OXIDATION OF CYSTEINE IN NON-AQUEOUS MEDIA*

THE “SULFENIC ACID” AS THE PRIMARY OXIDATION PRODUCT1

BY GERRIT TOENNIES

(From the Lankenau Hospital Research Institute, Philadelphia)

(Received for publication, June 24, 1937)

Since the postulate of Hammett (1930), that oxidation products of the sulfhydryl group are inhibitants of natural growth, has been variously confirmed (Hammett et al., 1936; Zirpolo, 1935; Voegt- lin, 1937), it seems important to make available model compounds of any as yet unknown intermediate stages in the oxidation of -SH. That a compound of the type -SOH or -S(O)H may be an important intermediary therein is indicated by the work of many investigators. Mathews (1924) had suggested for the oxidation of cysteine to cystine the mechanism R-SH + 0 + R-SOH and RSOH + R-SH + R-S-S-R + HSO, rather than the conventional formulation BR-SH + 0 + R-S-S-R + HzO. Meyerhof (1923) submitted evidence for the existence of a peroxide-like derivative of cysteine, and Dowler (1928) described conditions under which cystine is not oxidized by iodine, while cysteine is oxidized beyond the cystine stage so that the latter can hardly be considered an intermediary in the oxidation of the former, while assumption of the R-SOH level as the first oxidation stage would be in harmony with the observation. Findings by Brand and associates (1935) as well as by Medes (1937) on the catabolism of cysteine and cystine lend themselves to similar interpretations, and the results of the numerous thermo- dynamic studies of the reduction potential of cysteine (cf. Borsook et al. (1937)) also suggest that the actual mechanism of cysteine oxidation may follow a path analogous to the formulation of Mathews rather than that of the single electron transfer R-S:H -+ R-S* + l + H+ implied in a direct oxidation to cystine.

* Parts of this investigation have been reported at the meetings of the American Chemical Society, before the Division of Biological Chemistry, at New York, April, 1935, and at Pittsburgh, September, 1936.

t Aided by a grant from the Blanche and Frank Wolf Foundation, Inr. 27

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28 Oxidation of Cysteine

However, a cysteine derivative of the composition R-SOH has not been isolated. Simonsen (1933) believed to have evidence for its presence in the products of the oxidation of cysteine with iodine, but did not give any details, and the assumption of an -SOH compound of relatively high stability by Pirie (1933) has been shown to be erroneous (Toennies, 1937, c). Work of Schoberl and Eck (1936) on amino-free -SH compounds indicates a strong tendency of the -CHBOH group to turn with loss of HzS into an aldehyde group, a conception which is shared by Crowder and Harris (1936) on the basis of their observations on the behavior with alkali of the disulfide group and its oxidized derivatives in wool, while the dismutation of cystine-disulfoxide in alkaline solution (Lavine, 1936) suggests the tendency of the -SOH derivative of cysteine to react spontaneously according to the equation BR-SOH + R-S02H + R-SH.

It is the object of the present paper to render an account of experiments with a method by which cysteine apparently can be oxidized to an -SOH derivative. Although the goal of isolating the compound has not been attained so far, products have been obtained of which it appears to be, in the form of a sulfate, the main constituent. The method consists in oxidizing cysteine, dissolved as the perchlorate in isoamyl alcohol, with an equimolar amount of permonosulfuric acid in isoamyl alcohol in the presence of an excess of sulfuric acid. The oxidation takes place almost instantaneously, and immediately a white precipitate forms which, according to analytical evidence to be detailed, contains sulf- oxycysteine’ as a sulfate, presumably together with varying amounts of sulfinic and sulfonic acids and of cystine and its disulfoxide.

EXPERIMENTAL

Some Salts of Cysteine and Their Solubilities-l-Cysteine hydro- chloride (Merck) and free I-cysteine prepsred herefrom (Toennies and Bennett, 1935-36) were employed. Ordinary ethyl alcohol

1 To designate a compound of the composition HOOC-CH(NH,)- CHz-SOH the term sulfoxycysteine, rather than cysteine sulfenic acid, is preferred, as the -SOH group in the present compound has no noticeable acidic properties. However, no decision as to whether the structure of the group corresponds to -SOH or -S(O)H is implied.

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G . Toennies 29

(92 per cent) dissolves about 0.5 mole of cysteine hydrochloride per liter, but when 2 mM of free cysteine were treated with an equivalent amount of a 0.217 N solution of sulfuric acid in ethyl alcohol, copious crystallization occurred after initial dissolving; acid equivalent weight 171, calculated for cysteine + HzS04, 170; yield 69 per cent. As the hydrochloride is not suitable for oxida- tion experiments on account of susceptibility of chloride to oxida- tion, and as the sulfate is not soluble in alcohol, the perchlorate was investigated. Cysteine dissolved by an equimolar amount of a solution of perchloric acid (68 per cent) in ethyl alcohol formed a relatively stable system in 0.2 M solution: [ar]np = +6.6”, decreas- ing slowly ([a]nB = +5.4” after 10 days). Solutions of similar stability have been prepared by means of solutions of perchloric acid in isopropyl and isoamyl alcohol as well as in acetic acid. No change in acidity, even after months, appeared in the alcoholic solutions of perchloric acid (0.6 to 0.8 M). Acetic acid solutions seem remarkable for the high rotatory power of cysteine ( [a]ng = +18.5”) and for its stability (the optical rotation of a 0.50 M

cysteine perchlorate solution, a! = +2.23” in a 2 dm. tube, remained unchanged during a period of 4 weeks). The unusual stability may be of interest as an expression of the high acidity potential of the perchloric acid-acetic acid system (Hall, 1931).

