cent solution of this cystine in N hydrochloric acid gave an opti- cal rotation [cI]~~.O Hg (5461.h = -248.5”; its iron content was 0.004 per cent.

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K. Shinohara and M. Kilpatrick 243

difference between the values obtained by the titration of the blank and test solutions, thus minimizing errors caused by loss of iodine by evaporation and reduction due to impurities in the acetic acid.

TABLE I

E$ect on Iodine Consumption of Aging of Cystine Solutions in Nitrogen Atmosphere at 25 f 9”

Composition of reaction Mixture -4 (M per I. X Composition of reaction Mixture I3 (M per 1. X 10%): l-cystine 20.0,* HCl 192.5, iodine 25.0 f 104): Z-oystine 10.0, HCl 192.6, iodine 25.0 f

0.1, KI 120.5, acetic acid 4000, sodium ace- 0.1, KI 120.5, acetic acid 4000, sodium ace- tate 4000, temperature 25.0” * 0 01” tate4000, temperature 25.0” * 0.01”

Time clapsol

min.

2 15 30 45 60 90

120 150 180 210 240 300 360

IZ consumed per 1. (M X 104) in solution aged

Time drtPSC3l

30 25 44 52 66 min. days days days days __- ~-

min.

0.50 0.78 1.66 1.69 2.14 2 1.84 2.89 2.89 3.33 3

2.09 2.64 3.78 4.34 5 3.41 4.53 5.13 15

3.51 4.15 5.19 5.38 5.92 17 4.82 5.46 6.71 6.81 7.34 30 6.10 6.69 7.96 8.10 8.62 45 7.21 7.89 9.09 9.25 9.85 60 8.23 8.90 10.14 10.30 10.89 90 9.22 9.83 11.03 11.23 11.71 120 .0.1410.69 11.78 12.06 12.56 150 l.7012.14 13.33 13.58 14.14 180 L3.0713.54 14.63 14.85 15.32 210

240 300

I I I I I 360

1% consumed per 1. (M X 104) in solution aged

30 24 min. days d:;s __-~

0.83 0.69

0.64 1.51

1.33 1.41 1.73 1.94

2.13 2.44 2.73 2.86 3.22 3.54 3.46 3.83 4.28 4.13 4.53 4.97 4.73 5.16 5.58 5.27 5.70 6.22 5.89 6.31 6.81 6.94 7.25 7.82 7.76 8.26 8.81

65 days

0.67 0.92

1.54 1.64

2.00 2.17 2.49 2.64 2.69 3.06 3.70 3.92 4.43 4.65 5.15 5.34 5.78 6.00 6.37 6.59 6.91 7.22 8.02 8.28 8.91 9.23

* The concentration of cystine somewhat exceeds its solubility, and a small amount of cystinc crystals separated out about 4 hours after the reac- tion mixture was prepared. However, this does not influence the initial consumption of iodine.

Table I indicates that the longer the cystine solution has stood, the greater is the iodine consumption at the initial stage. In the later stage of the reaction, the rates are practically the same. The effect observed with the cystine solutions preserved in an atmos- phere of nitrogen is of the same order of magnitude as the effect observed in the case of the cystine solutions kept in flasks contain- ing air.

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244 Stability of Cystine in Acid Solution

The lead acetate solution showed neither precipitate nor darken- ing, which proves that hydrogen sulfide was not produced in an appreciable amount.

In order to learn whether the change takes place in the dark, an experiment was carried out with 0.01 M I-cystine in 0.2 M hydro- chloric acid, with the experimental conditio:ns the same as those described except that the flask containing the I-cystine was covered with tin-foil. At intervals, portions of the solution were removed and titrated directly with 25 X lo4 N iodine solution until the starch-iodine color lasted for about 30 seconds. During the first 7 days no change was observed. From the 7th to the 80th day (at which time the experiment was discontinued) the increase in amount of iodine solution required was found proportional to the time elapsed.

