the optical activity of cystine.* such widely differing figures have been reported for the

THE OPTICAL ACTIVITY OF CYSTINE.*

BY JAMES C. ANDREWS.

(Prom the Department of Physiological Chemistry, University of Penns~luania, Philadelphia.)

(Received for publication, January 27, 1925.)

Such widely differing figures have been reported for the optical activity of cystine that it is a matter of considerable doubt as to whether the different figures represent different degrees of racemization or different conditions under which the determination was made. For example, values for specific rotation as low as -200” were obtained by Gaskell (1) while Rothera (2) reported values above - 250” and most of the available data range between. Moreover, Hoffman and Gortner (3) have shown that active cystine is easily raccmized by heating in 20 per cent hydrochloric acid, conditions well duplicated in the ordinary method of its preparation. It was the purpose of this investigation, therefore, to study the reasons for these variations and so to stand- ardize conditions that values from different samples of cystine shall accurately indicate their relative degrees of racemization or, the degree of racemization being constant, the relative concentration of cystine.

The insolubility of isoelectric cystine necessitates its being dissolved as either an acid or an alkali salt, but only its salts wit,h acids are sufficiently stable to permit accurate study. Such salts hydrolyze with great ease and a large excess of acid is necessary. Possibilities of a number of reactions, therefore, present themselves and all of these may influence the composition of the asymmetric molecule and, as a result, its optical activity. For example, we may have simple dissociation of the cystine salt, and, in addition, a variety of reactions resulting from the

* An abstract of this paper was presented before the December, 1924, meeting of the American Society of Biological Chemists at Washington. D. C.

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148 Optical Activity of Cystine

tendency of cystine or its salts to form addition compounds with either the excess of acid or water. These compounds are of the oxonium type discussed in a series of papers by Kendall (4, 5) and his collaborators and the extent of their formation is a function of the discrepancy in acidity or basicity between the cystine compound and the acid present in excess. In addition the extent of hydration of all the ions present may conceivably influence the effect of the active body on polarized light through the mechanisms to be discussed below.

EXPERIMENTAL.

A carefully mixed sample of pure cystine, analyzing 11.66 per cent of N by Kjeldahl, was used. All measurements were made in a Schmidt and Haensch polariscope, using white light and a monochronometer, and all data here recorded were taken with yellow light. A 4 dm. tube was used and with any one sample, successive readings were taken until not less than five or six checked within 0.02’. In most cases, except wit’h the more concentrated solutions, the readings checked to the same O.OlO. All readings were taken at 29.0 & 0.2’33. The use of this temperature was dictated by the conditions prevailing in the room in which the polariscope was kept. The maximum temperature variation allowed while readings were taken cor- responded to a negligible variation in the value of [a],.

The experiment,al procedure used, unless otherwise noted, was as follows: 2 gm. samples of cystine were dissolved in a small amount of the acid. The solution was then made up to 100 cc. with the same acid. After the specific rotation was determined two series of dilutions were made, one with the same acid and the other with water or with a salt solution. In this way the series of acid dilutions offered decreasing cystine concentrations at practically constant pH whereas the water or salt dilutions preserved the same ratio between total acid and cystine. The use and effect of specific salt dilutions will be discussed below. Each successive dilution reduced the cystine concentration to 50 per cent of its previous value. These series were continued unt,il the final concentration was 0.0625 gm. of cystine per 100 cc. of solution. At this concentration a difference


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J. C. Andrews 149

of 0.01’ in the reading of the polariscope corresponds to a variation of 4 units in the value of [cr], and further dilution was con- sidered impracticable. Blank determinations on all diluting fluids gave negligible corrections.

The choice of acids was necessarily very limited. A large number were tried but only those discussed below dissolved enough cystine to be practicable.

The absence of mutarotation was evidenced by the fact that, once the tube has been brought to constant temperature, the reading remained constant.