Oxidation of Cysteine in Alcoholic Media. Preliminary Experi- ments-studies on solutions of permonosulfuric acid in organic media, described elsewhere (Toennies, 1937, a), showed that the peracid reacts Matively slowly with alcoholic -OH groups, In striking contrast herewith is the rapid reaction with the analogous -SH group, shown by the experiments summarized in Table I. The experiments indicate that cysteine takes up 1 atom of active oxygen with great ease, while the reactivity toward a 2nd and a 3rd atom appears to be progressively less. Similar studies on the oxidation of cystine have been previously reported (Toennies, 1934).

Since polarimetric observations under similar conditions indi- cated the instability of the primary oxidation products-which had also been found in the similar oxidation of cystine (Toennies, 1934) -a search was made for conditions under which the latter might spontaneously precipitate and thus be stabilized. When, instead of ethyl alcohol, isopropyl alcohol or mixtures of isopropyl alcohol

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Oxidation of Cysteine

and ether were used as the medium, oxidations with a 1: 1 ratio of cysteine and H$306 produced almost immediately small amounts of precipitates consisting apparently of mixtures of intermediary oxidation products in combination with sulfuric acid. Yield as well as composition became more favorable when isoamyl alcohol became the medium. Before detailing the results the general procedure of preparation and the system of analysis will be de- scribed. Preliminary to the oxidation the desired amount of finely pulverized cysteine is dissolved by being shaken with the equivalent amount of perchloric acid in a volume of isoamyl

TABLE I Rate of Oxidation of Cysteine by Permonosulfuric Acid in

Acid-Alcoholic Medium The solvent is 92 per cent ethyl alcohol. To 4 cc. portions, containing

3.3 to 3.7 mM of HklO, and the specified amounts of H2S06, 1 cc. portions of a 0.18 M cysteine perchlorate solution were added. After 55 seconds* 50 cc. of water containing starch and 1 mM of KI were added and the liberated iodine was titrated at once with 0.024 N NazSIOa so that the titra- tions were completed about 20 seconds later. Each experiment was performed in duplicate.

Molar ratio ?I!@& Cyfhine

4.33 2.30, 2.41 3.25 2.18, 2.23 2.17 1.65, 1.59 1.08 1.08, 0.98*

HG3Os reduced per molecule cysteine

* In the last experiment the titration was begun after only 25 seconds.

alcohol which will produce about twice the cysteine concentration intended for the oxidation itself. Into this solution, while it is being vigorously shaken, is poured, as rapidly as possible, an approximately equal volume of another isoamyl alcohol solution containing the desired amounts of HzS06 and H&304. Both solu- tions have been cooled, previous to the reaction, to about -lo”, and the reaction mixture, in a glass-stoppered flask, is shaken for several minutes in a freezing mixture and then at room temperature until the thick milky reaction product, which in most cases begins to form within 5 to 10 seconds after the reactants have been com- bined, curds into a homogeneous granular precipitate. This

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G. Toennies 31

precipitate is strongly acid, water-soluble, and extremely hygro- scopic. However, by washing it-on a Buchner funnel in an atmosphere of carbon dioxide (Roeder, 1934) or, more effectively, by digesting, centrifuging, and decanting-with isoamyl alcohol and dry ether (each about six times) and drying in a vacuum desiccator in the presence of PzOs and mineral oil, it is obtained as a loose powder of pure white appearance.

Analytical Methods

Acidity-Suitable samples (e.g. 40 mg.) are titrated directly with 0.05 N NaOH from a microburette, after addition of 1 drop of alcoholic methyl red solution, to the first color change dis- tinguishable within less than 1 minute on rapid titration. The red color returns rather rapidly, and the reaction responsible for this reappearance of acidity is brought to completion by several minutes of boiling. The first end-point, calculated as milli- equivalents of acid per 100 mg. of substance, gives “initial acidity” (ad), and the difference between the final and the first end-point, similarly calculated, “formed acidity” (a,).

Iodine-Reducing and Iodide-Oxidizing Power-The amount of iodine consumed upon oxidation (presumably to cysteic acid) as well as the amount of iodine liberated upon reduction (to cystine) are determined under conditions previously described (Toennies and Lavine, 1936; Lavine, 1936), suitable cg. quantities being used. The former, expressed as milli-atoms of oxygen (mM of IZ) consumed by 100 mg. of substance is termed “reducing value” (o+) and the latter, similarly expressed as oxygen liberated per 100 mg., “oxidizing value” (o-), Since it has been shown (Toennies and Lavine, 1936; Lavine, 1936) that under these analytical conditions cystine and cystine disulfoxide, as well as the sulfinic acid obtained from the latter, are oxidized to cysteic acid, while the disulfoxide and the sulfinic acid, but not cysteic acid, are reduced to cystine, the ratio 0+/o- (r) may be taken as a measure of the relative level of oxidation (cf. Toennies and Lavine (1933) Table I) of the oxida- tion product. The ratio r would be, for cystine 00, for the disulf- oxide 1.5, for the sulfinic acid 0.667, and for sulfoxycysteine pre- sumably 4.0. Interpretation is, of course, complicated when mixtures of these and other possible intermediate compounds are obtained.

Sulfur Determinations-Total sulfur was determined on cg.