Effect of Iron and Copper on Aging of I-Cyst&e Solutions in Nitrogen Atmosphere

Since it was suspected that the presence of metals like iron and copper might have some effect upon the aging, experiments were carried out with kcystine solutions 1 X 10V4 M in copper sulfate and ferric chloride respectively. The solutions were preserved in a nitrogen atmosphere, and upon examination of the metal-con- taining and corresponding metal-free solutions at the end of 137 days, no effect of iron or copper upon the aging was detected. The 1-cystine used in the previous experiments was not entirely free from iron, as mentioned before.

Change of I-, i-Cyst&e, and Cysteine in Acid Solution in Nitrogen Atmosphere at 80’

As already pointed out, the change of cystine in acid solution at room temperature is too slow to be studied conveniently and to allow identification of the reducing substance formed. In order to accelerate the change, therefore, experiments were carried out at 80” with I- and i-cystine. The i-cystine was prepared by heat- ing I-cystine in 15 N sulfuric acid. The preparation was optically inactive and dissolved in dilute hydrochloric acid, giving no more than a very slight opalescence. The solution was used after filter- ing off the insoluble matter.

Three solutions were prepared: one 0.01 M I-cystine in 0.2 M hy-

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K. Shinohara and M. Kilpatrick

drochloric acid, one 0.01 M i-cystine in 0.2 M hydrochloric acid, and one 0.02 M cysteine in 0.2 M hydrochloric acid, A portion of solution was placed in the inner chamber of a double-walled flask of about 250 cc. capacity, the outer chamber being connected with a small flask containing boiling benzene, and also with a condenser. Thus the inner flask was const.antly surrounded by the vapor of boiling benzene and the temperature of the solution was found t,o he 79.5” f. 0.2”. The flask was covered with tin-foil. Nitrogen purified as in the previous experiment was passed through the solution at a rate of 200 cc. per hour, and introduced into cadmium sulfate (or barium chloride-bromine mixture) and barium hydrox- ide solutions successively. At intervals the following tests were made.

(a) Modified Nitroprusside TesP-2 cc. of 0.2 M zinc acetate solution then 2 cc. of 1 M ammonium hydroxide solution were added to 5 cc. of test solution cooled to 25”. To the solution 0.5 cc. of 5 per cent sodium nitroprusside solution was added and the final mixture was shaken. The color was compared with the colors of a series of standards.

(b) Cobalt Complex Te&-2 cc. of 0.2 M cobaltous chloride solu-

2 It was found by the authors that the addition of zinc salt in a sufficient amount, before sodium nitroprusside, stabilizes the red color which will be produced when a sulfhydryl compound is present, and moreover it prevents hydrogen sulfide from giving the color reaction. This is probably due to the formation of zinc sulfide. Acetone and pyruvic acid do not give color in this test. Various organic sulfhydryl compounds including cysteine and glutathione are also precipitated by zinc acetate and ammonia; neverthe- less, the precipitate gives the color reaction upon the addition of sodium nitroprusside solution, while the supernatant liquid gives not more than an almost invisible pink color. The color of the supernatant liquid is probably due to the slight solubility of the zinc precipitate. The minimum concen- tration of cysteine which can be detected by this test is approximately 4 X

IO-” M. The intensity of color of the precipitate produced in the test is roughly

proportional to the concentration of cysteine. When the test solution is free from other sulfhydryl compounds, the concentration of cysteine can be roughly estimated by comparing the color of the precipitate with a series of colored precipitates produced from cysteine solutions of various concen- trations.

3 Cobalt complexes of cystine have been studied by Michaelis and his collaborators (3-6), and by Kendall and Holst (7). Readers are referred to their papers. According to the authors’ investigation, cysteine can be

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246 Stability of Cystine in Acid Solution

tion were added to the 5 cc. of the test solution cooled to 25” and the mixt,ure was then made strongly alkaline by the addition of 4 cc. of 0.5 N sodium hydroxide solution. After thorough shaking 1 cc. of 3 per cent hydrogen peroxide solution was added, and the cobal-tic hydroxide was removed by filtration or centrifuging. The brownish yellow color of the solution was st,able for more t.han 12 hours. The final color was compared with the color of standards prepared in the same way.

