THECo60 y RADIOLYSIS OF CYSTEINE IN DEAERATED AQUEOUS SOLUTIONS our density data and their density Curve 4 shows the heats of fusion of n-paraffins. The heat of fusion increases with increasing carbon number and approaches the value for the heat of fusion of extended chain crystals of polyethylene of high molecular weight. The difference of this curve from a similar one shown by Hendus and Illcrs17is due to our use of crystallographic specific volume of paraffins. In summary, the heat of fusion measurements support the conclusions drawn from the density measurements : Both (001) surface structure and -surface area must be considered as major factors in interpretation of crystals grown from solution. Each series of polyethylene
185
crystals of constant fold length has a different heat of fusion-specific-volume relation displaced from the curve for melt-crystallized polyethylene to lower heats of fusion. Acknowledgments. The work performed a t Rensselaer was supported by The National Aeronautics and Space Administration. Authors wish to express their appreciation to Mr. J. llitchel, Jr., and Mr. C. E. Day of duPont de Xemours and Co., for giving the informat.ion about the infrared determination of carbonyl content of oxidized polyethylene. The heats of fusion were determined in cooperation with Miss C. M. Cormier and Mr. C. L. Gruner.
The Cobalt-60 y Radiolysis of Cysteine in Deaerated Aqueous Solutions at pH Values between 5 and 6 by Verna Gaye Wilkening, Manohar Lal, Meta Arends, and D. A. Armstrong Department of Chemistry, The University of Calgary, Calgary, Alberta, Canada
(Received June 23, 1967)
The major products arising from the Cosoy radiolysis of loF2M cysteine (CySH) solutions a t pH’s in the range 5-6 are: cystine ((CyS)?), alanine (CyH), H2S, and hydrogen with yields of 3.4, 2.6, 2.5, and 1.1 molecules/100 ev, respectively. The reactions, which were proposed independently by El Samahy, White, and Trumbore and Armstrong and Wilkening in 1964 are consistent with the results. Nitrate ions and acetone repress the alanine and H2S yields by identical amounts. This and other observations indicate that: M cysteine solutions; (b) reaction 4, eaq- CySH + Cy (a) G,,,- = 2.5 0.2 in SH-, is the only significant reaction of eaq- with cysteine a t pH 5-6; and (c) that all Cy radicals undergo reaction 5, Cy CySH CyH CyS. The product of the reaction between eaq- and nitrate oxidizes two cysteine molecules to cystine with the concomitant formation of nitric oxide. In the p H range employed, reaction 8, HzOz 2CySH + 2Hz0 (CyS)2 is relatively slow and as a consequence there is a postirradiation oxidation of cysteine,
+
*
+
-f
+
+
Introduction
+ enq- +Cy + SHCySH + Cy +CyS + CyH H + + eaqH CySH
The radiation chemistry of the sulfhydryl amino acid cysteine is currently a subject of considerable interest. This arises from. the importance of cysteine in biological processes’ and its extreme sensitivity to radiation Recent studies6-’ Of the Y Of aqueous solutions of cysteine have provided evidence for the reactions
+H C.IISH + H CySH + OH CySH
---j ---j
4
+ Hz Cy + HzS CyS + HzO CyS
(1) (2) (3)
+
+
(4) (5) (6)
(1) J, T. Edsall and J, Wyman, “Biological Chemistry,” Academic Press, Inc., New York, N. Y., 1958. (2) 6. L. Whitcher, M. Rotheram, and N. Todd, Nucleonics, 11,
30 (1953). . . (3) P. Markakis and A. L. Tappel, J . Am. Chem. SOC.,82, 1613 (1960). (4) J. E. Packer, J . Chenz. SOC., 2320 (1963). ( 5 ) A. El Samahy, H. L. White, and C. N. Trumbore, J . Am. Chem. Soc., 86,3177 (1964).