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32 Oxidation of Cysteine

quantities by a modification of the KMn04 method of Blix (1928) in which, instead of decomposing the excess KMn04 by HCI, it is reduced by methyl alcohol to MnOn, the latter is filtered off and thoroughly washed, and BaS04 is precipitated in the filtrate. The amount of ionizable sulfate present in the reaction products was determined by an adaptation of the volumetric benzidina micromethod of Fiske (1921). The results are again calculated as mM of HzS04 per 100 mg., “sulfate content” (sod), and (by difference) “organic sulfur content” (s,) as milli-atoms of organic sulfur per 100 mg. Other analytical criteria used in later stages of the investigation will be described as the occasion arises.

Products Obtained by Oxidation in Isoamyl Alcohol

A number of preparations obtained and analyzed by the method outlined are summarized in Table II. A comparison of the ana- lytical results with those calculated for some of the possible oxida- tion products shows that, while mixtures of several of these pos- sibilities are evidently obtained, it seems probable that a sulfate of the composition HOOC-CH(NHz)-CHZ-SOH + HzS04 is the chief constituent. Table II further shows that the results of indi- vidual experiments are not reproducible within close limits and that when the ratio of oxidant and substrate was deliberately varied (Preparations 13 to 15) erratic variations in the composition of the reaction product resulted. But since the results as a whole suggested that the desired compound, as a sulfate, is formed in substantial amounts, further efforts were directed toward the possibilities of (a) isolating the sulfoxy compound from the mixtures obtained or (b) changing the conditions for the reaction in such a manner that the sulfoxy compound would result in a more nearly pure form.

When the crude precipitates described in Table II were treated with small amounts of 14 M (saturated at 0’) HCl, solution with subsequent crystallization occurred. The crystalline precipitates were placed on a porous plate, “washed” by adding single drops of 14 M HCI, which was absorbed by the plate, until the substance was nearly free of SO;, and analyzed after drying in vacua in the presence of a saturated solution of NaOH. This treatment fur- nished supporting evidence for the conclusion that the -SOH compound is the main oxidation product, inasmuch as the analyt-

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G. Toennies 33

ical values, including those for acidity, of the products obtained by replacement of H&SO+ with its two acid groups, by HCI were again closely related to those calculated for the corresponding R-SOH salt. At the same time no pronounced or reproducible improvement in purity resulted. The best hydrochloride product obtained (by treating 185 mg. of Oxidation Product 7, Table II, at - 18’ with 0.70 CC. of 14 M HCl; yield 38 mg.) showed ai = 0.578, af = 0.187, o+ = 1.235, o- = 0.286, and r = 4.32, compared with the corresponding calculated values of 0.577, 0.191, 1.153, 0.288, and 4.00 for R-SOH + HCl.

Variations in Conditions of Oxidation

Reaction Medium-Attempts to use higher alcohols other than isoamyl did not give promising results. A reaction product obtained in methylisobutyl carbinol was quite similar to those described in Table II; and, in addition, uncomfortable explosive tendencies appeared in solutions of permonosulfuric acid in higher secondary and tertiary alcohols (cf. Toennies (1937, a)). Among other (non-alcoholic) media considered, acetic acid or acetonitrile, while suitable solvents for cysteine perchlorate, did not cause precipitate formation by sulfuric acid upon oxidation; di-n-butyl ether dissolves 70 per cent perchloric acid in the presence of 99 per cent sulfuric acid, but the resulting solution does not dissolve cysteine, and a similar result was obtained with di-n-butyl sulfate.2 Evidently the limiting conditions for the suitability of the medium are, apart from non-reactivity, a polarity sufficiently high to permit solution of the salt cysteine perchlorate, and at the same time not high enough to prevent the precipitation of sulfates of the reaction products. In this connection conditions, incidentally discovered, for the formation of an acid sulfate of cystine may be of interest. When 10 cc. of a 0.35 M cystine perchlorate solution in acetonitrile (Toennies and Lavine, 1933) are carefully combined with 2 to 3 cc. of 15 M H2S04, and a 0.75 M solution of perchloric acid in isoamyl alcohol is slowly added until a turbidity remains, a crystallization of dense aggregates forms on standing. After thorough washing with ether and drying it showed on titration an acid equivalent weight of 111; calculated for cystine + 2H~S04, 109.

* A generous sample was kindly supplied by Professor C. Barkenbus of the University of Kentucky.

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TABL

E II

Oxida

tion

of

Cyste

ine

by

Perm

onos

ulfur

ic Ac

id

in

Isoam

yl Al

coho

l T

Reac

tion

mixt

ure

Reac

tion

prod

ucts

“t,$-

NO

.

(1)

- (

.-

:yste

ine

pedll

0- rate

- F I

T

(2)

‘errno

n*

sulfu

ric

acid

(3)

Ratio

(3

) : (2

)

(4)

(5)

(6)

IrptT

l.

5 0.

24

0.23

5 0.

98

6 0.

29

0.27

5 0.

95

7 0.

255

0.24

5 0.

96

8 0.

265

0.25

0 0.

96

9 0.

265

0.25

0 0.

96

11

0.29

5 0.

280

0.95

13

0.

223

0.18

1 0.

81

14

0.22

2 0.

229

1.03

15

0.

232

0.27

1 1.

17

7 I

Yiel

d by

B

veigh

t of

-.

cyst&

e us

ed

g (1

4)

g

per

cent

0

139

119

i 11

1 w (D

10

7 -*

137

i?

68

94

99

_-

- : (

1 n

L

3xidi

zinl

value

(0

-J

(10)

Orga

nic

sulfu

r co

nten

t (4

(12)

Sulfa

te

cont

ent

(.m)

(13)

H,SO

, (

mxid

ation

ed

uctio

n ra

tio

(I)

(11)

2yste

ine

used

R&

&ll&

co+)

(9)

Form

ed

(a/) (8)

- 7

.-

1. am

.

