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Cystine cleavage in alkaline medium
ARCHIVES
OF
BIOCHEMISTRY
AND
Cystine
BIOPHYSICS
Cleavage
C. DE MARCO, From the Institutes
of Biological
51-55 (1963)
100,
in Alkaline
R/I. COLETTA
Chemistry,
AND
Medium’ D. CAVALLINI
University of Rome, Rome, Italy; Modena,
and University
of Modena,
Italy
Received August 6, 1962 Cystine incubated at 38°C. in 0.033 fi NaOH and in the presence of Cu++ ions is extensively degraded. The following products have been found and quantitatively determined: cysteinesulfinic acid, alaninethiosulfonic acid, cysteic acid, S-sulfocysteine, acetic acid, ammonia, COz, and bhiosulfate. Cystine cleavage is initiated by an a,p-elimination reaction with production of the persulfide COOH-CHSH,CH2-S-SH called thiocysteine. The possible side reactions giving rise from t,his substance to the other sulfur compounds are discussed.
The study of the alkaline decomposition of cystine, catalyzed by copper ions, seems of particular interest since some of the detected reactions may represent non-enzymic models for the biological breakdown of cystine. Actually few of the products of the alkaline decomposition are known as metabolic products of cystine.
In previous research (1, 2) the oxidative degradation of cystine in alkaline medium and in the presence of copper ions was studied. Among the products of the reaction the following compounds were identified : alaninethiosulfonic acid, cysteinesulfinic acid, S-sulfocysteine, cysteic acid, ammonia, and thiosulfate; the oxygen consumption was found to he more than 2.F, moles/mole cystine. The essential role of copper ions and alkalinity has been emphasized in the reported papers. The omission of copper ions or its substitution by other metals, as well as the shift toward neutral pH, leads to a negligible decomposition of rystine. The addition of ethylenediaminetetraacetic acid prevented the effect of copper ions indicating the necessity for the reaction of copper in an ionized form (1, 2). The reaction scheme that was tentatively proposed did not account adequately for all the compounds arising from cystine, and me have therefore reinvestigated the reaction, paying particular attention in identifying the initial products. In the interpretation of the results we have now taken into account another possible mechanism of cystine cleavage recently demonstrated (3) and t’he particular reactions that some of the products could undergo.
EXPERIMENTAL Fifty micromoles of cystine were incubated with 1 pmole CuClz in 3 ml. of 0.033 A7 NaOH in a thermost.ated wat,er bath at 38°C. wit.h mechanical agitation. At, various times of incubation, aliquots of the reaction mixture were collected and the possible reaction products were determined by the following procedures. Cystine was determined by t,he Folin-Marenzi reaction (4). Thiosulfntes were determined by t.he cyanolysis reaction according to Yijrbo (5). For the analysis of cysteic acid, cysteinesulfinic acid, S-sulfocysteine, and alaninethiosulfonic acid, the incubation mixture was acidified with Dowex 50-H+ to remove the unreacted cystine, and then was submitt’ed to unidimensional paper chromatography using eollidine-lutidine as solvent. The compounds were located by ninhydrin, estimation and quantitative was made by of the chromatographic strips. densitometr) Standard values have been obtained from pure synthetic compounds submitted to the same analytical procedure. Alaninethiosulfonic acid was also quantitatively determined by the cyanolysis reaction described by Siirbo for thiosulfonates (5). Am-
1 This investigation was supported in part. by t,he Consiglio Nazionale delle Ricerche. 51
52
DE MARCO,
COLETTA
