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(36) F. Stuhl, J Chem. Phys., 59, 635 (1973). (37) R. Zellner and I. W. M. Smith, Chem. Phys. Left., 28, 72 (1974). (38) I. W. M. Smith and R. Zellner, “Symposium on Chemical Kinetics Data for the Lower and Upper Atmosphere”, Warrenton, Va., Sept 1974; Int. J. Chem. Kinet., Symp., 1, 341 (1975). (39) W. Hack, K. Hoyerman, and H. Gg. Wagner, Ber. Bunsenges. Phys. Chem., 78, 386 (1974). (40) R. A. Cox, R. G. Derwent, and P. M. Hob, Chemosphere, 4, 201 (1975). (41) R. A. Perry, R. Atkinson, and J. N. PMs, Jr., J. Chem. Phys., 64, 3237 (1976). (42) J. A. Kerr, M. J. Parsonage, and A. F. TrotmanDickenson, “Strengths of Chemical Bonds”, in “Handbook of Chemistry and Physics”, 56th ed, The Chemical Rubber Company, Cleveland, Ohio, 1975-1976. (43) D. D. Davis, R. E. Huie, and J. T. Herron, J. Chem. Phys., 59, 628 (1973). (44) 0.L. Singleton, S . Furuyama, R. J. Cvetanovic, and R. S. Irwin, J. Chem. Phys., 83, 1003 (1975). (45) J. S. Gaffney, R. Atkinson, and J. N. Pitts, Jr., J. Am. Chem. Soc., 97, 6481 (1975).
(46) D. L. Singleton and R. J. Cvetanovic, J. Am. Chem. Soc., 98, 6812 (1976). (47) D. D. Davis and R. B. Klemm, Int. J. Chem. Kinet., 5, 841 (1973). (48) N. R. Greiner, J. Chem. Phys., 53, 1284 (1970). (49) R. Atkinson and J. N. Pitts, Jr., J. Chem. Phys., 63, 3591 (1975). (50) R. Atkinson, R. A. Perry, and J. N. Pitts, Jr., Chem. Phys. Lett, 38, 607 (1976). (51) R. Atkinson, R. A. Perry, and J. N. PMs, Jr., J. Chem. Phys., in press. (52) S. W. Benson, “Thermohmical Kinetics: Methods for the Estimation of Thermochemical Data and Rate Parameters”, Wiley, New York, N.Y.. 1968. (53) R. Atkinson and R. J. Cvetanovic, J. Chem. Phys., 56, 432 (1972). (54) K. N. Egger and S.W. Benson, J. Am. Chem. Soc., 88, 241 (1966). (55) R. Shaw, F. R. Cruickshank, and S.W. Benson, J. Phys. Chem., 71, 4538 (1967). (56) E. J. Prosen, W. H. Johnson, and F. D. Rossini, J. Res. Nafl Bur. Stand. 36. 455 (1946). (57) L. M. Leyland, J. k. Majer, and J. C. Robb, Trans. Faraday Soc., 86, 898 (1970).
Radiolysis of Adenine in Dilute Neutral Aqueous Solution George Gorin,“ C. Lehman, C. A. Mannan, L. M. Raff, and S. E. Scheppele Department of Chemistry, Oklahoma State University, Stiiiwater, Oklahoma 74074 (Received August 16, 1976) Publication costs assisted by the National Institutes of Health
Solutions of adenine, 0.2 to 2 mM, have been radiolyzed at constant dose rate in air-equilibrated and in oxygen-saturatedphosphate buffer, pH 7. The radiochemical decomposition yield is 1.g4 f 0.1 molecules/ (100 eV). The progress of the radiolysis was monitored by a highly specific colorimetric method for determining the adenine, and by measurements of the absorption spectrum at wavelengths between 290 and 220 nm. The results show that the radiolysis products absorb in the wavelength range investigated;this explains why previously reported values of G(-Ade), which had been calculated from the rate of decrease of the absorbance with dose, are lower than that reported here. The spectra are unusual in that isosbestic points develop at 280 and 238 nm and persist to >50% decomposition. Analysis by thin-layer chromatography and mass spectrometry shows that urea is one of the products, and it confirms the formation of 8-hydroxyadenine and of 4,6-diamino-5formamidopyrimidine, which had been reported previously. None of these products interfere with the colorimetric determination of adenine.