1.32

1.

21

1.10

1.

09

1.23

2.

54

1.25

4.

90

1.20

9.

46

2.25

1.

81

1.42

1.

30

1.42

1.

30

1.40

1.

30

?a.-e

q. pe

r 10

0 na

g.

n.-t-

q.

pe,

100

mg.

0.78

4 0.

134

0.78

0 0.

125

0.76

6 0.

156

0.75

5 0.

134

0.77

2 0.

129

0.82

7 0.

171

dli-a

tom

0

Pm

100

mg.

0.82

5 0.

825

0.82

5 0.

805

0.81

8 0.

777

0.66

2 0.

742

0.92

8

dii-a

ton

0 Pm

10

0 m

g.

0.25

2 0.

245

0.27

6 0.

261

0.26

6 0.

306

0.25

9 0.

346

0.33

2

ailli-a

tom

loo”

“,,.

0.44

8 0.

464

0.51

0

mdl

pe

r 10

0 m

&l.

0.38

3 0.

358

0.35

3

3.27

3.

37

2.99

3.

09

3.08

2.

54

2.56

2.

14

2.80

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Calcu

late

d va

lues*

fo

r HO

OC-C

H(NH

z)-CH

Z-SO

H +

H~SO

I ...

.....

0.85

1 [H

OOC-

CH(N

Hz)-C

H&=&

+

2HzS

01.

......

. 0.

918

[HOO

C-CH

(NHZ

)-CH&

==&O

+

2H&O

,. ...

.. 0.

885

[HOO

C-CH

(NHz

)-CH&

==S2

02

+ 2H

2SOa

...

...

0.85

5 HO

OC-C

H(NH

z)-CH

*-SOz

H +

H2SO

d. ...

...

1.19

5 HO

OC-C

H (N

Hz)-C

H,SO

zH

......

......

....

0.65

4

0.14

2 0.

851

0.21

3 4.

cJ6

0.42

6 0.

426

0.00

0 1.

147

0.00

0 m

0.

459

0.45

9 0.

148

0.88

5 0.

221

4.99

0.

443

0.44

3 0.

213

0.64

1 0.

427

1.56

0.

428

0.42

8 0.

000

0.39

9 0.

598

0.67

0.

399

0.39

9 0.

000

0.65

4 0.

981

0.67

0.

654

0.00

0

- - -

* Th

e va

lues

in

Colum

ns

9 an

d 10

ar

e ba

sed

on

oxida

tion

to

cyst

eic

acid

an

d re

ducti

on

to

cyst

ine

resp

ectiv

ely.

The

“form

ed

acidi

ty”

value

s (C

olum

n 8)

ar

e ba

sed,

fo

r th

e di

sulfo

xide,

on

th

e ex

perim

ents

of

Lavin

e (1

936)

wh

o fo

und

that

in

ac

id

solu

tion

appr

oxim

ately

1

equiv

alent

of

ac

id

is

form

ed

from

1

mol

ecul

e of

di

sulfo

xide,

an

d fo

r th

e -S

OH

corn

- 0

poun

d on

th

e as

sum

ed

reac

tion

3R-S

OH

-+

R-S-

S-R

+ R-

SOzH

, i.e

. fo

rmat

ion

of

1 eq

uivale

nt

of

acid

fro

m

3 m

olec

ules

of

th

e ne

utra

l co

mpo

und.

Ad

ditio

nal

evide

nce

for

this

reac

tion

will

be

given

fu

rther

on

. Fo

r th

e m

onos

ulf-

e

oxide

[H

OOC-

CH(N

H*)-C

H&=S

nO

a pr

imar

y hy

droly

sis

into

2R

-SOH

, an

d su

bseq

uent

re

actio

n of

th

e lat

ter

z

acco

rdin

g to

3R

-SOH

+

R-S-

S-R

+ R-

SOIH

is

ass

umed

. E F.

z

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36 Oxidation of Cysteine

Function of Sulfuric Acid-An oxidation similar to those of Table II, except that by the use of an HzSOa solution of low HzSOd content (cj. Toennies (1937, a)) the H2SOI concentration of the reaction mixture was kept at 0.07 M, gave the following result: yield by weight 44 per cent of cysteine, ai = 0.707, a/ = 0.011, of = 0.693, o- = 0.090, r = 7.7, soI = 0.250, and ss (disulfide) = 0.12. The latter value was obtained by Shinohara’s modification of Folin’s phosphotungstic acid method (Shinohara, 1935-36), on the assumption--later confirmed (see below)-that R-SOH in the presence of bisulfite reacts rapidly according to R-SOH + NaHS03 -+ R-S-S03Na + HzO. The amount of R-SOH obtained in this oxidation is obviously small (cj. a,, OK, and r; a calculated composition of 0.205 mM of HzS04, 0.11 mM of cystine, 0.03 mM of R-SOH, 0.05 mM of R-SO,H, and 0.25 mM of R-SO,H accounts reasonably well for the analytical data), indicating that the presence of sulfuric acid has a profound effect on the amount of R-SOH intercepted. On the basis of this and the preceding experiments the following equations may serve as a tentative picture of what happens in the oxidations.

R-SH + 0 -+ R-SOH (1, fi)

R-SOH + 0 -+ R-SOzH (1, b)

R-SOH + HS-R + R-S-S-R + Hz0 (1, c)

R-S-S-R + 0 --) R-SO-S-R (1, 4

and further analogous oxygen additions which would lead to cys- teic acid, disulfoxides, etc. But also

It-SOH + HzSO, + R-SOH-H$O, (2)

and similarly for cystine and the other oxidation products, pre- sumably excluding those of an acid nature, as the sulfinic and cysteic acids, which, if formed, may be expected to precipitate as such.

