NOTEB
94 1
it is probably more significant that rate constants of the right order of magnitude are predicted than that agreement with existing experimental data is achieved. The transition state structure depicted by BEBO calculations should not be interpreted too literally13 and the results presented here do not constitute unequivocal proof that disproportionation reactions proceed via a "head-to-tail" transition state. However, the general picture of a loose .transition state in which the disproportionating radical has developed very little a-bond character is strikingly similar to the main aspects of the model proposed by Benson.2
Acknowledgment. This work was sponsored by the MDAC Independent Research and Development Program.
The Photolysis of Aqueous Solutions of Cystine in the Presence of Benzyl Chloride by C. J. Dixon and D. W. Grant Department of Pure and Applied Chemistry, Huddersfeld College of Technologg, Huddersfeld, England (Receiued June 23, 1969)
Although many products from the photolysis of aqueous solutions of cystine (HOOCCH(NH2)CH2S . ) 2by 254-nm radiation have been identified,l evidence for the nature of the intermediates involved is sparse. Electron spin resonance signals from the irradiated solid amino acid have been attributed by Bogle, et a1.,2to the presence of HOOCCH(NH2)CH2S (CyS radicals. These authors, however, admit that the signals do not exclude the presence of CySS radicals. The present communication describes work carried out to obtain evidence for the presence of CyS ., CySS , and C y . radicals in aqueous solutions of cystine irradiated with 254-nm radiation. If these radicals are actually produced, then CyS-benzyl, CySS-benzyl, and Cy-benzyl might be found among the products of the photolysis of cystine in the presence of benzyl chloride (BzCl), vix.
-
a )
BzC1 hu_ Bz. CyS. CySS * Cy*
+ C1.
+ B z - +CySBz + I32 +C ~ S S B Z *
+ B z * +CyBz
(1) (2)
(3)
(4) Evidence for reaction 1 has been obtained by Porter and Strachan13who detected the characteristic benzyl radical absorption at 319 nm when benzyl chloride was irradiated with 254-nm radiation in glassy solvents at -197".
Experimental Section Water used was twice distilled, the second distillation being from alkaline permanganate. L-Cystine (CySSCy), L-cysteine (CySH), and DL-lanthionine (CySCy) were supplied by Koch-Light Laboratories, Bucks, England. DL-cu-Alanine (CyH) and bibenzyl were supplied by British Drug Houses Ltd., Poole, England. 1-Amino-2-chloropropionic acid (CyC1) , 1,l'-diaminoadipic acid (CyCy),S l-amino-2-benzylpropionic acid (CyBz),e S-benzyl thiocysteine (CySSBz),' S-benzyl cysteine (CySBz),B 1-amino-l'-oxo-2,2'-dithiodipropionic acid (AODT-DPA), 9 and dialanine trisulfide (CySSSCy)'O were prepared as described in the literature. Benzyl chloride was prepared by the chlorination of toluene, followed by fractional distillation. Gasliquid phase chromatography revealed only one peak. All solutions prior l o irradiation mere thoroughly degassed on a vacuum line which incorporated an oil diffusion pump. Irradiation of stirred solutions (50 ml) was carried out at ambient temperature in 100-ml silica flasks using a 120-W low-pressure mercury vapor lamp (Model T/i\15/369E ; Thermal Syndicate Ltd., Wallsend, England) operated from a stabilized power suppIy. Wavelengths below 254 nm were filtered out using an aqueous solution of 0.1 M acetic acid. Cystine solutions (1 mM) in 0.1 M HCl, and cystine (1 mM)benzyl chloride (3.5 mM) solutions in 0.1 M HC1 were irradiated for respective times of 60 and 187 min to ensure the same photon absorption initially by cystine in each solution. The presence of S-benzyl cysteine and S-benzyl thiocysteine in irradiated cystine-benzyl chloride solutions was detected in the following way. Two 10-ml aliquots were concentrated to a few drops, applied to Whatman 33431 paper, and subjected to descending chromatography (EtOH-H20-H2S04, SO :20 : 0.5 by vol). After drying, one-half of the paper