OSCARGAWRON A N D JOSEPH FERNANDO
2906 [ C O T 1 RIBUTIOV
FRIIM 1 l I E
DEPARTMENT OF
CHESIIISTRY,
DUQUESSEUNIVERSITY,
VOl. 53 PITTSUUKCJH
19, P E N N A . ]
Kinetics of the Cyanide-Cystine Reaction BY OSCARGAWRON AND JOSEPH FERNANDO RECEIVED JANUARY 9, 1961
A spectrophotometric method was used to study the kinetics of the cystine-cyanide reaction in 0.04 M potassium hydroxide at pH 12.5. As expected for nucleophilic displacement, the reaction is bimolecular and cyclization of the thiocyanato product is much faster than the reverse reaction. Activation parameters a t 35" for the reaction are: E., 16.8 kcal./mole; AH*, 16.1 kcal./mole and AS*, -7.4 e.u. The entropy of activation is of the same order of magnitude as is that for the reaction of CX- with S8 but some 20 e.u. less than that for the reaction of sulfite ion with cystine, indicating the activated complex for cystine-sulfite to be more crowded than that with cyanide ion.
Of the several mechanisms by which scission of titration1* and by amperometric titrationla indicated the preparations to be 97.5% and 98.3y0 pure, respectively. a sulfur-sulfur bond occurs, nucleophilic displace- A portion of an aqueous suspension of cystine was treated ment a t a sulfur atom is of particular interest be- with hydrogen sulfide," filtered, and washed with distilled cause of the variety of nucleophiles which may be water and dried in VQCUO, a t looo, over phosphorus pentused and the variety of products which may be ob- oxide. The cystine so treated assayed 1 0 0 . 2 ~ oby formol tained. As a typical nucleophilic displacement, the titration. 2-.iimino-thiazoline-4-carboxylic acid was prepared by action of cyanide ion on various compounds con- the procedure of Schoberl and HamrnI' but the product was taining a sulfur-sulfur bond has been studied. isolated from 7oyo alcohol as per Wood and Cooley.18 Our These investigations have been concerned primarily product decomposed a t 234-237" and exhibited [ a ]2 5 -118', both of these values being higher than those rewith equilibrium aspects of the cyanide ion in- ported by Wood and Cooley. Cystine disulfoxide was preduced splitting. Kinetic investigations having pared by the procedure of Toennies.l* been limited to the reaction of cyanide ion with Kinetic Procedure.-Kinetic runs were followed by specSs, and with thiosulfate2 and in keeping trophotometric determination of appearance of product. At time intervals a n aliquot of the reaction with the nucleophilic displacement ~ o n c e p t ~mixture , ~appropriate was diluted 50 to several-hundred fold with 0.040 both of these reactions showed bimolecular kinetics. M potassium hydroxide. The dilution effectively stopped This investigation was undertaken to provide the reaction and the optical density of this solution a t 235 additional kinetic information on the reaction of mp then was determined in a Beckman DU instrument. cyanide ion with the sulfur-sulfur bond and in Optical density readings were converted t o cysteine molarity particular on the reaction of cyanide ion with bv means of the equation disulfides. Cystine was chosen for study because the stoichiometry of the reaction is known and the epsilons represent molar extinction coefficients, Th reaction goes to completion due to cyclization of The represents 2-amino-thiazoline-4-carboxylic acid and the the product, S-cyanocysteine, to 2-aminothiazoline- other symbols have their usual meanings. The optical denkarboxylic a ~ i d . ~Cystine +~ was also chosen be- sity a t zero time was calculated from the molar extinction cause the reaction of cyanide ion with proteins is coefficients of cystine and cyanide and the initial concentrations of each reactant. The values of extinction coeffiof general and because splitting of the cients employed, determined in 0.040 AI potassium hydisulfide bond of cystine is pertinent to the bio- droxide, are: ens-, 4.97 X 103; CTh, 2.60 X lo3; €R8SR, chemically important sulfhydryl-disulfide exchange 0.417 X lo8 and E C A - , 0.250 X los. Reaction mixtures varying in concentration of reactants r e a ~ t i o n . ~The kinetics of the reaction of cyanide 0.005 Af to several-hundredths molar were made up by with cystine also have been studied previously10 from mixing at zero time equal volumes of cystine and cyanide in the presence of excess cyanide and the reaction solutions of appropriate concentration. Both cystine and was found to be first order with respect to appear- cyanide solutions were prepared freshly prior to each run. Cystine solutions were prepared by dissolving the necessary ance of cysteine.