Several implications are inherent to this conception. If Equa- tion 1, a represents the primary reaction, success in obtaining its product as the insoluble sulfate depends on the competitive situa- tion between the reaction of Equations 1, b, 1, c, 1, d, etc., on the one hand and the reaction of Equation 2 on the other. There- fore, lowering of the concentration of R-SH and HzS06 (main-

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G. Toennies 37

taining their ratio at unity) while keeping the concentration of H&S04 constant should favor the reaction of Equation 2 at the expense of the reactions of Equations 1, b, etc., and the same effect should result from increasing the H&04 concentration while the concentrations of substrate and oxidant remain constant.

Bisulfates in Place of Sulfuric Acid-Furthermore, inasmuch as cysteine, etc. are present in solution as -onium ions, held in this state by the acid function of perchloric acid,

CHI-SH CH,-SH

I I H-C-NHJ+ + HClO, ---) H-C-NHs+ + -CIO,

I boo-

I COOH

(3)

the actual reaction which leads to the sulfate precipitates may bc thought of as one of the -onium ions with the bisulfate ion,

HOS-R--SHa+ + -SO,H --f HOS-R-NH,+ -SO,H C-L)

rather than with HzS04 molecules, as suggested by Equation 2. Since HzS04, as a relatively weak acid compared with HClO, (cf. e.g. Hall (1931)), is presumably incompletely ionized, even in its first acid group, in a medium of low dielectric constant such as isoamyl alcohol, substitution of a soluble bisulfate for the free acid could be expected to favor the rapidity of the precipitation reaction indicated by Equation 4. It was surprising to find that when the cysteine oxidation was carried out under conditions where most of the sulfuric acid was replaced by monoamylamine bisulfate3 (0.245 M cysteine perchlorate, 0.25 M H2S06, 0.09 M

HZSO,, and 1.50 M C&NH3+ -SO,H) no precipitation whatever

* Monoamylamine bisulfate was easily prepared by adding the amine (Sharples Solvent Corporation, 96 per cent according to titration) slowly to an equimolar amount of 99 per cent H&404 frozen in freezing mixture. After it was heated on the steam bath in order to dissolve some lumpy material (the neutral sulfate, which is insoluble in the base or in amyl alcohol), a clear and almost colorless syrup resulted which had a density of about 1.22 and contained 6.24 milli-equivalents of -HSO, per cc. according to titration of an aqueous dilution. The total acid content obtained by titration after the amine was expelled by boiling with excess alkali corre- sponded to 13.01 milli-equivalents per cc. and was equal to that calculated from the total H$SOd used. This syrupy substance is miscible with such solvents as amyl alcohol or acetonitrile.

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38 Oxidation of Cysteine

occurred. Another experiment, in which monobutylamine bi- sulfate was used (0.17 M cysteine perchlorate, 0.17 M HzSOr,, 0.27 M

HzS04, 1.00 M C4H9NH3+ -S04H), likewise gave no precipitate whatsoever in spite of the relatively high concentration of fret HzS04. For lack of actual knowledge about the state of ionization and dissociation of H2S04 and its salts in isoamyl alcohol no explanation, but merely the hypothesis of complex formation being involved, can be advanced to account for this unexpected result. That the presence of the amine in itself does not prevent the precipitate formation is indicated by the appearance of a precipitate upon addition, to the reaction mixture of the above reported oxidation in the presence of amylamine sulfate, of an amount of 0.8 M HClOk in isoamyl alcohol equivalent to the amount of bisulfate present. Thus apparently free H804, reformed from the bisulfate by the action of the stronger HClO,, is necessary for the precipitation to occur. Also there is no evi- dence that an oxidation of the amine (present in the NH3+ form, see below under “Evidence for identity of sulfoxycysteine”) by the peracid could interfere with the oxidation of the cysteine by the latter.4

Phosphoric and Permmophosphoric Acids-Since H3P04 showed a precipitate-forming action similar to that of HzS04, it was neces- sary to ascertain whether or not results would justify its substitu- tion for HzS04. In order not to have two different precipitating agents present, viz. H3P04 and HzS04 resulting from H&05, the latter was replaced by permonophosphoric acid, HsP06. The concentrations were 0.23 M cysteine perchlorate, 0.23 M HzPOs, 1.15 M H3P04, and the medium isoamyl alcohol containing 10 per cent acetonitrile, introduced with the permonophosphoric acid (Toennies, 1937, b). The heat of reaction was distinctly greater than in the corresponding HzSOs experiments and precipitation began within 2 seconds. The precipitate (yield 50 per cent of

4 Rather was it found that the smyl alcoholic solution of the peracid is rendered more stable by the presence of the amine sulfate. At room temperature a solution of 0.075 M HzSOs and 0.055 M H&SO4 showed, after 2 and after 24 hours, decreases in peracid (cf. Toennies (1937, a)) of 22 and 95 per cent respectively, while the same solution, containing in addition CaHnNHs+-S04H in 0.53 M concentration, showed decreases of only 17 and 53 per cent over the same periods.