Optical activity is expressed in terms of specific rotation, [cu],. In all cases the usual formula is used

where a = the angle measured, 1 = the length of the polariscope tube in decimeters, and c, the concentration of cystine in gm. per 100 cc. of solution.

EXPERIMENTAL DATA.

Hydrochloric Acid Series.

The standard (2.000 gm.) sample of cystine was dissolved in hydrochloric acid solutions ranging in concentration from 0.5 to 2.5 M and the two series of dilutions with acid and water were made. (See Table I and Fig. 1.)

The data in Table I are typical. With most acids the curves representing the effect of water dilution (constant ratio of cystine: acid) and acid dilution (constant pH) diverge in the same way as do t,hose of hydrochloric acid. It should be noted that in acid dilution curves, a slight maximum value of [a], becomes evident at 1.5 M HCl and above, while a gradual slope is obtained with 0.5 M HCI so that the most constant values of [cu], are those resulting from acid dilution of the cystine hydrochloride from 2.0 to 0.5 gm. in 1.0 M HCI. The significance of these curves will be more fully discussed below. It is also interesting to observe that at high dilut,ions, values of [LY]~ of from -200’ to -270” were obtained. Aside from the possibility of various degrees of racemization, changes in the conditions of the determination, such as are de-


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Optical Activity of Cystine

scribed above, amply account for the wide range of values previously recorded in the literature.’

In order to recheck the divergence at low concentrations of cystinc a further series of determinations was made in which a stock solution was used containing 0.5 gm. of cystine per 100

TABLE I.

- bli’ of Solutions of Cystine Hydrochloride. Eflect oj Variations in Concentration of Cystine Salt and of Hydrochloric Acid.

1-‘-A

100 cc. Acid ,HHz- 1 Acid Hz0 Acid .4cid solution. c&l;: dilu- dilu- dilu- Lx dilu- c%-

Acid dilu- :I??

tion. tion. tion. tion. tion. tion. tion. tion. tion. ~___-~~~__~~--

elm.

2.00 222.4 222.4 215.5 215.5 210.0 210.0 207.7 207.7 203.0 203.0 1.00 221.8 231.0 215.5 223.8 211.0 220.0 208.7 216.0 206.7 215.7 0.50 218.5 240.5 215.0 233 5 209.5 228.0 209.0 223 0 207.5 228.0 0.25 217.0 251.0 214.0 243.0 209.0 237.0 208.0 233.0 207.0 236.0 0.125 210 260 205 254 208 248 204 242 204 242 0.0625 204 270 200 268 204 256 200 / 252 200 252

TABLE II.

-[CL]:” of Solutions of C?Jstine Hydrochloride, C = 0~05 Gm. per 100 Cc.

Xormality of HCI

0.0125 265 0.0526 237 0.1128 230 0.1930 225 0.4625 205 0.9125 195 1 .3625 192 1.8125 190 2 2625 185

cc. of 0.125 M HCl. Portions of this solution were diluted ten times with water and with varying concentrations of HCl and

1 Similar variations were obtained with the sulfuric and oxalic acids, although the actual values are different. 2.00 gm. of cystine per 100 cc. in 1.00 M sulfuric and oxalic acids gave, respectively, values for [aID of 189.6 and 221.5. Attempts to run a series of determinations with cystine nitrate proved impractical because of gradual oxidation of the cystine.


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J. C. Andrews 151

rotations were taken. (See Table II and Fig. 2.) These figures completely substantiate the results previously obtained with high dilutions of cystine.

Phosphoric Acid Series.

Owing to the lower degree of ionization of phosphoric acid higher concentrations were necessary than in the experiments previously described. The data recorded in Table III and il- lustrated in Fig. 3 were obtained with 2.00 and 4.00 M solutions.

The remarkable results obtained from the acid dilutions at low cystine concentrations led at once to careful rechecking, but in both casts the form of the curve was fully substantiated.

Picric Acid Series.