Volume 7.9, Number 1 January 1968
V. WILKENING, M. LAL,M. ARENDS,AND D. ARMSTRONG
186 2cys
2CySH
----f
+ HzOz
(CyS), (CyS)z
+ 2Hz0
(7) (8)
I n this scheme the following representations are used: Cy, the radical .CH2CH(NH3+)CO2-;CyH, alanine; CySH, cysteine; and (CYS)~, cystine. It is quite obvious that a detailed study of all product yields must be performed if the foregoing mechanism is to be substantiated. An earlier paper' from this laboratory reported product yields from 1 N HC104 solutions. The objective of this investigation was to determine the yields of the major products at pH's in the range 5.0-6.0. Particular attention was given to the identification of the products arising from the reaction sequence 4 and 5. For reasons which will become apparent, the rate of the thermal reaction between 0.9 X M hydrogen peroxide and lo-, M cysteine in air-free solutions was also investigated. Dose ( e v / r n e ) XiO-'e
Experimental Section The thermal reaction between cysteine and hydrogen peroxide in air-free solutions was studied by the followM neutral solution of ing technique. A 1.8 X hydrogen peroxide was prepared by the radiolysis of aerated triply distilled water. A measured aliquot of this was then placed in a 10-ml cell, which was attached by a capillary tube to a similar cell containing an equal volume of 2.0 X lo-, M cysteine. The connecting tube was also attached to a standard 1-cm spectrophotometer cell. After dearation of the two solutions by several cycles of freezing and pumping, the entire system was sealed off from the vacuum line. The two solutions were then mixed and the reaction followed spectrophotometrically for several hours by measuring the optical density due to cystine at 248 mp. I n a number of these experiments, the concentration of cystine at the end of the reaction was also checked with the amino acid analyzer (see below). The details of the experimental procedures used in the radiolysis experiments have already been given.' Hydrogen and hydrogen sulfide were determined by the analytical techniques already described. The yields of cystine, alanine, and residual cysteine were followed by means of a Technicon amino acid analyzer. Cystine was also determined by its absorption at 248 mp. Xass spectrometric analyses of gaseous products were kindly performed for us by Dr. A. Hogg, Department of Chemistry, University of Alberta.
Results and Discussion Products and Radical Yields. Yield-dose plots for the amino-containing products of radiolysis of lo-, M cysteine solutions at pH 5.6 are shown in Figure 1. The concentrations of all products varied linearly with dose over the range 0-4 X lo1* ev/ml. The G values, obtained from the best straight lines through The Journal of Physical Chemistry
Figure 1. Yield-dose plots for the amino-containing products of radiolysis of M cysteine solutions at p H 5.6 and at 23': 0 , cysteine consumption; 0, cystine b y amino acid analyzer; X, cystine b y determination of OD at 248 mp; and A, alanine.
the yield-dose plots, are given below with their standard deviations G(CyH) = 2.6 k 0.2 G(H2S) = 2.5 k 0.2 G(HJ = 1.1 rfr 0.2 G((CyS)Z) = 3.4 i 0.1 G(-CySH) = 9.3
f
0.2
The fact that the alanine and HzS yields are equal, within experimental error, supports the view that reaction 5 is the exclusive fate of the Cy radicals formed in reactions 2 and 4. If alanine and cystine are the only major products containing the amino group, stoichiometry requires that G(-CySH) = 2G((CyS)d
+ G(CyH)
(i)
From the above results one obtains G(-CySH)
2(3.4)
+ 2.6 = 9.4
which demonstrates excellent material balance for the amino products when compared with the observed yield of G(-RSH) = 9.3 f 0.2. The present H2S yield agrees well with 2.5 reported by El Samahys for 5 X (6) D. A. Armstrong and V. G. Wilkening, Can. J. Chem., 42, 2631 (1964). (7) V. G.Wilkening, M. Lal, M. Arends, and D. A. Armstrong, ibid., 45,1209 (1967). (8) A. El Samahy, Ph.D. Thesis, University of Delaware, Newark, Del.. 1964.
THECosOy RADIOLYSIS OF CYSTEINE IN DEAERATED AQUEOUS SOLUTIONS (a) 0.03
5
10
TIME
15 ( Hrs.)
20
25
Figure 2. Reaction time curves for the HzOa-cysteine reaction: (a) synthetic HzOz-cysteine mixture; and (b) postirradiation rea,ction in an irradiated cysteine solution (see text); different size points identify separate experiments.