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G. Toennies 39

weight of cysteine) gave the following data, ai = 0.535, af = 0.000, of = 0.523, o- = 0.031, r = 16.9, po4 = 0.254, and ss = 0.100, which suggest absence of R-SOH (no a/, cf. foot-note to Table II) and formation of large proportions of cystine and cysteic acid. This result, which is in sharp contrast with those of the analogous H2SOsH2S04 experiments, may be explained either by the higher oxidizing power of H3P06 (indicated by the heat of reaction) which causes the oxidation to pass rapidly beyond the R-SOH stage, or by a lower precipitation affinity of HaPOd. That the latter is a factor is suggested by the outcome of another experiment in which HzS05 was the oxidant and H3P04 the quantitatively predominat- ing precipitant. The concentrations used were 0.25 M cysteine pcrchlorate, 0.25 M H$Ob, 1.24 M H,PO4, and 0.07 M HzS04; and the result, yield 40 per cent of weight of cysteine, ai = 0.665, af = 0.000, of = 0.727, o- = 0.041, r = 17.7, so4 = 0.198, ~04 = 0.065, and ss = 0.138, indicating not only that again cystine and cysteic acid are the prevailing products, but also that HzS04 has stronger precipitating tendencies than HsPO+ since it predomi- nates in the precipitate even though its total concentration (0.25 M

from HzSOb and 0.07 M free HzS04) amounted only to 26 per cent of that of H3P04.

Variations in Concentrations-When the superiority of HzS04 and H&SO5 over the corresponding phosphorus compounds had thus become evident, the effect of changes in the ratio precipitant to substrate-oxidant, discussed in a previous paragraph (“Func- tion of sulfuric acid”), was investigated. However, lowering the substrate-oxidant concentration to one-tenth of its former value (0.025 M), while sulfuric acid remains 1.25 M, apparently makes the whole system too dilute, since no precipitate appeared in the oxidation. Even when the medium at these concentrations was 10 per cent isoamyl alcohol and 90 per cent ether-a mixture in which cysteine perchlorate remains soluble-only a small amount of precipitate was formed. It thus remained to see what the effect would be of increasing the ratio of precipitant to substrate-oxidant by an increase of the sulfuric acid concentration. Solutions of 4 M HzS04 in isoamyl alcohol could be obtained by running the H&O4 (99 per cent) very slowly (about 0.4 cc. per minute) into the alcohol which was maintained at less than 0’. The resulting solution is of oily consistency and, even at low temperature, subject

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Oxidation of Cysteine

TABLE III

Oxidation of Cysteine in Isoamyl Alcohol in Presence of 4 M Sulfuric Acid

Of the symbols not previously explained nh, means amino nitrogen according to Van Slyke, factor 0.926 being used; ssi disulfide according to Shinohara (193536), the dry substance being dissolved in the bisulfite solution; ss/ additional disulfide, formed spontaneously in the aqueous solution of the substance; sh+ maximal amount of cysteine which reacts with the substance; a- acid liberated in the reaction with excess cysteine. The technique and interpretation of these determinations are discussed in the text.

Reaction Oxidation Product 34 Oxidation Product 35

Cysteine, HClO,, M ................ 0.104 0.099 H2SOs, M. ......................... 0.106 0.098 HZSO,, “. ......................... 4.0 4.0 Volume, cc ........................ 38.5 107 Temperature, “C .................. About -10 -5

Precipitate Found :alculsted Found C -

hlculsted

Yield, by weight, per cent ......... so,, mdf ........................... s., milli-atom. ..................... nhl, mar .......................... ai, m.-eq .......................... a/, (‘ ......................... ssi,mnr ............................ ss/, (( ........................... o+, milli-atom. ....................

0-p I‘ ..... ................ r .................................. sh’, m.u. ......................... a-, m.-Ed.......................... -

87 0.387 0.387

0.802 0.800 0.133 0.133 0.050 0.050 0.092 0.092 0.904 0.890 0.250 0.270 3.62 3.30 0.276 0.276

86 0.389 0.447 0.451 0.800 0.123 0.042 0.094 0.915 0.248 3.69 0.276 0.042

-

(

-

0.389 0.457 0.457 0.825 0.123 0.042 0.094 0.866 0.278 3.11 0.283 0.043

--

Calculated composition

H#O,. ..................................... Cystine .................................... Sulfoxycysteine ............................. Cystine disulfoxide ......................... Cysteine sulfinic acid., .....................

7nH per nut per 100 mg. 100 mp.

0.387 0.389 0.050 0.042 0.214 0.240 0.062 0.043 0.026 0.047

-

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G. Toennies

to esterification and gradual darkening. However, since shortly after the preparation titration showed the calculated acidity, the solutions were used soon after they were obtained. For the oxidations both halves (substrate and oxidant) were made of approximately equal HzS04 concentration, by addition of the required amount of H&SO4 to the cysteine perchlorate solution in isoamyl alcohol, while in the other half HzS06 in concentrated solution was added to the previously prepared H&Sod-isoamyl alcohol mixture. Two experiments of this type were performed, both at -5” to - 10’; one in which the solutions were mixed as usual and one in which the HzSOb solution was rapidly emptied into the cysteine solution which was being stirred at high speed (about 800 R.P.M.). The latter method was tried as it was thought that uneven distribution, due to imperfect mixing of the viscous solutions, might cause local deviations from the 1: 1 ratio between substrate and oxidant and thus favor the formation of those compounds which are higher and lower in the oxidation scale than the sulfoxy compound. The results of the two experiments arc summarized in Table III. They indicate in the first place that rapid stirring has no profound effect on the nature of the reaction product. Secondly, they show markedly different analytical values from those of Table II. Whether or not the higher ratio precipitant to oxidant-substrate has resulted in a better yield of the desired compound cannot be stated with certainty as not all of the analytical criteria reported in Table III were applied to the earlier products. Since, for the time being, additional experi- mentation on the problem could not be undertaken, the work was brought to a temporary conclusion by expanding the scope of analytical tests applied to the oxidation products and finally putting the question of the presence of sulfoxycysteine to a crucial test.