containing the products from one aliquot was developed with 0.2y0 ninhydrin in ethanol-acetic acid-collidine (25 : 5 : 1 by vol) to reveal a spot at Rala (RFvalue relative to alanine) of 1.25. A band at Rala1.2-1.3 was cut from the undeveloped piece of paper and eluted with glacial acetic acid. High voltage electrophoresis (20 kV, pH 1.85) of the (1) W. F. Forbes and W. E. Savige, Photochem. Photobiol., 1, 1 (1962). (2) G. S. Bogle, V. R. Burgess, W. F. Forbes, and W. E. Savige, ibid., 1 , 277 (1962). (3) G. Porter and E. Strachan, Trans. Faraday Soc., 54, 1595 (1958). (4) E. Fischer and K. Raske, Be?., 40,3717 (1907). (5) A. Bertha and J. Maier, Ann., 498,60 (1932). (6) P. E. Gagnon and B. N o h , Can. J . Res., 27B,742 (1949). (7) G. W.Stapleton and J. M. Swan, Aust. J . Chem., 15, 570 (1962). (8) H. Suter, Hoppe-Seylers, 2.Physiol. Chem., 20, 562 (1895). (9) C. J. Dixon and D. W. Grant, Biochem. J., 105,8c (1967). (10) W. E. Savige, J. Eager, J. A. Maclaren, and C. M. Roxburgh, Tetrahedron Lett., 3289 (1964).
Volume 74, h'umber 4 February 19, 1970
942 eluate yielded two ninhydrin-positive spots with mobilities relative to alapine of 0.42 and 0.44, colored brown and green, respectively. The brown and green spots were shown to be S-benzyl thiocysteine and Sbenzyl cysteine, respectively, by subjecting authentic mixtures of these compounds to the above procedure. For quantitative analysis 4-ml aliquots were evaporated to dryness, dissolved in a few drops of methanol, and applied to Whatman 3MM paper for electrophoresis (1 hr a t 20 kV, pH 1.85). The paper containing the separated products was then dried a t 70" for 20 min. The section of paper containing all products except alanine was sewn to another sheet of Whatman 3MR4 paper (46 X 57 cm) and descending chromatography (EtOH-H20-HClj 80 :20 :0.5 by vol) was carried out in a direction a t right angles to that of the electrophoresis. The products were located by the ninhydrin-cadmium acetate technique'l modified by the replacement of acetone with a similar volume of ethanol-acetic acidcollidine (25 : 5 : 1 by vol). Developed spots were eluted with 10 ml of methanol and extinctions at 506 nm measured in a Unicam SP600 spectrophotometer. Since alanine is well separated from the other products by electrophoresis alone, it was determined in eluates from paper strips after spot development with the unmodified ninhydrin-cadmium acetate technique.'l Amino acid concentrations in all eluates were obtained using standard extinction curves of authentic samples similarly treated. Ammonia yields in irradiated solutions were determined by the Conway microdiffusion technique.I2 Bibenzyl, benzyl alcohol, and benzyl chloride were determined by extraction of the appropriate solutions with 25 ml of benzene, and subsequent analysis of 1-p1 aliquots of the extracts with gas-liquid phase chromatography on a Silicone SE column operated a t 175" for bibenzyl, and at 100" for benzyl chloride and benzyl alcohol. The intensity of absorbed light, determined using the ferrioxalate actinometer,l3 was 2.0 & 0.15 X 1 O I 8 photons sec-l.
NOTES zyl is good evidence for the presence of benzyl radicals in the irradiated solutions, it might be argued that these radicals do not produce the benzyl derivatives via reactions 2 and 3 but rather via reactions with cystine itself, e.g.
+ CySSCy --+ B z . + CySSCy Bz.
----f
The Journal of Physical Chemistry
(5) (6)
Reactions 5 and 6 can be ruled out on the basis of their high endothermicities ( = 92 kJ mol-'). Thermal reaction between benzyl chloride and the photolysis product cysteine (CySH) to produce CySBz was shown to be unimportant a t ordinary temperatures. Therefore, the production of the benzyl derivatives can be attributed with some confidence to reactions 3 and 2. However, it might also be argued that the radicals CyS and CySS . are produced not by direct photolysis of cystine itself, but rather through reactions of chlorine atoms with cystine, vix.