10-3 M unbuffered cysteine solutions. To the best of our knowledge, the alanine yields have not been quantitatively determined in our pH range by any other workers. Whitcher, Rotheram, and Todd2 concluded from their work that the oxidation of cysteine by H202was negligibly slow in strongly acid solutions. This conclusion was substantiated by Packer4 and by our own more recent study.’ On the other hand, El Samahys reported that reaction 8 was very rapid in neutral solutions. The rate of this reaction was therefore determined in air-free M cysteine solutions in the pH range obtaining in this study. Figure 2a shows a plot of optical density, due to the cystine absorption a t 248 mp, us. time for duplicate experiments. The initial M was close to peroxide concentration of 0.9 X that which would have built up in the longer radiolysis experiments if 110 reaction with cysteine occurred. At the end of 24 hr, the reaction was 7 5 4 0 % complete, and proceeding at an almost imperceptible rate. The kinetics of the reaction was not examined in very great detail, but further experiments showed that it was much slower a t lower concentrations of cysteine and/or peroxide. It appears to be first order in peroxide and of fractional order in cysteine. The reaction time curve in Figure 2a demonstrates that the oxidation of cysteine by hydrogen peroxide will be nowhere near complete under the conditions of this study, unless the irradiated samples are allowed to stand for several hours before analysis. If the solutions are analyzed immediately after a short irradiation a t a very high dose rate, it should be possible to observe a postirradiation oxidation of cysteine. To confirm this, two samples of dearated M cysteine were subjected to a very high dose rate from a Philips Model MG150B/10 150-kvp constant potential X-ray machine
187
for a period of 10 min (the usual radiations with the 100-curie Coao source lasted from 2 to 10 hr). At the end of these irradiations, the optical density had changed by 0.24 f 0.02 unit. Further changes were then followed over a period of 24 hr. As shown in Figure 2b, the reaction time curve for the postirradiation oxidation is quite similar to that in Figure 2a. The initial peroxide concentration for the radiolysis is not very different from that obtaining in the synthesized H202-CySH solution. As a standard procedure, all irradiated samples were allowed to stand for about 24 hr before analysis with the amino acid analyzer. Further changes in cystine yield beyond this time will lie within the over-all experimental error of our determinations. For practical purposes, it may therefore be assumed that 0.5 f 0.1 (see below) molecules of cystine per 100 ev are formed in reaction 8. The mechanism given in the Introduction requires that eap-, H, and OH each eventually produce a thiyl radical. Consequently the total yield of cystine from reactions 7 and 8 should be given by the relationship G((CyS)J =
+ GOH+ Geaq-I + G ‘ ~ z ~ (ii> 2
l/2{G~
Upon rearrangement one obtains
GH
+ GOH+ Ge,,-
= 2{G((CyS)t)
- G M ~ l ~ l(iii) ]
The experimental value of G((CYS)~) is 3.4, and, allowing for the scavengingg of some of the precursors of may molecular peroxide in the radiation tracks, GMHeO, M cysteine solutions. In supbe taken as 0.50 in port of this estimate, we may note that Packer4 obtained GMHzOz = 0.6 in 4.4 X lo-* M cysteine solutions a t p H 4, where reaction 8 would be much slower and G M ~ ? ocould Z be measured directly. Thus from expresGH GOH,is sion iii, the total radical yield, Ge,,found to be 5.8. Unfortunately, the lability of the hydrogen atom on the SH group precludes isotopic labeling and the molecular hydrogen yield cannot be measured directly in cysteine solutions. Navon and Steinlo have estimated that Icl is about 109 M-I 8ec-l and cysteine is also highly reactive toward eaq-.ll I n view of these observations, it is evident that cysteine will compete quite efficiently with radical combinations which give rise to molecular hydrogen. By analogy with nitrite0 and other highly ? be reduced from 0.45 to a reactive solutes, G M ~may M cysteine solutions. Since value as low as 0.25 in the total hydrogen yield from these solutions is 1.1 molecules/100 ev, the yield from processes involving cysteine should be 0.65 to 0.85. This tends to be higher
+ +
(9) See Figure 5.6 of A. 0.Allen, “The Radiation Chemistry of Water and Aqueous Solutions,” D. Van Nostrand Co., Inc., New York, N.Y.,1961. (10) G . Navon and G. Stein, Israe2 J . Chem., 2 , 151 (1964). (11) R. Braams in “Pulse Radiolysis,” M. Ebert, J. P. Keene, A. J. Swallow, and J. H. Baxendale, Ed., Academic Press, London, 1965.