Evidence for Identity of Suljoxycysteine

The amino N determination on Oxidation Product 35 (Table III) shows substantial equivalence between -NH2 groups and organic sulfur and thus supports the conclusion (Toennies, 1934) that the amino group in its acid form is protected against oxida-

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42 Oxidation of Cysteine -- H . .

tion by the absence of free electron pairs5 (R:N:H, while the

ii H H

basic form R: i : is susceptible to addition of oxygen, R: i : 0 and

ii subsequent changes).

ii

If the gradual formation of cystine and acidity which occurs in solution is due to spontaneous changes of the compound R-SOH, acid groups and cystine molecules may be expected to appear in equal numbers (cj. foot-note to Table II). Fig. 1 shows that the ratio of uf and ss/ is not unity but approximately 1.31 in the case under investigation. If the higher ratio is tentatively attributed to the presence of cystine disulfoxidc-which in acid solution forms acid groups and cystine molecules in a 3: 1 ratio (Lavine, 1936)- the calculated values for sulfoxycysteine and cystine disulfoxidc of Table III result. If these are correct the postulat,ctl reactions

R-SOH + R-SH --+ R-S-S-R + H,O (5)

aud It-S@-lt + II-SH + 11-S-S-R + R-SOrH C(i)

require the calculated values given in Table III under sh+ and a- for the amounts of cysteine consumed and acid simultaneously liberated. The cysteine consumption was determined by dis- solving weighed samples corresponding to a calculated content of 0.07 to 9.08 mM of -SOH + -SzOz-- in ‘25 cc. flasks with freshly standardized neutral cysteine solution corresponding to 0.10 mM of -SH. The remaining -SH was determined on 4 cc. portions by the technique of Shinohara (1935). The amount of cysteine found consumed, calculated per 100 mg. of substance, (sh+) was 0.284, 0.270, and 0.275 mM in three independent experiments and the amount of acid liberated in the reaction (a-), obtained by titration and the subtraction of the value of ai (OBOO), was 0.038, 0.050, and 0.042 milli-equivalent. Thus the .results are in sub- stantial agreement with the amounts of -SOH and -SzO2-

H . .

6 Just as ammonia in the H:N:H form is stable under oxidizing condi-

H tions in the Kjeldahl method.

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G . Toennies

calculated from the spontaneously formed amounts of cystine and acid. However, it was noted that while in a comparable experi- ment with cystine disulfoxide the maximal amount of cysteine

FIG. 1. The spontaneous formation of cystine and acidity from an oxida- tion product in solution. 106.2 mg. of Oxidation Product 35 (Table III) were dissolved to 100 cc. with water, and periodical determinations were made of -S-S- by the Folin-Shinohara method (Shinohara, 1935-36), with 4 cc. samples made up to 25 cc. for the determination, and of the acidity by titrating 10 cc. samples with 0.05 N NaOH from a microburette to the intermediate gray color of brom-cresol purple (pH 6). The two resulting curves are labeled “acid pH 2” and “-S-S- pH 2” (the acidity of this solution was about pH 2.3 in the beginning). The curves “-S-S- pH 5” and “- S-S- pH 10” are the result of determinations made on solu- tions of similar concentrations which contained 20 mM of sodium acetate and 6 mM of acetic acid (pH 5.2) and 1.263 milli-equivalents of NaOH per 100 mg. (pH 10) respectively. The time of determination for the -S-S- values was defined as the moment of addition of the bisulfite, on the assump- tion-borne out by the constancy of the values obtained-that the spon- taneous changes are arrested by the action of the bisulfite, R--S&-R + NaHS08 + R-S-SOaNa + R-SOzH (Lavine, 1936) and presumably R-SOH + NaHS03 -+ R-S-SOsNa + HIO.

had reacted 2 minutes after its addition, in the case of the present substance the final value was not attained until after about 3 hour, the reaction being only about 80 per cent complete after 2 minutes.

As a further consequence and final means of verification of the

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44 Oxidation of Cysteine

assigned composition may be taken the amount of cystine resulting from the reaction expressed by Equations 5 and 6. To this end 127.8 mg. of Oxidation Product 35 and 43.6 mg. of cysteine were dissolved in 20 cc. of water previously brought to the sensitive intermediate gray shade (pH 4.9) of a 3 : 2 mixture of brom-cresol blue and methyl red (Kolthoff, 1929). Within 25 minutes a total of 1.080 mM of NaOH was added to neutralize the acid liberated in the reaction. After 24 hours standing in the refrigerator, the pH (4.9) remaining unchanged, 61.35 mg. of cystine were obtained by filtration and drying. It was identified by its optical rotation in a 0.5 per cent solution in N HCl. The value found [a]:: = -231” f lo, compared with the one calculated for pure I-cystine (Andrews, 1925; Toennies and Lavine, 1930), [a]:: = -235”, indicates a purity of the cystine precipitate of 98 per cent. The filtrate was made up to 100 cc. The amount of iodine liberated (cf. “Methods”) by 20 cc. fractions was 0.0236, 0.0257 mM. The filter paper on which the crystallized cystine had been collected was treated with 5 cc. of N H&S04 in order to dissolve residual cystine. The acid washings, made up to 50 cc., contained, accord- ing to a determination with phosphotungstic acid (Shinohara, 1935-36) 0.0304 mM of disulfide, while the filtrate itself by the same method showed 0.142 mM of disulfide. In order further to identify this portion 50 cc. of the filtrate were evaporated to dry- ness, leaving a residue, dried over PZOS, of 62.9 mg. The solution of this residue in N HCl (total volume 6.70 cc.) showed ai!& = -0.49” (1 dm. tube). These analytical data are the basis of the following computations, which are referred to the unit of 100 mg. of substance. The calculated values arc based on the analytical data given in Table III.