Introduction This investigation is one of a series aimed at elucidating the chemical reactions that take place when ionizing radiation interacts with living organisms.lJ Our attention has been directed, in particular, to the bases which occur in DNA, inasmuch as this substance indubitably plays an important role in the development of radiation i n j u r i e ~ . ~ A previous paper2 describes our general approach and methods, and their application to the radiolysis of cytosine; here we report their extension to the radiolysis of adenine. Adenine possesses an intense and fairly sharp absorption maximum at 260 nm, which decreases on irradiation. The changes can be easily measured with relatively good accuracy, and such measurements thus provide a very convenient means of following the progress of the reaction. This approach was first utilized by Scholes et a1.,4 who came to the conclusion that the products of radiolysis did not absorb at 260 nm; on that basis they calculated the radiolytic decomposition yield, G(-Ade), to be 1.1molecules/(100 eV). G values ranging from 0.65 to 1.2 have been reported subsequently by other investigator^.^^^ However, these results are inconsistent with the reports that some of the radiation products have a substantial As we have shown in a previous absorbance at 260 nm.5~6,8 paper,2if the products have an appreciable absorbance and this fact is neglected in calculating the value of G from spectrophotometric measurements, the result inevitably will be lower than the true value. The Journal of Physical Chemistry, Vol. 8 1: No. 4, 1977
These considerations prompted us to’reinvestigate the question, and to use a highly specific colorimetric method to determine the adenine.g Moreover, we undertook to measure the complete spectrum of the irradiated solutions, not only the absorbance at the maximum. The results show that the radiolysis products do indeed absorb at 260 nm, so that the decrease in absorbance does not measure the amount of adenine decomposed. The spectra of the irradiated solutions exhibit a rather strange and interesting feature, namely, two isosbestic points, at which the absorbance remains sensibly constant, to at least 50% decomposition. The development of one isosbestic point at 236 nm had been noted earlier by Uliana and Creac’h,lobut they did not offer an explanation of the phenomenon. We shall show what conditions are necessary for this phenomenon to develop. Experimental Section Adenine (Sigma and Schwarz Biochemicals), [8-14C]adenine (New England Nuclear), and isoguanine (Sigma) were commercial samples. 8-Hydroxyadenine and 4,6,diamino-5-formamidopyrimidine were synthesized.’l The solutions were prepared in 0.05 M phosphate buffer, pH 7.0. The radiation source was a “Co Gammacell-200 (Atomic Energy of Canada); dose rates were 3300-2800 rads/min.12 The course of the radiolysis depended on the adenineloxygen ratio. In air-equilibrated solutions, the
Radiolysis of Adenine in Solution
305
concentration of oxygen is about 0.25 mM, in solutions saturated with oxygen at 1 atm it is 1.3 mM.13 The solutions containing 2 mM adenine were saturated with oxygen by bubbling the gas continuously through them, at the rate of 1-2 mL/s;14 the spectra obtained under these conditions differed substantially from those which developed in initially air-equilibrated solutions without replenishing the oxygen. In solutions containing 0.2 mM adenine, the same results were obtained for air-equilibrated samples as in those saturated with oxygen. Usually, the volume of the irradiated samples was 5 mL, the surface area 2 cm2. The adenine concentration was determined by the colorimetric method developed by Davis and M ~ r r i s .The ~ procedure is as follows: to a 3-mL sample containing 0, the fractional change in absorbance will define the lower limit to G(-S):
G(-S)
> (Ao* - ADh)/&
es
(3) In order to interpret the spectra of a system in which only some, or possibly none, of the radiolysis products are known, the above relationships must be applied in reverse. If it is found experimentally that, over some wavelength
1001
I
I
X,nm
Figure 1. Spectra of irradiated 0.2 mM adenine solutiins, air equilibrated: (curve A) 0 krad; (B) 15.5 krads; (C) 31.0 krads; (D) 62.0 krads; (E) 93.0 krads.
TABLE I: Fractional Change in Absorbance at Various Wavelengths ( 0 . 2 mM Adenine after 62.0 krads) WaveWavelength, nm (Ao*A-0 f 6 2 h ) / length, nm (A,*A0 - +**)/ 280 260 255 250
0.0 0.288 0.27 5 0.238
245 240 238
0.177 0.050 0.0
range, the fractional absorbance is independent of A, i.e.