Experimental Materials.-Fisher AR potassium cyanide from freshly opened bottles was used. Fresh solutions were prepared prior to each run and were assayed with silver nitrate." Reagent grade cysteine hydrochloride monohydrate and cystine were purchased from Nutritional Biochemicals Corp. and used as such. Purity determinations by formol (1) A. J. Parker a n d N. Kharasch, Chem. Revs.,59, 583 (1959). (
(2) P. D. Bartlett and R. E. Davis, J . A m . Chem. Soc., 80, 2513 1938). f:l) 0 . Foss, Acto Chem. Scand., 4, 404 (1950). (4) 0. Foss, Kgl. Norskc Vadenskob. Sclskab. Skriffer, 1946, No. 2
(lW7). ( 5 ) A. Schoberl and M. Kawohl, Angem Chem., 64, 643 (1952). ( 6 ) A. SchBberl. M. Kawohl and R . H a m m , Ber., 84, 571 (1Y51). ( 7 ) 0 . Gawron, J. Keil and A. J. Glaid, 111, Biochim. Biophys. A d a , 19, 170 (1956). (8) Ti'. R . Cuthbertson and H . Phillips, Biochem. J.. 39, 7 (1945). (9) For a recent review see E. V. Jensen, Science, 130, 1319 (1959). (10) T. I l a t a a n d S. Matsushita, Meni. Res. Znsr. Food Sci., Kyoto U n i v . , S o . 9, 1 9 (1955). (11) I. If.Koltboff and E. B. Sandell, "Textbook of Quantitative 1952, pp. Inorganic Analysis." The .\lacmillan Co., New York, N. Y., 4;18-400.
~
quantity of cystine in the calculated quantity of 0.1 M base and diluting to 100 ml. with 0.040 M potassium hydroxide. Cyanide solutions were directly prepared in 0.040 124 potassium hydroxide. The potassium hydroxide solution was thoroughly deoxygenated prior to use by passing purified nitrogen through the solution for 45 minutes. To minimize access of air to reaction mixtures during the run, the initial reaction mixture was apportioned into three glass-stoppered tubes, each tube being filled as completely as possible, and a given tube was used t o follow the reaction for ca. one-third of the reaction time. Aliquots of the reaction mixture were
(12) In t h e case of cystine, excess standard alkali and formaldehyde were added initially and a potentiometric titration with standard hydrochloric acid was carried out. (13) I. M. Kolthoff and W. Stricks, J . A m . C h m . SOL.,7 2 , 1952 (1950). (14) This treatment which removes cystine disulfoxide's "as carried out although cystine disulfoxide could not be detected a n a l y t i ~ n l l y ~ ~ in t h e cystine. (15) €3. Sorbo, Biochim. B i o p i t y s . Acta, 2 2 , 570 (1956). (16) T. F. Lavine, J . BioL. Chem., 113,581 (1936). (17) A. Schoberl and R. H a m m , Ber., 81,210 (1948). (18) J. L. Wood and S. L. Cooley, J. Biol. Chem., 218, 449 (1956). (19) G. Toennies, i b i d . , 113, 580 (1936). For a discussion of the structure of "cystine disulfoxide" see B. J . Sweetman. Nalf4uc. 183,7 4 4 (1R59).
KINETICSOF THE CYANIDE-CYSTINE REACTION
July 5, 1961
V
I
I
0.01
000
2907
I
0.03
0.02 [ C Y ST INS].
Fig. 1.-Effect of initial cystine concentration (molarity) on initial rate (moles X liter-' X mim-1) at 35' in 0.04 M potassium hydroxide. removed under nitrogen. Under the conditions of the reaction, reactants and products were stable for the time periods employed in following the reaction.
Results and Discussion 1 A+B, -C+D 2 3
a.7
rCYAHlDt1 l d .
Fig.2.-Effect of initial cyanide ion concentration (molarity) on initial rate (moles X liter-1 X min.-1) at 35' in 0.04 M potassium hydroxide. 0.3
The results are consistent with formulation of the reaction as
1.1
0.q
000
1
1
(1)
D+E (2) where A, B, C, D and E represent, respectively, cystine, cyanide ion, cysteine, P-thiocyanatoalanine and 2-aminothiazoline-4-carboxylicacid. Steady state treatment yields the kinetic equation d(C) dt
~
-
- C)
k1ka(Ao C)(BD ks kzC
+
(3)
For the early stages of the reaction, k3 >> k2C, hence eq. 3 reduces to the bimolecular rate expression 4. Equation 3 also reduces to the bimolecular rate expression 4 if k) > kz and hence that thiocyanate ion could not be found in reaction mixtures21v22 constant = klt ( 5 ) (20) After 120 min., under the reaction conditions employed, destruction of cysteine becomes noticeable.