Cysteine used for reaction,. 0.232 lllM I‘ calculated to be required.. . 0.283 “

Acidity, after reaction with cysteine.. 0.845 m.-eq. << before “ “ ” 0.800 ” ‘L increase, found.. 0.045 ‘I << ,‘ calculated.. 0.043 “

Iodide-oxidizing power, before reaction with cys- teine............................................. 0.248 rnhf 1~

Iodide-oxidizing power, after reaction with cys- teine............................................ 0.096 “ “

Iodide-oxidizing power, decrease, found ............ 0.152 ‘I “ ‘I ,I “ calculated ....... 0.142 “ “

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G. Toennies 45

Evaporation residue of filtrate Na2SOI, calculated.. . 0.389 mM = 55.2 mg. Cystine, calorimetrically.. 0.111 “ = 26.8 “ R-SOJVa from R-&0,-R, calculated. 0.043 “ = 7.5 “

‘I ” original assumed R-SOZH.. 0.047 “ = 8.2 ”

Calculated.. .%? “ Found............................................... 98.4 “

Assuming the value [a]ng = -33” (Lavine, 1936) for the specific rotation of the sulfinic acid and assuming that the amount of the latter present in the evaporation residue lies between (per 100 mg. of substance) 0.090 mM (according to original calculation) and 0.043 mM (resulting from disulfoxide only), one obtains a calculated correction of +0.03” f 0.01’ and, therefore, the rota- tion to be assigned to cystine as = -0.52’ f O.Ol’, or a value for its specific rotation of [cY]& = -0.52’ X 6.70/0.017 = -205’, compared with a calculated value of -235”. On this basis the minimum amount of I-cystine present would be 87 per cent of the calorimetric value, i.e. 0.097 mM (per 100 mg.).

l-Cystine identified

Main portion, according to weight and optical rotation. O’l&4 “ “ residue on filter, calorimetrically. 0.024

Filtrate, calorimetrically 0.111 mM ‘I polarimetrically 0.097 “ 1

mean value.. 0.104

Total, after reaction with cysteine.. . . . . . 0.332 Found (calorimetrically) before reaction with cysteine.. 0.042 -. Increase,found......................................... 0.290

‘I calculated..................................... 0.283

The result of this experiment confirms the assumed composition as far as R-S-S-R, R--&02-R, and R-SOH are concerned, since the values obtained for the changes in acidity, iodide-oxidiz- ing power, and cystine are substantially within the range of the calculated ones. Uncertainty must be admitted regarding the fraction designated as R-SO&I in the original calculation (Table III), on account of the considerable discrepancies between the calculated and found values for ai, o-, and o+. Numerous observa- tions encountered in the course of this work point toward the possible existence of a cysteine derivative R-SOZH, isomeric with the sulfinic acid which results from alkaline decomposition of the disulfoxide (Lavine, 1936), but having neither acid- nor iodide-

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Osidation of Cysteine

oxidizing properties; a compound which also may be involved in the acid decomposition of the disulfoxide (Lavine, 1936). However, less than 10 per cent by weight of the product under investigation is involved in this uncertainty since 93 per cent (including HzS04) may be considered identified as l-cystine or oxidation products of I-cysteine-cystine which by combining with I-cysteine are converted into Lcystine. The largest part of this cystine-forming fraction combines with cysteine without liberation of acid; a condition which, together with the other analytical data, is satisfied by the level of oxidation that corresponds to the addition of 1 oxygen atom to the cysteine molecule. Whether the compound is present in its anhydride form (R-StO-R, cystine monosulfoxide) or as sulfoxycysteine (R-S(O)H or R-SOH) cannot be definitely stated, although the latter form is favored by the analytical values and seems by far the most likely according to the expected ease of its formation in the interaction of equimolar amounts of cysteine and peracid.

SUMMARY

The possibility of oxidizing cysteine to the sulfenic acid level by rapid oxidation in non-aqueous media has been studied. Per- monosulfuric acid acting in isoamyl alcohol upon cysteine perchlo- rate causes immediate precipitation of sulfate-containing products in which the desired compound appears to be present in varying amounts. The effect of experimental variations has been studied and the identity of the chief oxidation product as a cysteine derivative corresponding to the sulfenic acid level has been estab- lished by a quantitative demonstration of the reaction with cyst&c which conforms with the equation R-SOH + R--SH -+ R-S-S-R + HzO.

BIBLIOGRAPHY

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G. Toennies 47

Fiske, C. H., J. Biol. Chem., 47,59 (1921). Hall, N. F., Chem. Rev., 8, 191 (1931). Hammett, F. S., Protoplasma, 11, 382 (1930). Hammett, F. S., and associates, Am. J. Cancer, 28, 554 (1936), and

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(1937, 6); J. Biol. Chem., 120, 297 (1937, c). Toennies, G., and Bennett, M. A., J. Biol. Chem., 112, 497 (1935-36). Toennies, G., and Lavinc, T. F., J. Biol. Chem., 89, 153 (1930); 100, 463

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Gerrit ToenniesOXIDATION PRODUCT

"SULFENIC ACID" AS THE PRIMARYNON-AQUEOUS MEDIA: THE OXIDATION OF CYSTEINE IN

1937, 122:27-47.J. Biol. Chem. 

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