(A,* - AD*)/AO* f f ( h )
(4)
it can be inferred to a very high degree of probability that tzX= 0. This condition was fulfilled in the case of cytosine? which is therefore especially favorable, but rather special. Figure 1 and Table I show representative results, obtained by irradiating 0.2 mM adenine. It may be seen that two well-defined isosbestic points develop at 280 and 238 nm. Clearly, the fractional change in absorbance varies strongly with A, and eq 4 does not hold in the present case. It follows immediately from eq 1that the condition for an isosbestic point is ES*
= EX*
(5)
It should be noted that eq 5 is equivalent to that derived by Cohen and FischerlGin their general treatment of the isosbestic phenomenon; radiolytic reactions are a special case. The development of (one or more) isosbestic points constitutes by itself unequivocal proof that the radiolysis products absorb. Moreover, it defines the values of exX at the isosbestic wavelengths; they are 1100 at 280 nm, and 5600 at 238 nm, respectively. However, the results so far discussed do not allow us to determine the value of G(-Ade). Obviously nothing can be learned about this value from measurements at the isosbestic points, and at the other wavelengths the value of exh is not known; as was shown above, all one can obtain from such data is the lower limit to G(-Ade). The results of this analysis are shown in Figure 2, line C; the apparent values of G extrapolate to about 1 molecule/(100 eV) at zero dose. As we have mentioned, Scholes et al.4 obtained, by the same method of analysis, a G value of 1.1. In other words, our experimental results are in close agreement with theirs, and we differ only in the interpretation; this hinges entirely on the question of how strongly the products absorb at 260 The Journal of Physical Chemistry. Vol 81, No. 4, 1977
Gorin et ai.
306
TABLE 11: Chromatography and 10-eV Mass Spectra of Radiolysis Products Thin-layer chromatography Rf value Spot System System no. A B lg
2g 3g 4 5 68 7 8
9
0.70 0.61 0.58 0.54 0.51 0.43 0.39 0.38 0.35
0.51 0.63 0.42 0.52 0.51 0.50 0.32 0.55 0.27
Yielda
-3 2 1 0.15 0.5 0.15 0.3 0.4 0.3 0.2 0.2
Rf value
40
I20 80 DOSE, k r
160
Figure 2. (Left ordinate, curve A) Absorbance of irradiated 0.2 mM adenine solution at 260 nm. (Right ordinate, line B) G(-Ade) calculated from results of the Davis-Morris method; (line C) apparent G(-Ade) calculated from (dAZ6'/dD) assuming cZz6' = 0.
The Journal of Physical Chemistry, Vol. 8 1, No. 4, 1977
Mass / charge
Probe t:mE, C
17g 45 46 60g 86 88 96 108g 111 125g 129 135g 151g 153g
30 30 30 55 130 130 130 130 210 120 130 130 150 140
Principal ionsC
Compound
system A
Probe temp,
Adenine
0.70 0.51 13400
adenine IIf
0.43 0.50
sys-
tem B
mle OCd 135(100) 130 108 (31) Urea 0.61 0.63 60(100) 55 17 (15) 8-Hydroxy- 0.58 0.42 10IOOe 151 (100) 220
200
nm. In order to resolve this question, we took recourse in an independent, highly specific method for determining adenine, the colorimetric test devised by Davis and M o r r i ~ .The ~ inventors of this procedure demonstrated that 14 purine and pyrimidine derivatives did not interfere with the determination of adenine, and we have ascertained in this work that this also is the case for three additional compounds, 8-hydroxyadenine, 4,6-diamino5-formamidopyrimidine, and isoguanine. The results of applying the Davis-Morris procedure to irradiated adenine solutions are illustrated by line B in Figure 2, which is a linear least-squares fit to the calculated values of G(-Ade). In this instance, extrapolation to zero dose gives the value Go(-Ade) = 1.84 molecules/(100 eV). Eight independent determinations of Go(-Ade) averaged 1.94 f 0.1; Le., the reproducibility is quite satisfactory. It is of course to be expected that this result should be larger than the values reported