(c) Iodine Titration-12.5 cc. of the test solution, cooled to 25”, were titrated with 0.01 N iodine solution. It was.found possible to determine by titration with iodine the cysteine concentration (within f4 per cent) of solutions containing both cystine and cysteine in known amounts. From a chart constructed from the titration of known solutions, the cysteine concentrations listed in Table II were read off.

(d) Determination of Optical Rotation-The solution was ex- amined in a 20 cm. tube at 25” f l”, a mercury lamp being used.

qualitatively differentiated by this test from other similar compounds. I’yruvic acid, thioacetic acid, and thiourea do not give any color. Thio alcohols and thiophcnols produce a reddish brown precipitate but no color in the solution. Thiocarboxylic acids, like thioglycolic acid, give a deep brown color to the solution; nevertheless the color can be differentiated from that of cysteine before the addition of hydrogen peroxide. Moreover, the color produced by the thiol acid fades on standing overnight. Gluta- thionc produces practically no color before the addition of hydrogen perox- ide, after which a brownish pink color is produced in the solution. Disul- fides including cystine give entirely colorless solutions. Histidine is the only one among many amino acids that gives a yellow-brown color to the solution. However, the color turns to pink on oxidation by air or hydrogen peroxide. Hydrogen sulfide produces a black precipitate but the solu- tion remains colorless. When any thiol compound is mixed with cyst,inc, the characteristic color of the cysteinc complex is produced. This is prob- ably due to the reduction of cystine to cysteine by the thiol compound as reported by Kendall and Holst with regard to cystine and glutathione mix- tures (7). Hydrogen sulfide mixed with cystine gives a brown which differs from that given by cysteine. As Mauthner (8) reported, the reduction of cystine by hydrogen sulfide is very slow, and it is questionable if the brown coloration is due to cysteine formation. The color intensity produced in this test is proportional to the cysteine concentration, provided the colbalt salt, exists in slight excess.

The cobalt complex test, therefore, can be used, both qualitatively and quantitatively, for the determination of cysteine without serious errors under such conditions as prevailed in the authors’ experiment.

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TABLE II

A. Change of i- and I-Cysline Solutions in Nitrogen Atmosphere at 79.5 f. 0.2”

Composition of the solution: 0.01 M i- or I-cystine in 0.2 M HCl. The concentrations are measured in moles per liter X lo5 throughout.

Optical rotation (X - 1). m2& Cysteine concentration by iodine titration Time

elapsed

0

105 200 270 320 320 340 320

2.Cystine 3

Cystine 3

85 175 240 290 260 300

-Cysthe 1

__-

0 18

50 125 195 240 270 250 240

I-Cy;tirre

1.31 1.30 1.23 1.15 1.11 1.03 0.93 0.91 0.87

i-Cystine 2

-.

1.31

1.18 1.09 0.99 0.90 0.85 0.82 0.85

-Cystill<,

0 20 98

156 225 252 285 300 380

days

0.0 0.25 1.0 2 3 4 5 6 7

1.31

1.21 1.09 0.99 0.92 0.89 0.85

Cysteine concentration

Modified nitroprusvide test Cobalt complex test

+

++

6 17 32 45 50 65

Time elapsed

dayi?

0.0

0.25 1.0 2 3 4 5 G 7

.-

i- Cystine

0 20

120 240 350 420 340 480

Cystinr

0 20

200 320 400 350 480 480

-

1

-

, 1 -Cy?ine

0 20 50

160 220 280 380 320 320

!-Cystine 3

0

100 200 280 320 360 440

-Cystine

0 17 40

120 220 320 360 320 320

‘-Cystine 3

: I

0

100 200 200 320 360 400

B. Change oJ Cysteine Solution in N,itrogen Atmosphere at 79 i 0.d”

Composition of the solution: 0.02 M cysteinc in 0.2 M HCI.