monia was determined performing the incubation in glass-stoppered Conway cells incubated at 38°C. with slow mechanical agitation. The cells contained in the center well 1 ml. of 1 IV H&Sod; the ammonium sulfate formed was then estimated by the Kessler reagent or by the ninhydrin reaction, the results obtained by these two procedures being in good agreement. Preliminary experiments showed that in 0.033 N NaOH at 38°C. ammonia diffuses very rapidly. For CO* determination, 1 ml. of the alkaline incubation mixture was transferred in Warburg vessels containing in the side arm 0.3 ml. of 5 N HzSOI. After temperature equilibration, t,he sulfuric acid was mixed with the contents of the main cavity, and the COZ evolved within 20 min. was recorded. Acetic acid was determined by distillation of 0.5 ml. of the incubate over sodium citrate in Conway cells (6). On aliquots of the incubation mixture, the “cold cyanolysis reaction” (in which cyanide is allowed to react for 10 min. at room temperature) (7) was also performed. Since in this reaction various compounds, as polpsulfides or persulfides, could give rise to thiocyanate, the results obtained are reported below as micromoles of SCN-. Analyses for keto acids were made according to Cavallini and Mondovi (8), and analyses for sulfites according to Fromageot et al. (9). For the
AND
CAVALLINI
detection of sulfate, BaCl? was added to the acidified incubat,ion mixture. RESULTS
The amounts of the various compounds formed in the reaction as a function of time are reported in Fig. 1. Table I gives the mean values, from five determinations, of the quantities of the compounds which were present in the incubation mixture after 2 hr. at 38°C. Analyses for keto acids, sulfites, and sulfates always gave negative results. The first product which we have been able to detect is ammonia. Thus after 5 min. of incubation, when no other compound is practically detectable and only 3 wmoles cystine has disappeared, 1 pmole ammonia is formed, and thereafter the ammonia production is linear with time. Carbon dioxide and acetic acid also appear in the first 10 min. of incubation and increases parallel to ammonia. After 2 hr. of incubation, from 1 mole cystine there were formed 1 mole each of ammonia. COZ, and acetic acid; these three compounds thus account stiochiometrically for a half molecule of cystine, apart from the sulfur.
‘x \
X Cystme
\
X
\
FIG. 1. Micromoles of various compounds present at different times of incubations in a reaction mixture containing 50 pmoles cystine and 1 rmole CuCL in 3 ml. of 0.033 N NaOH. Temp. : 38°C. CSA = cysteinesulfinic acid; ATS = alaninethiosulfonic acid; S-S-Cy = S-sulfocysteine; and, SCN = thiocyanate arising from cold cyanolyzable sulfur (See text for details).
CYSTIKE TABLE
I
MICROMOLES OF COMPOUNDS PRESENT, AFTER 2 HR. OF INWBATION AT 38”, IN A REACTION MIXTURE CONTAINING 50 ~MOLES OF CYSTINE AKD 1 ~MOLE OF CUCI,~ IN 3 XL. OF 0.033 N KAOH Mean
values
from five repeated (see text for details).
Cystine SH, CO? Scetic acid Cysteinesulfinic acid Alaninethiosulfonic acid Cysteic acid S-Sulfocysteine Thiosulfate SCN- (cold cyanoliaable &to acids Sulfite Sulfate
sulfur)
experiments
17.5 27.5 26.4 21.2 10.3 10.7 1.9 2.2 4.5 1.9 0.0 0.0 0.0
On the other hand, within the limits of experimental error, the sulfur of the cystine which disappeared after 2 hr. of incubation (65 pat’oms) has been recovered in different organic or inorganic compounds, i.e. cysteinesulfinic acid 10.3 patoms sulfur, alaninethiosulfonic acid 21.4, cysteic acid 1.9, X-sulfocysteine 4.4, thiosulfates 9, and cyanolyzable sulfur (polysulfides) 3.8 for a total of 50.8 patoms. Of these sulfur coinpounds cysteinesulfinic acid is the most readily formed: It rises parallel to ammonia in the first 30 min. and then slows down, conincident with the appearance of alaninethiosulfonic acid. Cysteinesulfinic acid and alaninethiosulfonic acid are the sulfur compounds produced in greater amounts during the reaction; S-sulfocysteine, cysteic acid, and thiosulfates appear later in the incubation mixture and must be regarded as originating from side reactions. DISCUSSION