(21) Thiocyanate ion was tested for with ferric iron and hydrochloric acid using the reaction conditions suggested by I. M. Kolthoff and E. B. SandelI, ref. 11, p. 456. (22) When N,N*-diacyl-cystines were treated with cyanide under the same reaction conditions, thiocyanate ion was found.
2908
OSCARGAWRON ASD
JOSEPH
FERXANDO
VOl. 53
pared to cyanide ion, since in the sulfite reaction reaction rates with sulfite ion increase with positive charge on cystine ion, but decrease to negligible values when bisulfite ion is the reacting species.24 Bimolecular rate constants in 0.040 M potassium hydroxide also were determined a t several different temperatures, and a plot of log k v s . 1/Tis presented in Fig. 4. The activation parameters a t 35' are: E a 16.8 kcal./mole, A H * 16.1 kcal./niole and AS* -7.4 entropy units. The entropy of activation is larger by a few units than the corresponding value for the reaction of cyanide ion (in methanol) with elemental sulfur,2 but considerably smaller, by 20 e.u., than the value for the reaction of sulfite ion with cystine di-anion.24 The latter difference is in agreement that cyanide ion is a stronger thiophile than the sulfite ion2 and presumably is due to the fact that the sulfite-cystine activated I I I 1 complex is sterically more crowded than the cy3.10 3.10 3 ,so 340 anide-cystine activated complex. XlOS The kinetics of the reaction between cyanide Fig. .l.-Teniperature dependence of bimolecular rate ion and N,N'-dif~rmyl-cystine~~ were also investiconstant, k l ; rate constants calculated from runs initially gated, the reaction being followed by amperocontaining 0.01 d l cystine and 0.02 M cyanide. metric titration of thio1.13,28At 35' and pH 12.4, indicating that the cyclization reaction is also con- a bimolecular rate constant of 1.5 i 0.3 liter X siderably faster than the displacement of the thio- mole-' X min.-' was found. The faster rate of reaction with the di-formyl compound may be atcyanato group with mercaptide ion, ;.e. tributed to the electron-withdrawing effect of the RSCN + RS- ---+R-S-R + SCNacyl groups, which may make the disulfide bond The above bimolecular rate constant for the more susceptible to nucleophilic attack. The rate cystine-cyanide reaction a t pH 12.423is, of course, of reaction of N,Kl-diformyl-cystine with sulfite that for the reaction of cyanide ion with the di- ion is similarly faster, by a factor of three, than the anion of cystine and is of the same order of magni- rate of reaction of cystine with sulfite.24 In caltude as that for the reaction of sulfite ion with the culating the reaction rate constant, only initial di-anion of cystine, 3 liters x mole-l X min.-' a t data were used since thiocyanate ion appears 23°.24 It is also of interest to note that the effect subsequently in the reaction and because the reof pH on the reaction rate is, as expected, complex: action does not go to completion ?io thiazoline preliminary investigation a t 40' yielded the bi- was detected in the reaction mixture, presumably molecular rate constants: 1.01 (PH 12.4), 0.79 because cyclization of /3-thiocyanato-N-formyl(pH 11.5), 0.79 (PH 10.3) and a t 99' and pH 4.75, alanine to the thiazoline is prevented by the low -. 9 76,25,26The'rate constant a t 99' and PH 4.75 nucleophilicity of the acylated nitrogen atom.?' indicates the rate of reaction between hydrocyanic Acknowledgments.-Support of this work by acid and cystine zwitterion to be quite slow com- grant G-8933 from the National Science Foundapared to the reaction between cyanide ion and tion is gratefully acknowledged. % e' also wish to cystine di-anion. This may be attributed to the thank Dr. John Keil for carrying out preliminary lower nucleophilicity of hydrocyanic acid as com- experiments on the kinetics of the cyanide-cystine (23) T h e pH of the reaction mixture was measured with t h e glass reaction. electrode. (24) R. Cecil and J. R. McPhee, "hdvances in Protein Chemistry," L-01. XIV, Academic Press, Inc., New York, N. Y., 1959, p. 303. ( 2 5 ) T h e reaction was conducted in sealed tubes; a t 40' t h e reaction is very slow. (26) H . Fraenkel-Conrat, J . A m . Chenz. SOL., 63, 2533 (1941),has demonstrated the stoichiometry under these conditions.
(27) J . S . Fruton and H. T . Clnrke, J . Biol. Chem., 107, 667 (1034). (28) An aliquot of t h e reaction mixture was adjusted t o pH 5.6 with acetate buffer and, alter hydrocyanic acid was removed with nitrogen an amperometric titration was carried out. (29) T h e reaction of cyanide with di-acylated ck-stines is under further study in our laboratory.