Picric acid, although too insoluble to permit the investigation of a complete series, was nevertheless studied. 100 cc. of a saturated solution (0.0505 M) dissolved about 0.15 gm. of cyst.ine. From this solution, water and acid dilutions were then ma.de in the usual way. (See Table IV and Fig. 4.) The constant values obtained by diluting with water make this system dis- tinct from any other studied.

Trichloroacetic Acid Series.

Experiments with this acid were confined to 1.00 and 2.00 M

solutions. The data, recorded in Table V and Fig. 5, introduce a new condition in which the acid curve retains its normal form while dilution with water causes the specific rotation to fall to a minimum value and then rise in the usual manner.

Sulj’osalicylic Acid Series.

Sulfosalicylic acid (CsH3(OH)(S03H)(COOH)) (6) was selected as an example of an organic acid with a degree of ionization com- parable to that of hydrochloric acid. 0.5 and 2.00 M solutions were used. These data presented no new features except that change in concentration of the acid was almost entirely without effect on the optical activity. 2 gm. of cystine per 100 cc. of 0.5 and 2.00 M acid gave, respectively, values for [cy]:’ of 221.0 and 221.5.


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TABLE III.

-[a]:” of Solutions of Cystine Phosphate. Effect of Variations in Concentrations of Cystine Salt and of Phosphoric Acid.

C cystine per 100 cc. solution.

gm.

2.0 1.0 0.5 0.25 0.125 0.0625

- [a]i” in 2.0 x HaPOa.

Acid dilution.

203.4 203.4 195.9 195.9 205.0 217.7 194.3 204.5 205.0 228.5 193.5 215.5 204.0 239.0 193.0 223.0 204 250 192 232 204 256 192 248

Hz0 dilution.

- [a]i” in 4.0 7.f 1I3P01

T-

Acid dilution. HsO dilution.

TABLE IV.

-[a]:’ of Solutions of Cystine Picrate. Effect of Variations in Concentration of Cystine Salt and of Picric Acid.

C cystine per 100 cc. solution. - [a]E” in 0.0505 M picric acid.

Acid dilution. I Hz0 dilution.

Brn.

0.15 207 207 0.075 203 206 0.0375 200 207

TABLE V.

-bl:8 of Solutions of Cystine Trichloroacetate. E$ect o/ Variations in Concentration of Cystine and Trichloroacetic Acid.

C cystine per 100 CD. solution.

iPa. 2.00 1.00 0.50 0.25 0.125 0.0625

Acid dilution. HrO dilution. Acid dilution. Hz0 dilution.

232.0 232.0 247.1 247.1 231.2 227.0 247.5 232.2 231 .O 228.5 248 .O 227.0 226.0 234.0 245.0 225.0 216.0 240 238 230 208 248 236 232


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2.0 p) ‘C .-

+

c I.5 \,

2 C

.a I.0 <

z x

5 0.5 (j

: b

0

FIQ. 1.

24

%

u”

1.5 P

C .? *

z I.0 )

5 u

: 0.5 u

la-- za-- ---

0 -200 -225 -2so

FIG. 3.

-200 -225 -250 -275

Ed’,” 0 -200 -210 -220

FIG* 2. FIG. 4.

FIG. 1. Specific rotation of cystine hydrochloride. 1 a = 2.5 M HCl, acid dilution; lb = 2.5 M HCl, water dilution. 2 a = 2.0 M HCl, acid dilution; 2 b = 2.0 M HCl, water dilution. 3 a = 1.5 M HCl, acid dilution; 3 b = 1.5 M HCl, water dilution. 4 a = 1.0 M HCl, acid dilution; 4 b = 1.0 M HCl, water dilution. 5 n. = 0.5 M HCl, acid dilution; 5 b = 0.5 M HCl, water dilution.

FIG. 2. Specific rotation of cystine hydrochloride at high dilution (0.05 gm. per 100 cc.). Effect of HCl concentration on specific rotation.