The results obtained are in agreement with a reaction mechanism proposed by Swan (10) and Dann et al. (11) for the degradation of cystine in alkaline medium and recently demonstrated in our laboratory for pyridoxal-catalyzed cystine cleavage (3). Cystine degradation would be initiated by the ionization of the a-hydrogen atom, followed by the p-elimination of an unstable
53
CLEAVAGE
persulfide for which we have proposed the name thiocysteine (3). According to the scheme, the other products would be pyruvic acid and ammonia. Under the present experimental conditions, however, pyruvic acid must be completely degraded into acetic acid and COZ. It should be recalled that also during cystine cleavage catalyzed by pyridoxal (3), a reaction which takes place through 01, Pelimination, pyruvic acid was in part recovered as CO,. Ammonia, COZ, and acetic acid are produced in equimolar amounts and account for a half molecule of cystine degraded; also, this finding is in agreement with the above reaction scheme. The fate of thiocysteine, which is a very unstable compound, could be in different directions: Under some conditions it has been reported to be split into elementary sulfur and cysteine (3). However, under the present experimental conditions the release of elementary sulfur has never been detected. Thiocysteine could also be split into cystine and a disulfide according to the following reaction: 2R-S-SH
+ R-S-S-R
+ H&z
We cannot exclude the fact that, at least to a small extent, this reaction takes place in alkaline medium. We have indeed detected some cold cyanolyzable sulfur, which can be accounted for by thiocysteine itself R-S-SH
+ CN-
--f R-SH
+ SCN-
or by the disulfide originating from it: s2-- + CY ---ts-- + RCNThe disulfide could undergo a further oxidation to thiosulfate, which we also have detected in the incubation mixture. Kevertheless, this sequence of reactions could hardly explain the production of cysteinesulfinic acid. We must then consider another reaction which thiocysteine may undergo, i.e., hydrolysis favored by the presence of metal ions (12) leading to hydrogen sulfide and cysteinesulfenic acid. R-S-SH
+ H?O + R-SOH
+ H,S
Cysteinesulfenic acid would then be readily converted to cysteinesulfinic acid by dismutation, and hydrogen sulfide oxidized
54
MARCO,
DE
COLETTA
to thiosulfate. The production of alaninethiosulfonic acid could be also explained by a transsulfuration reaction taking place between thiocysteine and cysteinesulfinic acid (7, 13). R-S-SH
+
R-SOIH
+
R-SOzSH
+
R-SH
Cysteic acid could arise from oxidation of cysteinesulfinic acid. The formation of Ssulfocysteine could also be due to an interreaction of thiosulfate with cysteine (14). The thiosulfate produced could arise by the oxidation of H2S in the presence of CL++ (15) ; however, also S-sulfocysteine (16) and alaninethiosulfonic acid (unpublished data) under the present experimental conditions split off thiosulfate.
CAVALLINI
The breakdown of cystine under the present conditions is accompanied by a high oxygen uptake; since the LY,p-elimination is an anaerobic process, the oxygen uptake must be due to oxidative side reactions, like the oxidation of cysteine arising from thiocysteine, or oxidation of hydrogen sulfide to thiosulfate, or cysteinesulfinic acid to cysteic acid. The effect of Cu++ ions as a catalyst for the reaction (1, 2) should be again emphasized. This suggests that some of the findings obtained in previous studies on the alkaline degradation of cystine (11, 17, 18) may be due to the presence of traces of cupric ions in the solutions. REFERENCES
CONCLUSIONS
The data here reported indicate that in alkaline medium copper ions catalyze the cleavage of cystine by an LY, p-elimination reaction, with production of thiocysteine, according to the following scheme CH2-
AND
1. DE MARCO, C., MONDOV~, B., AND CAVALLINI, D., Ital. J. Biochem. 7, 158 (1958). 2. POVOLEDO, D., DE MARCO, C., AND CAVALLINI, D., Ital. J. Biochem. 7, 78 (1958). 3. CAVALLINI, D., DE MARCO, C., AND MONDOV?, B., Arch. Biochem. Biophys. 87, 281 (1960).