Time elapsed con

M per 2. x 105

0 +

Amount of FSaCOs in- crcascs with t,imc

days M per 1. x 105

0 -0.10 0 1 12 2 24 3 42 4 54 5 65 G 75 7 -0.14 85 51

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248 Stability of Cystine in Acid Solution

(e) Determination of Hydrogen XulJide (f) Determination of Carbon Dioxide Tests (e) and (f) on the issuing gas were carried out in the cus-

tomary manner. Upon the 4th day a yellow precipitate appeared at the bottom of

the flask containing the heated solution of l- or i-cystine, and there- after the amount increased wit’h time. The yellow precipitate was found, upon testing its solubility in carbon disulfide and examina- tion under the microscope, to be sulfur. The heated solution of cysteine, on the other hand, remained clear throughout the 7 day period. None of the solutions gave a test for sulfate.

Experiments in Alkaline Xolution

In order to supplement the experiments mentioned above, qualitative determinations of cysteine in alkaline solutions of cyst- ine were made. These solutions exhibited much greater rates of cysteine formation. For instance, after a few minutes of heating, a solution of I-cystine in 0.2 M sodium hydroxide gave distinct colors in both the modified nitroprusside and cobalt complex tests. At the end of 15 minutes of heating the colors were very intense. In a solution of I-cystine in ammonia the colors developed more slowly than in a solution in sodium hydroxide, but more rapidly than in a solution in hydrochloric acid. This relationship indi- cates that the rate of production of cysteine is dependent on the acidity of the solution. Thus the rate in a concentrated solution of hydrochloric acid (which has frequently been employed for the preparation of i-cystine from I-cystine (9,lO)) probably is very low.

That the hydrolysis of the disulfide to form a thiol compound is not peculiar to cystine, but is characteristic of disulfides, was dem- onstrated by testing diethyldisulfide and dithiodiglycolic acid in sodium hydroxide solution. Both compounds gave intense red color in the modified nitroprusside test after their solutions had been heated for several minutes. They were found, however, to be considerably more stable in 0.2 N hydrochloric acid solution than is cystine.

DISCUSSION

It is beyond doubt that the substance which rapidly reduces iodine, gives red color to the zinc precipitate in the modified nitro-

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K. Shinohara and M. Kilpatrick

prusside test, and characteristic color in the cobalt complex test, is cysteine. The agreement in the values of cysteine concentration determined by the three methods is regarded as satisfactory. Sep- aration of the products was not tried, because it was thought that the chemical processes ordinarily used for the isolation of cysteine salts or derivatives would decompose cystine in a similar manner, probably more rapidly and so that isolation would not serve as proof.

It has been the usual conception that the change in optical rota- tion is caused merely by racemization of I-cystine. The experi- ments presented here show that this is not the case. For the change in optical rotation is accompanied by the formation of cysteine, and if velocity constants are computed from the data in Table II, A for the change in optical rotation, and for the forma- tion of cysteine from I-cystine, it is seen that under the conditions of the experiment cysteine formation constitutes roughly 60 per cent of the total change. The possibility that I-cystine first race- mizes, and the resulting I-cystine in turn produces cysteine, is eliminated by the fact that the rates of cysteine production from both b- and i-cystine are approximately the sa,me. There is, however, no doubt that part of the cysteine is produced from i-cyst- ine previously formed from I-cystine.

A comparison of the amounts of hydrogen sulfide produced from cystine and cysteine in acid solution at 80” tends to favor the mechanism that the cystine first decomposes to cysteine which in turn gives hydrogen sulfide. In regard to the possibility that the hydrogen sulfide is the cause of the formation of cysteine (8), it should be pointed out that several workers (11-15) have carried out experiments using cystine with salts of heavy metals, and have found no indication of metallic sulfides before the production of mercaptides.