vohme 7.2, Number 1
January 1988
V. WILICENINQ, 34. LAL,M. ARENDS,AND D. ARMSTRONQ
188 than the normally accepted yield of residual hydrogen atoms, GH = 0.60 f 0.05, obtained with alcohols and formic acid as reactive solutes.12 If one assumes k1/k2 = 3.5 as in 1N acid solution^,^ an additional yield of -0.2 hydrogen atom/100 ev must form HzS, and the discrepancy in the apparent value of GH would be even larger. However, in these sulfydryl systems a t pH 5-6, relatively large experimental deviations (see Figure 3) were observed in the values of G(H2) and no real significance can be attached to the above observation. Nevertheless it should be noted that Littman, Carr, and Brady13 found reaction 9 H
+ CyS- +Cy + SH-
*
*
3,O-
G
-
2.0
(9) 1.0
-
to be very efficient in their study of the reactions of hydrogen atoms with aqueous cysteine. This observation argues against reaction 10 eaq-
+ CySH +CyS- + H
(10)
being of any great importance. Since the sulfur-hydrogen bond is stronger than the sulfur-carbon bond,I4 reaction 10 is also less probable than reaction 4 on thermochemical grounds. It is therefore extremely unlikely that reaction 10 could be responsible for an increase in the yield of residual hydrogen atoms in this system . On the basis of the proposed mechanism, reactions 1, 2, 4,and 5 are the only processes leading to the formation of hydrogen, H2S, and alanine, and it follows that G(H2)
+ G(H2S) or G(CyH) = GH + G, + G M ~ (iv) l
Hence, we find
GH
+ Ge == G(H2) + G(CyH) - G M ~ * 3.4
(v)
From this and the above value of the total radical yield, GOHis calculated to be 2.4, which compares favorably with values reported elsewhere.12 As the pH of the cysteine solutions is reduced, increasing proportions of the solvated electrons would be converted to hydrogen atoms by reaction 6. Since reaction 1 predominates over 2, it follows that this should cause the hydrogen yields to increase and those of alanine and H2S to decrease,Btheir values remaining identical with each other. The data presented in Figure 3 bear out these predictions. The uppermost points are values of GH G, calculated from expression v, using the hydrogen and alanine yields and assuming that G M ~is$0.30.'~ As the pH decreases from 3.0 to 2.0 GH G, is seen to increase abruptly from 3.4 to 3.8. The occurrence of this increase is well established from extensive work in other ~ystems.~BJ7Our values of GH G, are larger than those observed in most other systems,12J6J7 the discrepancies being greatest at the higher pH's. A portion of this increase may be attributed to the high reactivity of the SH group toward
+
+
+
The Journal of Physical Chemistry
*
1
I
I
I
1
1
I
I
6
5
4
3
2
I
0
PH
Figure 3. Effect of p H on product yields: $ , H2S; 0 , alanine; and 4 , HZ G, GH (calculated from expression v, see text). The,vertical lines through the H2 and H2Spoints represent the maximum deviations observed in the 3-10 replicate determinations. Standard deviations are given in the text.
+ +
all radicals and to the fact that we have employed a lov2M concentration, whereas radical yields have more frequently been determined at solute concentrations of M . However, the value of G,,,- obtained below (2.5 f 0.2) seems quite reasonable and the discrepancy appears to be due primarily to the high apparent value of GH,on which we have already commented. Competition Experiments. Trumbore and his coworkers6 have already shown that acetone and nitrate reduce the yield of H2S in cysteine solutions. They attributed this to competitions between reactions 11 and 12, viz. eaq-
+ (CH&CO eaq-
4
(CHs)&O-
+ NOs- -+ Nos2-
(11) (12)
and reaction 4. If the proposed mechanism is correct, these scavengers should cause equivalent reductions in the alanine yields, which to the best of our knowledge (12) See Proceedings of the Fifth Informal Conference on the Radiation Chemistry of Water, University of Notre Dame, Notre Dame, Ind., 1966,p 27 and references cited therein. (13) F. E. Littman, E. M. Carr, and A. P. Brady, Radiation Res., 7, 107 (1957). (14) H. Mackle, Tetrahedron, 19, 1159 (1963). (15) Note that the relatively small changes of -0.06 molecule per 100 ev in G M ~over 2 the pH range 0-6 [see C. H. Cheek, V. J. Linnenborn, and J. W . Swinnerton, Radiation Rea., 19, 636 (1963)l has been neglected. (16) See F. S. Dainton, et al., Trans. Faraday Soc., 62, 3170 (1966), and previous related papera. (17) E. Hayon, Trans. Faraday Soc., 61, 723 (1965).