previously. Equation 2 can now be employed to determine the value of cZz6O; the average of some 40 determinations was 5500 f 1100. This result is not very precise because it had to be derived from measurements of (Ao - A,) at low doses, which are inevitably subject to large errors. However the data leave no doubt that tg260is much greater than zero. Our general approach to the study of the radiolysis of biologically important compounds has been to focus attention on the decomposition yield of the original substance, because that is the phenomenon of critical importance from the biological point of view.lS2 The identity of the products is of lesser importance, although in some cases they may have a biological effect of their own.l7 In the present context, it was also pertinent to know whether the products might interfere with the Davis-Morris test. For these reasons, a partial analysis of the products was carried out by thin-layer chromatography and by mass spectrometry. The results are summarized in Table 11. Chromatography gave eight well-defined spots, besides that of adenine (spot 1). The observation confirms our
Mass Spectrometry _______
4600e 153 (100) 170 125 (32)
a (Counts in spot)/(change in counts in adenine spot); approximate values, averaged over the range 130-540 krads. Temperature at which ion first appears. Number in parentheses is the abundance of the ion relative to the principal peak; only ions with relative abundance >10 are listed. Temperature at which the molecular ion is most intense. e In agreement with Cavalieri and Bendich." f 4,6-Diamino-5-formamidopyrimidine. g Identified with known compound in bottom part of table.
supposition that to determine all the radiolysis products would be a laborious undertaking. We have identified one of the products (spot 2) as urea; to our knowledge, this product had not been reported previously. Since the urea is radioactive, it must come, at least in part, from the C-8 atom of adenine. Two other products are %hydroxyadenine (I, spot 31, and 4,6-diamino-5-formamidopyrimidine (11, spot 6); these products have been reported previ~usly.~,~ As noted above, I and I1 do not interfere with the Davis-Morris test. The mass spectrophotometric results cannot be interpreted unambiguously. However the mass spectra are consistent with the presence of the aforementioned products, and of some additional ones which we have not identified. I and I1 account for a major fraction of erZ6O. Their contribution can be estimated by substituting the appropriate values of G(UJ and from Table I1 and the value of G(-Ade) = 1.9 into eq 2, as follows: ~
+
~ 11) ~ = (1/1.9)(0.5 ( 1 X 10100 + 0.4 X 4600) = 3600
(6)
This leaves a remainder of some 1900 for the equivalent absorbance of all the other products, which account for about half of the adenine decomposed. The results thus provide a self-consistent and largely unambiguous interpretation of the radiolysis process. The
Ternary Nonelectrolyte Systems
only uncertainty concerns the identity of some of the products. It should be noted that, if any of the unidentified products gave some color in the Davis-Morris test, the value of G(-Ade) would be even higher than that which has been quoted above. This possibility cannot be absolutely excluded, but it is very unlikely in view of what is known about the radiolysis process and the unidentified products.
Acknowledgment. The work described above was aided by a Biomedical Sciences Support Grant from the National Institutes of Health to the Research Foundation, Oklahoma State University, and by NSF Grant MPS 75-18967 to L.M.R.
References and Notes (1) R. N. Rice, G. Gorin, and L. M. Raff, J. fhys. Chem., 79, 2717 (1975). (2) G. Gorin, N. Ohno, and L. M. Raff, J. fhys. Chem., 80, 112 (1976). (3) D. T. Kanazir, frog. Nucl. Acid Res. Mol. Biol., 9, 117 (1969).