FIG. 3. Specific rotation of cystine phosphate. 1 a = 4.0 M H,POa, acid dilution; 1 b = 4.0 III H,POa, water dilution. 2 a = 2.0 M H,POI, acid dilution; 2 6 = 2.0 M H,PO+ water dilution.

FIG. 4. Specific rotation of cystine picrate. 1 a = acid dilution series; 1 b = water dilution series.

153


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Salt Series.

2.50 M Hydrochloric Acid.

In some series, successive dilutions were made with solutions of a salt instead of the free acid, thereby diminishing the acidity while maintaining the same concentration of the negative radical. It seemed of considerable interest to ascertain whether the im- portant factor in determining the form and position of dilution curves was the total osmotic concentration, the negative ion concentration, the pH, or some totally different factor. In the first and second cases, the curve resulting from dilution with an equi- valent concentration of salt should nearly approximate the acid dilution curve; in case the pH of the solution was the controlling factor t,he salt dilution curve should closely approach that produced by water dilution.

A solution of 2.0 gm. of cystine in 100 cc. of 2.500 M HCl was made up and aliquots were diluted in the usual way with 2.50 Y NaCl, 0.50 M NaCl, and also, for further comparison, with 0.50 M sodium sulfosalicylate solution. (See Table VI and Fig. 6.) In order to facilitate comparison with the new curves, those previously described and recorded for 2.50 M HCl are again included. The depressing effect of NaCl on the position of the water dilution curve (on [LX],) is very evident. Sodium sulfosalicylate had a much smaller effect.

Trichloroacetic Acid.

Solutions of cystine in 1.00 and 2.00 M trichloroacetic acid were diluted with 1.00 and 2.00 M solutions of sodium trichloroacetate2 and with 1.00 and 2.00 M sodium chloride. (See Table VII and

2 The curves of dilution with sodium trichloroacetate are subject to a special source of error because of the instability of this salt, even at room temperatures. Sodium trichloroacetate hydrolyzes very easily into chloroform and sodium bicarbonate; the action goes rapidly to completion at temperatures near the boiling point, and the only practicable method of employing the salt is to make and use it in solution immediately, keeping the solution as cold as possible. The salt solution was made by mixing equal volumes of carefully standardized NaOH and CCl&OOH. The solutions were cooled in an ice bath and then mixed with rapid stirring. Even with these precautions, the solution smelled of chloroform before the series was completed.


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J. C. Andrews 155

Figs. 7 and 8.) There is a consistent tendency on the part of sodium trichloroacetate to raise the low values of [cY], which result from water dilution. This is in decided contrast to the opposite effect exhibited by sodium chloride.

TABLE VI.

-[cx]:” of Solutions of Cystine Hydrochloride. Effect of Variations in Concentrations o.f Cystine Salt and of Sodium Chloride and

Sodium Sulfosalicylate.

- [a]Eg in 2.50 M HCl. c cystine

per 100 cc. solution.

-

_-

-

.cid dilution I 1~0 dilution. I 2.50 M N&l dilution.

203 :0 203.0 203 0 206.7 215.7 201 .o 207.5 228.0 198.0 207 .O 236.0 199.0 204 242 203 0 200 252 208

0.50 M N&l dilution.

050YMNa rulfosalicylate

dilution.

203.0 213.0 224.0 233.0 242 256

A

-

gm. 2.0 1.0 0.5 9.25 0.125 0.0625

203.0 208.5 214 0 221 .o 227 236

1

TABLE VII.

-[orID of Solutions of Cystine Trichloroacetate. E$ect of Variations in Concentration of Cystine Salt and of Sodium Trichloroacetate and

Sodium Chloride. - 1 -[a], in 1.00 M

triohloroscetic acid -[a], in 2.00 M trichloroacetic acid

C cystine per 100 cc

solution.

Pm.

2.00 1.00 0.50 0.25 0.125 0.0625

-

(

Acid Hz0 Salt Acid HrO Dilution. dilution. dilution. dilution. Slution

Salt iilution. c

1.00 M N&l

lilution “N”B”cl”

lilution.