-
Cl. CI
+ c y s s c y +c y s s . + CyCl + c y s s c y +c y s . + CySCl
(7) (8)
Reactions 7 and 8 cannot be eliminated from bond energy considerations, but seem unlikcly because CyCl and CyS02H were not detected in electropherograms of the irradiated solutions. If reaction 8 occurs, then the sulfenyl chloride would be expected to undergo hydrolysis to the sulfenic acid which wouId disproportionate to the sulfinic acid, vix. CySCl
+ HzO
+CySOH
+ H+(aq)
2CySOH +CySH
+ CI-(aq)
+ CySOzII
(9) (10)
The failure to detect lanthionine (CySCy) in irradiated cystine solutions indicates that CySS radicals are not produced to any significant extent in the reaction
cys + c y s s c y +c y s c y + c y s s '
'
(11)
The evidence would thus appear to favor production of CyS. and CySS radicals in the photolytic steps
Results and Discussion Thermal hydrolysis of benzyl chloride (3.5 X M) in 0.1 M HC1 was shown to be unimportant at ambient temperature by the fact that even after 48 hr only 0.2 mol % of the chloride was converted into benzyl alcohol. M ) in the Irradiation of benzyl chloride (3.5 X 111) increased the presence or absence of cystine rate of hydrolysis, but the concentrations of alcohol produced (= M ) were not photochemically significant. It has been assumed, therefore, that hydroxyl radicals from the photolysis of benzyl alcohol3 can be ignored in the subsequent discussion. The most significant result (Table I) is the detection of CySBz and CySSBz in irradiated cystine-benzyl chloride solutions. Although the formation of biben-
+ CySBz + CyS.
C ~ S S B Z Cy.
cysscy
h', 2 c y s .
c y s s c y -2cyss.
+ cy.
(12) (13)
According to reaction 13 Cy. and CySS. radicals are produced in equal numbers, yet neither CjrHz nor CyCy was detected. This is readily explained by assuming that Cy radicals readily abstract hyd!ogen atoms from cystine and/or benzyl chloride to form alanine (CyH). If all Cy. radicals are produced by reaction e
(11) J. Heilmann, J. Barrollier, and E. Watzke, Hoppe-Seylers 2. Physio2. Chem., 309,219 (1958). (12) E. J. Conway and A. Byrne, Biochem. J . , 27,419 (1944). (13) C. G. Hatchard and C. A. Parker, Proc. Roy. Soc., A235, 518 (1956).
NOTES
943 ~~~~
~
Table I : Quantum Yields of Products from 50-ml Irradiated Cystine and Cystine-Benzyl Chloride Solutions (Light Intensity 2.0 ==! 0.15 X 10Ibquanta see-l)
______ c y s s c y : 1.0
__-Product‘ “3
AODT-DPA CYH cyssscy (B2-h CySBz CySSBz CyBz Ha CYSCY CyCl (CY-)z BzH -BzC1
3 . 7 x 10-2 5 x 10-3 7 x 10-4 8X
44 6 0.8 0.9
65 5 0.9 1 43 1 1
>O.ld >0.06 >0.ld >o. Id >o, Id
>4
x
> io-4
5 x 1O-Zb 4 x 10-3b 8X 8 X 2 x 10-zc 8 x 10-4b 8X >10-4b
10-6
>10-4
>10-4 d
650
a Cy- = HOOCCII(NH2)CH2-;Be- = CeH6CH2-. Not detected.
Based on quanta absorbed by CySSCy.