CH2-S-SH
S-S-C&
I
I
CH-NH2 I COOH
CH-NH2 I COOH
CH3
I
-
CH-NH2 I COOH
+
NH3
+
CO I COOH
CH3 + I COOH
Thiocysteine could be the source of all the other compounds which were detected in the incubation mixture, according to the following reaction sequence :
4. SHINOHARA, K., J. Biol. Chem. 112,683 (1935). 5. SBRBO, B., Biochim. et Biophys. Acta 24, 324 (1957). 6. CONWAY,
CHz- S -SH
CHz-SO,I CH-NH, I COOH
co,
SH
E. S., “Microdiffusion
Analysis
and
CYSTIXE Volumetric Error” & Son, London,
7. 8.
9.
10. 11.
p. 236. Grosby Lookwood 1957. CAVALLINI, I)., DE MARCO, C., MONDOV~, B., AND MORI, B. G. Enz~mologia 22, I61 (19GO). CAVALLINI, D., AND MONDOV?, B., Clin. Chim. Acta 2, 312 (1957). FROMACTEOT, C., CIIATAGNER, F., .&ND BERGERET, B., B&him. et Biophys. Acta 2, 294 (1948). SWAN, J. M., .yatzcye 179, 9G5 (1957). T~ANN, J. It., OLIVER, G. L., AND GATES, J. W., J. Am. Chem. Sot. 79, lG44 (1957).
CLEAVAGE
55
12. STRIKS, W., AND KOLTHOFF, J. M., dnal. Chem. 25, 1050 (1953). 13. DE MARCO, C., COLETTA, M., AND CAVALLINI, D., Arch. Biochem. Biophys. 93, 179 (1961). 14. SZCZEPKOWSKI, T. W., LVature 18.2, 934 (1958). 15. DE MARCO, C., COLETTA, M., AND CAVALLISI, D., Ezpe&ntia 18, lli (1962). 16. COLETTA, M., MARI, S., AND DE MARCO, C., Ital. J. Biochem. 10, 411 (1961). 17. ANDREWS, J. C., J. Biol. C’h,em. 80, 191 (1928). 18. THOR, C. J. B., AND GORTNER, R. il., .J. Biol. Chem. 99, 383 (1953).
OF
BIOCHEMISTRY
AND
Cystine
BIOPHYSICS
Cleavage
C. DE MARCO, From the Institutes
of Biological
51-55 (1963)
100,
in Alkaline
R/I. COLETTA
Chemistry,
AND
Medium’ D. CAVALLINI
University of Rome, Rome, Italy; Modena,
and University
of Modena,
Italy
Received August 6, 1962 Cystine incubated at 38°C. in 0.033 fi NaOH and in the presence of Cu++ ions is extensively degraded. The following products have been found and quantitatively determined: cysteinesulfinic acid, alaninethiosulfonic acid, cysteic acid, S-sulfocysteine, acetic acid, ammonia, COz, and bhiosulfate. Cystine cleavage is initiated by an a,p-elimination reaction with production of the persulfide COOH-CHSH,CH2-S-SH called thiocysteine. The possible side reactions giving rise from t,his substance to the other sulfur compounds are discussed.
The study of the alkaline decomposition of cystine, catalyzed by copper ions, seems of particular interest since some of the detected reactions may represent non-enzymic models for the biological breakdown of cystine. Actually few of the products of the alkaline decomposition are known as metabolic products of cystine.
In previous research (1, 2) the oxidative degradation of cystine in alkaline medium and in the presence of copper ions was studied. Among the products of the reaction the following compounds were identified : alaninethiosulfonic acid, cysteinesulfinic acid, S-sulfocysteine, cysteic acid, ammonia, and thiosulfate; the oxygen consumption was found to he more than 2.F, moles/mole cystine. The essential role of copper ions and alkalinity has been emphasized in the reported papers. The omission of copper ions or its substitution by other metals, as well as the shift toward neutral pH, leads to a negligible decomposition of rystine. The addition of ethylenediaminetetraacetic acid prevented the effect of copper ions indicating the necessity for the reaction of copper in an ionized form (1, 2). The reaction scheme that was tentatively proposed did not account adequately for all the compounds arising from cystine, and me have therefore reinvestigated the reaction, paying particular attention in identifying the initial products. In the interpretation of the results we have now taken into account another possible mechanism of cystine cleavage recently demonstrated (3) and t’he particular reactions that some of the products could undergo.