232.0 232.0 232.0 247.1 247.1 247.1 247.1 247.1 231.2 227 .O 232.0 247.5 232.2 253.0 228.8 224.8 231 .O 228.5 233.0 248 0 227.0 253.6 217.8 210.0 226.0 234 .O 238.0 245.0 225.0 255 6 215.0 205.0 216 240 244.0 238 230 258 216 206 208 248 260 236 232 266 225 218

Sulfosalicylic Acid.

The dilution of a solution of cystine sulfosalicylate with sodium sulfosalicylate and sodium chloride solutions gave results in keeping with what, might have been expected from the previous behav-


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- 0 -200 -225 -250

FIQ. 5.

: Q I-- -

u,-.-- -

45 3--

- RI\ 4-- --

\ PS D

5-- --

x

\

I31 0 -zoo . -225 -250

FIQ. 6. FIG. 8.

2.0

I.5

1.0

a!5

0

-200 -225 -250

FIG. 7.

FIG. 5. Specific rotation of cystine trichloroacetate. 1 a = 1.0 M CCl&OOH, acid dilution; 1 b = 1.0 M CCl&OOH, water

dilution; 2 a = 2.0 M CCl&OOH, acid dilution; 2 b = 2.0 M CC13COOH, water dilution.

FIG. 6. Specific rotation of cystine hydrochloride diluted as follows: (1) 2.5 M HCl dilution; (2) water dilution; (3) 2.5 M NaCl dilution; (4) 0.5 M NaCl dilution; and (5) 0.5 M sodium sulfosalicylate dilution.

FIQ. 7. Specific rotation of cystine trichloroacetate diluted as follows: (1) 1.0 M CCl&OOH dilution; (2) water dilution; and (3) 1.0 M CC13COONa dilution.

FIG. 8. Specific rotation of cystine trichloroacetate diluted as follows: (1) 2.0 M CCl,COOH dilution; (2) water dilution; (3) 2.0 M CCl,COONa dilution; (4) 1.0 M NaCl dilution; and (5) 2.0 M NaCl dilution.

156


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J. C. Andrews 157

ior of these salts. Sodium sulfosalicylate slightly raised the values of [(Y]~ obtained from the water dilution of cystine sulfosalicylate while sodium chloride lowered these values.

DISCUSSION.

Examination of these results reveals several fairly general characteristics.

1. At low concentrations of cystine (less than about 0.2 gm. per 100 cc. or 0.0083 molar) the value of [ar], seems to depend chiefly on the pH of the solution, low pH values giving low values for [(Y]~.

2. At higher concentrations of cystine than 0.2 gm. per 100 cc. the effect of various ions is almost entirely specific and peculiar to the particular ions present.

3. For any given acid, dilution with a constant concentration of that acid (at practically constant pH) results in practically constant values for [ar],. That this constancy is at least partially a result of constant hydrogen ion concentration is indicated by the fact that substitution of an equimolar concentration of the sodium salt for the acid results in marked changes in the form of the dilution curves.

The curves resulting from water dilution show the form charac- teristic of dissociation reactions and probably represent, in the main, the dissociation of the cystine salt into its ions. To what extent this salt has also combined with the excess acid to form the oxonium compound and to what extent the dissociation of this compound influences the form of the water dilution curve seems impossible to say from the present data. Hadrich (7) determined the effect of dilution on the optical activities of the salts of several alkaloids with strong acids and showed that the resulting curves in most cases, followed the course predicted on the basis of ionic dissqciation. It was unnecessary, however, for Hadrich to employ the large excesses of acid that were required in the present work and he was not, therefore, led to an investigation of the effect of varying amounts of other electrolytes. In the case of a number of other investigators, notably Rimbach (S), similar attempts to establish the exclusive influence of simple ionization on the rotation of active electrolytes were unsuccessful.