THECosOy RADIOLYSIS OF CYSTEINE IN DEAERATED AQUEOUS SOLUTIONS have not been measured at pH 5.5. Also, in the case of acetone, the (CH3)2CO-radical ion should be rapidly converted to the 2-hydroxyisopropyl radical, (CH3)2COH, which would react with the sulfydryl group of cysteine in the same way as the Cy radical, viz. (CH3)2COH
+ CySH
-t
(CH3)2CHOH
+ CyS
+ 2RSH +NO + 20H- + (RS)2
A
~
AA B
Y
(13)
The yield of thiyl radicals and of cystine should therefore be unaffected by the presence of acetone. Figure 4a shows the yields of cystine, HsS, and alanine M cysteine solutions containing acetone at from acetone: cysteine ratios in the range 0-10. As required by the mechanism, the cystine‘ yields are independent of the amount of acetone added. In addition, the reductions in the alanine and H2S yields are essentially equivalent. The apparent divergence at acetone :cysteine = 1and 2 is not regarded as being significant, since there is an experimental uncertainty of A0.2 molecule/ 100 ev in both the alanine and HzS yields. A gas-chromatographic analysis of irradiated solutions with an acetone:cysteine ratio of 5-10 showed a peak corresponding to the residual acetone and only one new peak occurring at the retention time of isopropanol. The 8-ft Poropak column was run at -66” and the amino acids were retained. Unfortunately, the sensitivity of the flame ionization detector did not permit an accurate determination of the isopropyl alcohol yield, but G(isopropy1 alcohol) appeared to be in the range of 2-3 as expected. As shown in Figure 4b, the competition for eaQbetween nitrate ions and cysteine also reduced the alanine and H2S yields by equivalent amounts. However, in this case G((CYS)~) increased from 3.4 toward a value of 4.7 as the nitrate concentration rose. The ) one half of the magnitude of this increase ( ~ 1 . 3 is value of G,,,- reported in ref 18 and other recent investigations (see footnote 12). Each Cy radical formed in reaction 4 produces one CyS radical in reaction 5 and subsequently one-half of a molecule of cystine. The above increase in G((CYS)~) can be explained if the product of reaction 12 gives rise to two CyS radicals or one molecule of cystine. This product is believed to be a hydrated form of ?;0219 or N032-,20 as indicated above. The observation of nitric oxide in the mass spectrum of the gaseous products from irradiated solutions containing both nitrate and cysteine provides strong support for the occurrence of
K0B2-
n
189
(14)
which also accounts for the formation of one molecule of cystine from each N02-. It should be noted that, under our conditions, nitrite ions, which are the final products of radiolysis of aqueous nitrate solutions,20 do not react with cysteine at a measurable rate. The rate constants for the reactions of hydrogen atoms with acetone and nitrate are considered to be -lo6 and -lo7, respectively.21 Since k, is estimatedl0
I
I
I
I
I
I
0
2.0
4.0
6.0
8.0
10.0
((CH,)2
CO) /( Cy S H )
(8).
5 0 -
A
A
A
h
(NO;)/(CySH)
(b).