The formation of cysteine might be explained by the following reactions :

R-S-S-R + Hz0 = intermediate compounds = R-SH + R-S--OH (1)

(like R-S-OH2-S-R)

BR-S-OH = R-SH + R-S-021% (2)

R-S-OH + R-S-02H = R-SH + R-S-0,H (3)

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250 Stability of Cystine in Acid Solution

Addition of the three equations gives Equation 4 which was pro- posed by Vickery and Leavenworth (11) t,o explain the formation of a heavy metal salt of cysteine and cysteic acid from cystine solu- tion containing the metal ions.

3R-S-S-R + 3H,Q = 5R-SH + R-SG-0J-I (4)

Kinetically, the reaction of Equation 4 would not take place as such, but would proceed step by step, as suggested by Equations 1, 2, and 3. It is of interest to notice that the reaction takes place without met’al ions, although slowly, to the large extent indicated in Table II, A.

The authors would like to express their gratitude to Mr. Robert McNeil of the McNeil Chemical Company for the grant of a fel- lowship to Kamenosuke Shinohara. They also wish to acknowl- edge a gift of purified I-cystine from Merck and Company, Inc., and to thank Drs. Stanley P. Reimann, Frederick S. Hammett, and Gerrit Toennies, of the Research Institute of the Lankenau Hospital, for their assistance.

SUMMARY

1. Cystine in hydrochloric acid solution is not &able and pro- duces a reducing substance, on standing, which can be detected by titration with iodine.

2. In an atmosphere of nitrogen, the solution undergoes a similar change. At room temperature, the change becomes det,ectable after an induction period of about 7 days. The change takes place in the dark.

3. Iron and copper ions apparently have no influence upon this change.

4. At SO”, the change is more rapid, becoming distinct by the end of 6 hours.

5. The reducing substance is mainly cysteine, the source of its production being the hydrolysis of cystine.

6. An alkaline solution of cystine undergoes a similar change at a much faster rate.

7. A modified nitroprusside test for the determination of or- ganic thiol compounds, and a cobalt complex test for the deter- mination of cysteine in the presence of cystine, have been described.

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K. Shinohara and M. Kilpatrick 251

BIBLIOGRAPHY

1. Shinohara, K., J. Biol. Chem., 96, 285 (1932). 2. Shinohara, K., J. Biol. Chem.., 97, xxii (1932). 3. Michaelis, L., and Barron, E. S. G., J. Biol. Chem., 83, 191 (1929). 4. Michaelis, L., and Yamaguchi, S., J. Biol. Chem., 83, 367 (1929). 5. Michaelis, I,., and Schubert, M. P., J. Am. Chem. Sot., 62, 4418 (1930). 6. Schubert, M. P., J. Am. Chem. Sot., 63, 3851 (1931); 64, 4077 (1932); 66,

3336 (1933). 7. Kendall, E. C., and Holst, J. E., J. Biol. Chem., 91, 435 (1931). 8. Mauthner, J., 2. Biol., 42, 184 (1901). 9. Hoffman, W. F., and Gortner, R. A., J. Am. Chem. Sot., 44, 341 (1922).

10. Andrews, J. C., J. Biol. Chem., 97, 657 (1932). 11. Vickery, H. B., and Leavenworth, C. S., J. BioZ. Chem., 86, 129 (1930). 12. Vickery, H. B., and White, A., J. Biol. Chem., 99, 701 (1932-33). 13. Simonsen, D. G., J. Biol. Chem., 94, 323 (1931-32). 14. Andrews, J. C., and Wyman, P. D., J. BioZ. Chem., 87, 427 (1930). 15. Preisler, P. W., and Preisler, D. B., J. Biol. Chem., 89, 631 (1930); 96,

181 (1932); J. Am. Chem. Sot., 64, 2984 (1932).

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Kamenosuke Shinohara and Martin KilpatrickSOLUTION

THE STABILITY OF CYSTINE IN ACID

1934, 105:241-251.J. Biol. Chem. 

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