307 (4) (5) (6) (7) (8) (9) (10) (11) (12)
(13)
(14) (15) (16) (17)
G. Scholes, J. F. Ward, and J. Weiss, J. Mol, BiOl., 2, 379 (1960). J. J. Conlay, Nature(London), 197, 555 (1963). J. J. van Hemmen and J. F. Bieichrodt, Radiat. Res., 46, 444 (1971). J. F. Ward in “Advances in Radiation Research”, Voi. 5,J. T. Lett and H. Adler, Ed., Academic Press, New York, N.Y., 1975, p 207. H. J. Rhaese, Biochim. Biophys. Acta, 166, 311 (1968). J. R. Davis and R. N. Morris, Anal. Biochem., 5, 64 (1963). R. Uliana and P. V. Creac’h, Bull. SOC. Chim. Fr., 2904 (1969). L. F. Cavaiieri and A. Bendich, J Am. Chem. Soc., 72, 2587 (1950). “Radiation Dosimetry: X-Rays and Gamma Rays with Maximum Photon Energies Between 0.6 and 60 MeV” (ICRU Report No. 14), International Commision on Radiation Units and Measurements, Washington, D.C., 1969. H. Stephen and T. Stephen, “Solubilities of Inorganic and Organic Compounds”, Vol. 1, Pergamon Press, Oxford, 1963, part 1, pp 87-88; W. F. Llnke, Ed., “Solubilities”, Vol. 11, American Chemical Society, Washington, D.C., 1965, p 1228. C. A. Mannan, M.S. Thesis, Oklahoma State University, 1972. L. Josefsson, Biochim. Biophys. Acta, 72, 133 (1963); P. Grippo, M. Iaccarino, M. Rossi, and E. Scarano, ibid., 95, 1 (1965). M. D. Cohen and E. Fischer, J. Chem. Soc., 3044 (1962). E,g., M. Poiverelli and R. Teouie, C. R. Acad S d faris Ser. C, 277, 747 (1973).
A Chromatographic Investigation of Ternary Nonelectrolyte Systems Jon F. Parcher” and Theodore N. Westlake Chemistry Department, University of Mississippi, University, Mississippi 38677 (Received May 17, 1976; Revised Manuscript Received November 8, 1976) Publication costs assisted by the University of Mississippi and the National Science Foundation
The “chromatographic”partition coefficients of eleven solutes at infinite dilution were measured as a function of composition in four binary solvents at 45 “C. The binary liquid phases were formed by utilizing a condensable component in the carrier gas. The molecular sizes of the components were disparate and the systems were used to test several nonelectrolytesolution theories. It is shown that Purnell’s “microscopic partitioning theory” was inadequate for most of the systems studied and that the Flory-Huggins theory provides a much better, although imperfect, description of the particular systems investigated.
In the last decade, gas-liquid chromatography has proven to be an accurate and convenient technique for measuring vapor-liquid equilibria data for nonelectrolyte systems. This is especially true in the low concentration ranges (infinite dilution) normally inaccessible by static vapor pressure measurements. The primary data usually obtained are the “chromatographic” partition coefficient and the Raoult’s law activity coefficient of the volatile solute in the stationary liquid phase. Binary liquid phases have also been investigated by several authors. There are two main purposes for this type of investigation. One is the attempt to control the selectivity of a chromatographic column by using mixed liquid phases and the other is an attempt to measure the equilibrium constants for the weak type of complexes, such as charge transfer, r bonding, or H bonding complexes, between the solute and one component of a binary liquid phase. The latter type of investigation has invoked a great deal of controversy, especially involving the comparison of complex formation constant data from chromatography, UV-visible spectrophotometry, and nuclear magnetic resonance spectrometry. Pilgrim and Kellerl reviewed the general area of mixed liquid phases in 1973 and pointed out the multiplicity of empirical and theoretical equations which “fit” the partition coefficient data. Dal Nogare and Juvet2first suggested a simple relation between the “chromatographic” partition coefficient of a
solute, i, in a binary solvent, A + S, and the composition of the liquid phase. Several author^^-^ have used the equation
where (tRi)j is the retention time of solute i in liquid phase j and Wj is the weight of component j in the mixed liquid phase. This equation was shown to be accurate for over 100 chromatographic systems by plotting (tRi)A+Svs. the weight percentage of one of the liquid phase components. Waksmundzki, Soczewinski, and S u p r y n o w i ~ zpro~~~ posed an equation of the form
In (ki)AtS = @A In (IZi)A+ $S In (2) where (kJj is the partition ratio (moles of i in the liquid phase per mole of i in the gas phase) of solute, i, in solvent j, and 0, is the volume fraction of component j in the liquid phase. The partition ratio is equal to the ratio of the retention time of a solute to the retention time of air or another inert solute. Thus, eq 2 predicts a linear relation between In tRi and the volume fraction of one of the liquid phase components. This equation was shown to be accurate for 17 chromatographic systems, although the authors laters modified eq 2 to include a term to account The Journal of Physical Chemistry, Vol. 81, No. 4, 1977