EXPERIMENTAL Fifty micromoles of cystine were incubated with 1 pmole CuClz in 3 ml. of 0.033 A7 NaOH in a thermost.ated wat,er bath at 38°C. wit.h mechanical agitation. At, various times of incubation, aliquots of the reaction mixture were collected and the possible reaction products were determined by the following procedures. Cystine was determined by t,he Folin-Marenzi reaction (4). Thiosulfntes were determined by t.he cyanolysis reaction according to Yijrbo (5). For the analysis of cysteic acid, cysteinesulfinic acid, S-sulfocysteine, and alaninethiosulfonic acid, the incubation mixture was acidified with Dowex 50-H+ to remove the unreacted cystine, and then was submitt’ed to unidimensional paper chromatography using eollidine-lutidine as solvent. The compounds were located by ninhydrin, estimation and quantitative was made by of the chromatographic strips. densitometr) Standard values have been obtained from pure synthetic compounds submitted to the same analytical procedure. Alaninethiosulfonic acid was also quantitatively determined by the cyanolysis reaction described by Siirbo for thiosulfonates (5). Am-
1 This investigation was supported in part. by t,he Consiglio Nazionale delle Ricerche. 51
52
DE MARCO,
COLETTA
monia was determined performing the incubation in glass-stoppered Conway cells incubated at 38°C. with slow mechanical agitation. The cells contained in the center well 1 ml. of 1 IV H&Sod; the ammonium sulfate formed was then estimated by the Kessler reagent or by the ninhydrin reaction, the results obtained by these two procedures being in good agreement. Preliminary experiments showed that in 0.033 N NaOH at 38°C. ammonia diffuses very rapidly. For CO* determination, 1 ml. of the alkaline incubation mixture was transferred in Warburg vessels containing in the side arm 0.3 ml. of 5 N HzSOI. After temperature equilibration, t,he sulfuric acid was mixed with the contents of the main cavity, and the COZ evolved within 20 min. was recorded. Acetic acid was determined by distillation of 0.5 ml. of the incubate over sodium citrate in Conway cells (6). On aliquots of the incubation mixture, the “cold cyanolysis reaction” (in which cyanide is allowed to react for 10 min. at room temperature) (7) was also performed. Since in this reaction various compounds, as polpsulfides or persulfides, could give rise to thiocyanate, the results obtained are reported below as micromoles of SCN-. Analyses for keto acids were made according to Cavallini and Mondovi (8), and analyses for sulfites according to Fromageot et al. (9). For the
AND
CAVALLINI
detection of sulfate, BaCl? was added to the acidified incubat,ion mixture. RESULTS
The amounts of the various compounds formed in the reaction as a function of time are reported in Fig. 1. Table I gives the mean values, from five determinations, of the quantities of the compounds which were present in the incubation mixture after 2 hr. at 38°C. Analyses for keto acids, sulfites, and sulfates always gave negative results. The first product which we have been able to detect is ammonia. Thus after 5 min. of incubation, when no other compound is practically detectable and only 3 wmoles cystine has disappeared, 1 pmole ammonia is formed, and thereafter the ammonia production is linear with time. Carbon dioxide and acetic acid also appear in the first 10 min. of incubation and increases parallel to ammonia. After 2 hr. of incubation, from 1 mole cystine there were formed 1 mole each of ammonia. COZ, and acetic acid; these three compounds thus account stiochiometrically for a half molecule of cystine, apart from the sulfur.
‘x \
X Cystme
\
X
\
FIG. 1. Micromoles of various compounds present at different times of incubations in a reaction mixture containing 50 pmoles cystine and 1 rmole CuCL in 3 ml. of 0.033 N NaOH. Temp. : 38°C. CSA = cysteinesulfinic acid; ATS = alaninethiosulfonic acid; S-S-Cy = S-sulfocysteine; and, SCN = thiocyanate arising from cold cyanolyzable sulfur (See text for details).