Particular interest attaches the few points determined for the


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picric acid series. If we assume the usual form of the water dilution curves to be the result of electrolytic dissociation of the cystine salt we must conclude that the evidence here points to complete lack of dissociation under the conditions of the experiment. This is noteworthy in view of the frequent USC of picric acid as a precipitant of organic bases.

In connection with these data, the work of Patterson (9) on the effect of internal pressure on optical activity is of interest. Patterson assumes that the shape of an asymmetric molecule is influenced by the internal pressure of the solution and that this shape, or some function of it, is directly responsible for its specific rotation. His data show, moreover, the particularly great influence of internal pressure at high dilutions of the active solute.

In the present work, similar influences undoubtedly come into play. The substance studied differs from those of Patterson’s work in being of electrolytic nature3 and in the present work changes in internal pressure are effected chiefly by changing the ionic content of the solution. The connection between electro- lyte concentrations and the molecular volume of the active solute (a direct function of internal pressure) is obvious. It is, therefore, not surprising that the ions, with their different degrees of hydration, cause the highly specific effects observed in the various salt dilution curves. It does not seem profitable to do more than indicate the direction in which the explanation for these data lies.

The above experiments permit the standardization of conditions for the determination of the optical activity of cystine. The very erratic values for [alo of cystine Which appear in the literature may be amply explained by the variety of conditions employed, although frequently no description of these has been included. The necessity for such particularization is shown by the data in the present paper. Inspection of the data presented above shows that the most constant and easily duplicable conditions for the determination of [Q]~ of cystine are to be obtained by the use of HCl of constant concentration (preferably 1.00 M), containing 1.0 gm. of cystine per 100 cc. of solution. A glance

3 Patterson worked chiefly with ethyl tartrate in water and various organic solvents.


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J. C. Andrews 159

at Table I shows that with this concentration of acid, the effect of variation in the proportion of cystine used is very slight. Con- venience will, in many cases, dictate the use of a lower temperature than that used in this work. Comparison of readings, taken at both 20” and 29”C., has showed an average temperature coefficient of - 1.7” [a], per 1°C. over this range of temperature.

SUMMARY.

The optical activity of cystine in the form of a variety of its salts with strong acids was studied. The effect of changes in such conditions as the concentration of cystine, of excess acid, and of added salts was determined. Various mechanisms in- fluencing the optical activity of cystine have been discussed and a probable method by which these influences are brought into play has been suggested. This method involves the effect of the varying degrees of hydration of different ions on the internal pressure of the solution and the consequent effect on the shape of the asymmetric molecule. Standard conditions for the determination of the optical activity of cystine have been out,lined.

BIBLIOGRAPHY.

1. Gaskell, J. F., J. Physiol., 1907-08, xxxvi, 142. 2. Rothera, C. H., J. Physiol., 190405, xxxii, 177. 3. Hoffman, W. F., andGortner, R. A., J. 9m. Chem. SOL, 1922, xliv, 341. 4. Kendall, J., Booge, J. E., and Andrems, J. C., J. Am. Chem. Sot., 1917,

xxxix, 2303. Kendall, J., and Booge, J. E., J. Am. Chem. Sot., 1917, xxxix, 2323.

5. Kendall, J., Davidson, A. W., and Adler, H., J. Am. Chem. Sot., 1921, xliii, 1481.

6. Umetsu, K., Biochem. Z., 1923, 442. cxxxv, 7. Hlidrich, H., 2. physik. Chem., 1893, xii, 476. 8. Rimbach, E., Z. physik. Chem., 1899, xxviii, 251. 9. Patterson, T. S., J. Chem., SOL, 1901, lxxix, 167, 477; 1902, lxxxi, 1097,

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James C. AndrewsTHE OPTICAL ACTIVITY OF CYSTINE

1925, 65:147-159.J. Biol. Chem.

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the optical activity of cystine.* such widely differing figures have been reported for the

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