13 and react in this way, then the alanine yield is a measure of the net amount of C-S fission produced on photolysis. The formation of CySSBz and CySBz merely reflects the relatively low reactivities of CySS . and CyS. radicals compared with Cy. radicals. The unreactive character of CySS . is due to the low bond energy Sz-H, approximately 84 kJ mol-’ less than that of S-H.I4 I n the presence of oxygen, alanine is no longer a photolytic product and is replaced by a substance whose chromatographic and electrophoretic behavior suggest that it is serine (CyOH).15 This oxygen effect is consistent with a radical mechanism for alanine production. Since CySS . radicals do not abstract hydrogen atoms they most probably react to form the trisulfide, vix. cyss.
x 10-8 M-----BzC1: 3.5 X 10-6 M Quantum yield Yield, mol X 107
CySSCy: 1.0 X 10-8 M---------Yield, Quantum mol X lo7 yield
+ c y s . +c y s s s c y
(14)
The similarity of the quantum yields of CySSSCy and alanine in the absence of benzyl chloride indicates that most CySS radicals probably react according to reaction 14. I n the presence of benzyl chloride there must be reactions(s) in addition to (13) leading to the production of CySS. radicals, since the quantum yield of alanine is significantly less than that of the sum of the quantum yields of trisulfide and CySSBz. The total amount of noncondensable gas from 50 ml M cystine irradiated in deoxygenated solution of for 85 min did not exceed 6 X mol. If all this gas were hydrogen, then its quantum yield would not exceed 4 X loW6, which suggest,s at first sight that AODT-DPA is not produced via photolytic rupture of a C-H bond. However, low hydrogen yields do not necessarily mean small numbers of hydrogen atoms in the system, since abstraction reactions might be much less important than the displacement reaction
H
Based on quanta absorbed by BzCI.
+ CySSCy +CySH + CyS.
(15)
A strict comparison between the quantum yields in the presence and absence of benzyl chloride is not possible since the relatively high conversions of substrates make the absolute values of quantum yields accurate to no better than i20’%. However, it is noteworthy that benzyl chloride would be expected to promote enhanced deamination of cystine (cf. Table I) since chlorine atoms generated radiolytically have been shown to deaminate cystine readily.16 If twice the bibenzyl yield is regarded as a measure of the minimum number of chlorine atoms which escape cage-recombination, the ammonia yield would increase by about 8.6 X mol in the presence of benzyl chloride assuming all chlorine atoms attack cystine. Since the observed increase is only 2 X mol (Table I), it would appear that only a relatively small fraction of the chlorine atoms actually attack cystine under the experimental conditions obtaining. Since AODT-DPA has been shown to be only a minor product in the radiolysis of oxygenated cystine solutions in 0.1 M HCl,” it would appear that chlorine atoms preferentially attack the 0-carbon atoms of cystine when the amino acid is in the protonated form. It is of interest to note that the quantum yields for ammonia and alanine in cystine (Table I) compare fairly well with those of 0.04 and 0.001, respectively, reported recently by Risi, et a1.,18 for nitrogen-saturated solutions of cystine at p H 1-2. (14) N. J. Friswell and B. G. Gowenlock, “Advances in Free Radical Chemistry,” Val. 2, Logos Press, London, 1967, p 26. (15) C. J. Dixon and D. W. Grant, unpublished work. (16) W. A. Armstrong and D. W. Grant, Can. J . Chem., 41, 1882 (1963). (17) D. W. Grant and D. J. Powles, unpublished work.
Volume 74, Number
February 19, 1970
NOTES
944 I n conclusion it ought to be mentioned that Box, et al.,19 have obtained evidence for the presence of radical-ions in single crystals of cysteine hydrochloride irradiated with ultraviolet light at 77°K. Although the possibility of radical-ion production in irradiated cystine itself cannot be ruled out, it would appear unlikely that CySBz and CySSBz are produced via radical-ion reactions in 0.1 M HC1, where rapid neutralization with hydrogen or chloride ions would occur.
Acknozuledgments. The financial support of the Wool Textile Research Council and the Huddersfield Corporation is gratefully acknowledged. (18) S. Risi, K. Dose, T. K. Rathinasamy, and L. Augenstein, Photochem. Photobiol., 6,423 (1967). (19) H. C. Box, H. G. Freund, and E. E. Budrinslci, J . Chem. Phys., 45, 809 (1966); the authors are grateful t o one of the reviewers for drawing their attention to this paper.