CYSTIKE TABLE
I
MICROMOLES OF COMPOUNDS PRESENT, AFTER 2 HR. OF INWBATION AT 38”, IN A REACTION MIXTURE CONTAINING 50 ~MOLES OF CYSTINE AKD 1 ~MOLE OF CUCI,~ IN 3 XL. OF 0.033 N KAOH Mean
values
from five repeated (see text for details).
Cystine SH, CO? Scetic acid Cysteinesulfinic acid Alaninethiosulfonic acid Cysteic acid S-Sulfocysteine Thiosulfate SCN- (cold cyanoliaable &to acids Sulfite Sulfate
sulfur)
experiments
17.5 27.5 26.4 21.2 10.3 10.7 1.9 2.2 4.5 1.9 0.0 0.0 0.0
On the other hand, within the limits of experimental error, the sulfur of the cystine which disappeared after 2 hr. of incubation (65 pat’oms) has been recovered in different organic or inorganic compounds, i.e. cysteinesulfinic acid 10.3 patoms sulfur, alaninethiosulfonic acid 21.4, cysteic acid 1.9, X-sulfocysteine 4.4, thiosulfates 9, and cyanolyzable sulfur (polysulfides) 3.8 for a total of 50.8 patoms. Of these sulfur coinpounds cysteinesulfinic acid is the most readily formed: It rises parallel to ammonia in the first 30 min. and then slows down, conincident with the appearance of alaninethiosulfonic acid. Cysteinesulfinic acid and alaninethiosulfonic acid are the sulfur compounds produced in greater amounts during the reaction; S-sulfocysteine, cysteic acid, and thiosulfates appear later in the incubation mixture and must be regarded as originating from side reactions. DISCUSSION
The results obtained are in agreement with a reaction mechanism proposed by Swan (10) and Dann et al. (11) for the degradation of cystine in alkaline medium and recently demonstrated in our laboratory for pyridoxal-catalyzed cystine cleavage (3). Cystine degradation would be initiated by the ionization of the a-hydrogen atom, followed by the p-elimination of an unstable
53
CLEAVAGE
persulfide for which we have proposed the name thiocysteine (3). According to the scheme, the other products would be pyruvic acid and ammonia. Under the present experimental conditions, however, pyruvic acid must be completely degraded into acetic acid and COZ. It should be recalled that also during cystine cleavage catalyzed by pyridoxal (3), a reaction which takes place through 01, Pelimination, pyruvic acid was in part recovered as CO,. Ammonia, COZ, and acetic acid are produced in equimolar amounts and account for a half molecule of cystine degraded; also, this finding is in agreement with the above reaction scheme. The fate of thiocysteine, which is a very unstable compound, could be in different directions: Under some conditions it has been reported to be split into elementary sulfur and cysteine (3). However, under the present experimental conditions the release of elementary sulfur has never been detected. Thiocysteine could also be split into cystine and a disulfide according to the following reaction: 2R-S-SH
+ R-S-S-R
+ H&z
We cannot exclude the fact that, at least to a small extent, this reaction takes place in alkaline medium. We have indeed detected some cold cyanolyzable sulfur, which can be accounted for by thiocysteine itself R-S-SH
+ CN-
--f R-SH
+ SCN-
or by the disulfide originating from it: s2-- + CY ---ts-- + RCNThe disulfide could undergo a further oxidation to thiosulfate, which we also have detected in the incubation mixture. Kevertheless, this sequence of reactions could hardly explain the production of cysteinesulfinic acid. We must then consider another reaction which thiocysteine may undergo, i.e., hydrolysis favored by the presence of metal ions (12) leading to hydrogen sulfide and cysteinesulfenic acid. R-S-SH
+ H?O + R-SOH
+ H,S
Cysteinesulfenic acid would then be readily converted to cysteinesulfinic acid by dismutation, and hydrogen sulfide oxidized
54
MARCO,
DE
COLETTA
to thiosulfate. The production of alaninethiosulfonic acid could be also explained by a transsulfuration reaction taking place between thiocysteine and cysteinesulfinic acid (7, 13). R-S-SH
+
R-SOIH
+
R-SOzSH
+
R-SH
Cysteic acid could arise from oxidation of cysteinesulfinic acid. The formation of Ssulfocysteine could also be due to an interreaction of thiosulfate with cysteine (14). The thiosulfate produced could arise by the oxidation of H2S in the presence of CL++ (15) ; however, also S-sulfocysteine (16) and alaninethiosulfonic acid (unpublished data) under the present experimental conditions split off thiosulfate.