Figure 4. Effect of solvated electron scavengers on t h e product yields: (a) acetone as scavenger; and (b) NOSas scavenger; (A, cystine, 0, alanine, and X H2S).
to be about lo8, the scavenging of H by acetone and nitrate ions should not have been appreciable over the range of acetone and nitrate concentrations shown in Figures 4a and b. Reasonably accurate estimates of G,-,,, k4/kll, and k4/kl2may therefore be obtained from reciprocal plots of the data of these two figures, based on expression vi. (18) C. J. Hochanadel and R. Casey, Radiation Res., 25, 198 (1965). (19) M. Anbar in E. J. Hart, “Solvated Electron,” Advances in Chemistry Series, No. 50,American Chemical Society, Washington, D. C., 1965. (20) M. Daniels and E. E. Wigg, J. Phys. Chem., 71, 1024 (1967). (21) H. A. Schwarz in “Advances in Radiation Biology,” Vol. 1, L. G . Augenstein and R. Mason, Ed., Academic Press, New York, N.Y.,1964,p 17.
Volume 78, Number 1 January 1068
V. WILKENING,M. LAL,M. ARENDS,AND D. ARMSTRONG
190 l/AG(H&) = { 1
+ h(CySH)/ks(S) ]
/'Ge&q-
(vi)
where (S) represents the scavenger concentration and k , its rate constant for reaction with eSq-. The intercepts of the reciprocal plots gave Ge,,- = 2.5 for the acetone and 2.6 for the nitrate results. These values fall in the same range, 2.3-2.7, as most other recent determinations.12Js The fact that they are equal within experimental error to G(CyH) and G (H2S) indicates that contributions to the H2S yields from reaction 2 cannot exceed the experimental uncertainty of 0.2 molecule/ 100 ev, and G, may reasonably be set at 2.5 ==! 0.2 in this system. The rate constant ratios were found to be k4/kll = 1.95 and k4/k12 = 1.03. These are both twice as large as the corresponding ratios originally reported by El Samahy, White, and Trumbore.6 The cause of this discrepancy has not been explored, but it is to be noted that the latter workers used much lower cysteine concentrations as well as different analytical techniques. By substituting the absolute values of k1122and in the above ratios, k4 is found to be 1.1 X 1OloM-' sec-'. This agrees reasonably well with 8.7 X log M-I sec-l obtained by Braams" using the pulsed radiolysis technique, and there can be little doubt that reaction 4 is the predominant process by which solvated electrons react in cysteine solutions at pH 5.0-6.0. M cysteine soluThe results reported above for tions provide confirmation for the radiolytic mechanism based on reactions 1 to 8. Product yields from lod3 M solutions are dependent on dose beyond 3 X lo'* ev/ml. However, from the linear yield-dose plots at doses below this, G(-RSH), G((CYS)~), and G(CyH) have been found to be 7.9,2.9, and 2.2, respectively. These yields
The Journal of Physical Chemistry
also conform to expression i, but are significantly lower than those observed in the low2M solutions. This is to be expected, since the competition with track reactions will not be as extensive and reaction 8 will be much slower in these more dilute cysteine solutions. G((CYS)~) is in excellent agreement with that of El Samahy in 9 X 10-4 M solutions.8 The hydrogen yield from M solutions rises with decreasing pH as anticipatedj6while the alanine yield falls. At pH 5.5 G(H2) has the same value, 1.1, in 10m3 M cysteine solutions as in M solutions. From this one must conclude that any in% from the reduction in solute crease in G M ~resulting concentration is compensated, within experimental error, by the loss of hydrogen atoms to track reactions with radical species. As pointed out in ref 7, the H2S yields reported by Armstrong and Wilkenings are high. Further work on these is currently underway. Acknowledgments. The authors wish to acknowledge many helpful discussions with Dr. C. N. Trumbore of the University of Delaware, Newark, Delaware. They are also indebted to the Defence Research Board of Canada for the purchase of the Technicon amino acid analyzer and for the financial support to V. G. W. and M. A. The investigation was also supported by U. S. Public Health Service Grant No. ROl-GM-14020OlMCH-B from the National Institute of General Medical Sciences, which provided the stipend for M. L.
(22) kll = 5.9 X 100 M-1 sec-1; E. J. Hart, S. Gordon, and J. K. Thomas, J. Phys. Chem., 68, 1271 (1964). (23) k a = 1.1 X 10'0 M-1 seo-1; J. K. Thomas, S. Gordon, and E. J. Hart, ibid., 68,1524(1964).