The Osmotic Pressure of Polyelectrolyte in Neutral Salt Solutions by Akira Takahashi, Narundo Kato, arid Mitsuru Nagasawa Department of Applied and Synthetic Chemistry. Nagoya University, Chikusa-Ku, Nagoya, Japan (Received June SO, 1969)
Most theories1b2on the second virial coefficient ( A 4 of spherical colloidal electrolytes so far published predict that the se:ond virial coefficient is proportional to l/C,O, where Cso is the concentration of added salt in mol/l. in the solvent with which the sample solution is in equilibrium. hforeover, the second virial coefficient is believed to be independent of molecular weight. These predictions arise from the fact that electroneutrality must be fulfilled in both the sample solution and the solvent, and, hence, the second virial coefficient is determined primarily by the Donnan distribution of diffusible ions between them. I n practice, there were several experimental studiessM5which supported the speculation. Our recent light scattering experiment using a linear polyelectrolyteJ6 however, showed that the invariance of A2 with molecular weight and the linearity between A2 and 1/C,O holds only at low ionic strengths, whereas at high ionic strengths A , depends on molecular weight and is linear with respect to l/.\/c,o. The second virial coefficient of linear polyelectrolytes in the salt-added system is determined not only by the electrostatic interaction between ions, but also by the intermolecular interaction of polyion We concluded6 that the deviation from the linear plot of A2 us. l/C,O as well as the molecular weight dependence of A2 observed at high ionic strengths arises from the The Journal of Physical Chemistry
intermolecular interaction between coils. If the concentration of added salt is low, the expansion of the polyion coil is so high that the effect of intermolecular interaction on A2 becomes practically independent of both molecular weight and added-salt concentration. Consequently, A2 appears to be linear with respect to the reciprocal salt concentration and independent of molecular weight. An ambiguity concerning the above conclusion still exists since in osmotic pressure measurements reported in the literatureJgA2 was always proportional to 1/C,O though not many measurements at high salt concentrations were made. We speculated6 that the polymer concentration used in the osmometry may be too high to obtain A z . The purpose of this work is to carry out careful measurements of osmotic pressure both in salt-free and salt-added systems of the same sample as used for light scattering and to compare the second virial coefficients determined by both methods.
Experimental Section
Polymer Sample. The F-8 fraction of sodium poly(styrenesulfonate) [Na-PSI (mol wt = 4.3 X lo6) used in a previous investigation6 was selected in this study. A measured amount of the sample was dissolved into NaCl aqueous solutions of specified concentrations in volumetric flasks. Osnaometer. The most important criterion in the osmometry of polyelectrolyte solutions in salt-added systems is to confirm the complete Donnan equilibrium between the sample solution and solvent separated by a membrane. To this end, a Zimm-Myerson osmometerX0was modified by incorporating two magnetic stirrers inside the cell. They were swung as pendulums with a magnet operated from outside of the cell. To avoid the contamination of the solution with ions, the whole cell was made of poly(methy1 methacrylate) resin, 6-10 Nylon, and glass. A stainless steel rod in the original design was replaced by a long-stem glass syringe, and the glass capillaries were fastened to the cell with synthetic rubber stoppers. (1) F. G. Donnan and E. A. Guggenheim, 2 . Phys. Chem., 162, 364 (1934). (2) T . L. Hill, Discussions Faraday Soc., 21, 31 (1956); J . Phys. Chem., 61, 548 (1957). (3) M. Nagasawa, A. Takahashi, M. Izumi, and I. Kagawa, J . Polym. Sci., 38, 213 (1959). (4) 2. Alexandrowicz, J . Polymer Sci., 43, 337 (1960); 56, 115 (1962). (5) H. Inagaki and H. Hirami, 2. Electrochem., 63,419 (1959). (6) A. Takahashi, T . Kato, and M. Nagasawa, J . Phys. Chem., 71, 2001 (1967). (7) T. A. Orofino and P. J. Flory, ibid., 63, 283 (1959). (8) H. Eisenberg, J . Chem. Phys., 44, 137 (1966). (9) D. T. F. Pals and J. J. Hermans, Rec. Trav. Chirn., 71, 458 (1952). (10) R.H. Zinim and I. Myerson, J . Amer. Chem. Soc., 68,911 (1946).