CAVALLINI
The breakdown of cystine under the present conditions is accompanied by a high oxygen uptake; since the LY,p-elimination is an anaerobic process, the oxygen uptake must be due to oxidative side reactions, like the oxidation of cysteine arising from thiocysteine, or oxidation of hydrogen sulfide to thiosulfate, or cysteinesulfinic acid to cysteic acid. The effect of Cu++ ions as a catalyst for the reaction (1, 2) should be again emphasized. This suggests that some of the findings obtained in previous studies on the alkaline degradation of cystine (11, 17, 18) may be due to the presence of traces of cupric ions in the solutions. REFERENCES
CONCLUSIONS
The data here reported indicate that in alkaline medium copper ions catalyze the cleavage of cystine by an LY, p-elimination reaction, with production of thiocysteine, according to the following scheme CH2-
AND
1. DE MARCO, C., MONDOV~, B., AND CAVALLINI, D., Ital. J. Biochem. 7, 158 (1958). 2. POVOLEDO, D., DE MARCO, C., AND CAVALLINI, D., Ital. J. Biochem. 7, 78 (1958). 3. CAVALLINI, D., DE MARCO, C., AND MONDOV?, B., Arch. Biochem. Biophys. 87, 281 (1960).
CH2-S-SH
S-S-C&
I
I
CH-NH2 I COOH
CH-NH2 I COOH
CH3
I
-
CH-NH2 I COOH
+
NH3
+
CO I COOH
CH3 + I COOH
Thiocysteine could be the source of all the other compounds which were detected in the incubation mixture, according to the following reaction sequence :
4. SHINOHARA, K., J. Biol. Chem. 112,683 (1935). 5. SBRBO, B., Biochim. et Biophys. Acta 24, 324 (1957). 6. CONWAY,
CHz- S -SH
CHz-SO,I CH-NH, I COOH
co,
SH
E. S., “Microdiffusion
Analysis
and
CYSTIXE Volumetric Error” & Son, London,
7. 8.
9.
10. 11.
p. 236. Grosby Lookwood 1957. CAVALLINI, I)., DE MARCO, C., MONDOV~, B., AND MORI, B. G. Enz~mologia 22, I61 (19GO). CAVALLINI, D., AND MONDOV?, B., Clin. Chim. Acta 2, 312 (1957). FROMACTEOT, C., CIIATAGNER, F., .&ND BERGERET, B., B&him. et Biophys. Acta 2, 294 (1948). SWAN, J. M., .yatzcye 179, 9G5 (1957). T~ANN, J. It., OLIVER, G. L., AND GATES, J. W., J. Am. Chem. Sot. 79, lG44 (1957).
CLEAVAGE
55
12. STRIKS, W., AND KOLTHOFF, J. M., dnal. Chem. 25, 1050 (1953). 13. DE MARCO, C., COLETTA, M., AND CAVALLINI, D., Arch. Biochem. Biophys. 93, 179 (1961). 14. SZCZEPKOWSKI, T. W., LVature 18.2, 934 (1958). 15. DE MARCO, C., COLETTA, M., AND CAVALLISI, D., Ezpe&ntia 18, lli (1962). 16. COLETTA, M., MARI, S., AND DE MARCO, C., Ital. J. Biochem. 10, 411 (1961). 17. ANDREWS, J. C., J. Biol. C’h,em. 80, 191 (1928). 18. THOR, C. J. B., AND GORTNER, R. il., .J. Biol. Chem. 99, 383 (1953).