Photochemtstry and Pholoblology, 1976, Vol. 27. pp.79-85
Pergarnon Press
Printed in Great Brltaln
FLASH PHOTOLYSTS OF AQUEOUS TRYPTOPHAN, ALANYL TRYPTOPHAN AND TRYPTOPHYL ALANINE H. TEMPLER and P. J. THISTLETHWAITE* Chemistry School, Melbourne University, Parkville, Victoria, 3052, Australia (Receiwd 3 July 1975; accepted 1 October 1975)
Abstract-Tryptophan has been flash photolysed in deoxygenated and air equilibrated solulions. Thc kinetics of the subsequent reactions of the tryptophyl radical and the hydrated electron havc bccn elucidated. Oxygen has been found to reduce the primary photoionization yield, indicating a triplet state precursor, but no evidence has been found for a biphotonic process. Appreciable differences in the photoionization quantum yields have been found for tryptophan, alanyl tryptophan and tryptophyl alanine.
photoionization is a single photon process involving a singlet state as precursor to the radical. Our own results support the idea of a single photon process. However, we have observed a decrease in radical yield in the presence of oxygen which points to triplet state involvement and is in contradiction to Bent and Hayon (1975). Work prior to that of Bent and Hayon on the effect of oxygen on the radical yield was in some cases rather ambiguous in that the possible contribution by hydrated electron to the absorbance at 510 nm, the absorption maximum of the neutral radical,was overlooked. Removal of ea; in the presence of oxygen could thus have accounted for the drop in 510nm absorbance observed by Paikhorpe et uI. (1973). As will be described, we have attempted to overcome this source of confusion. The observation of Pailthorpe et al. that the overall destruction yield increased in the presence of oxygen indicated that the triplet may be involved in the photochemical change but not necessarily as the electron ejecting species. The fate of the electron and radical after their formation is also uncertain. In oxygen free solutions the electron has been reported to undergo a back reaction with radical (Grossweiner and Usui, 1971) and elsewhere to decay by second order reaction with itself (Pailthorpe er al., 1973). Other data suggest that the electron should predominantly react with unphotolysed parent molecule (Braams, 1966; Finnstromm, 1971). Attempts have been made to correlate the quantum yield for permanent destruction with the quantum yields for radical and electron production (Pailthorpe et al., 1973). Such correlations require answers to the questions raised above. Grossweiner and Usui (1971) have sought to explain the low permanent photolysis yields in oxygen free solutions on the basis of a back reaction of electrons and radicals. An increased permanent photolysis yield found in the presence of oxygen could then in part result from interference with this back reaction due to e& scavenging by oxygen. It should, however, be pointed
INTRODUCTION
The photochemistry of tryptophan (trp) has been the subject of a number of investigations. The interest in trp arises because, as a photochemically labile amino acid, it is thought to play a crucial role in the light sensitivity of a number of peptides and proteins. Previous studies have utilized steady photolysis and EPR at low temperatures (Guermonprez et ul., 1967; Santus et a!., 1968, 1970, 1972; Vladimirov et d., 1968, 1970; Hklkne et ul., 1968; Pailthorpe et ul., 1972),flash photolysis (Grossweiner et al., 1963, 1971; Santus and Grossweiner, 1972; Joschek and Grossweher, 1966; Finnstromm, 1971; Subramanyan and Tollin, 1972; Pailthorpe et ul., 1973), and most recently, laser flash photolysis (Bent and Hayon, 1975). Despite the wide attention it has received, a number of aspects of the behaviour of trp remain unclear. There is general agreement that absorption of light in the 280 nm band initially leads to the ejection of an electron from the indole ring and the formation of a radical cation. In aqueous solutions at pH greater than zero this cation is thought to lose the proton from the indole nitrogen to form the neutral radical (Santus and Grossweiner, 1972; Bent and Hayon, 1975). Overall, the radical, like the trp molecule, may still have a net positive or negative charge according to the state of ionization of the amino and carboxyl groups. For pH between 4 and 9 the predominant species will be that of zero net charge. In low temperature glasses there is some evidence that the electron ejection is a consecutive biphotonic process with the probability of the triplet state acting as an intermediate (Santus et al., 1968, 1970; Vladimirov and Fesenko, 1968).The triplet has been detected by EPR in such glasses. A recent laser flash photolysis study by Bent and Hayon (1975), which appeared while the present work was being prepared for publication, has indicated that in room temperature solution the *To whom correspondence should be directed. 19
80
H. TEMPLER and P. J. THISTLETHWAITE
out that the permanent destruction yield observed by Pailthorpe et al. in the presence of oxygen was several times the radical yield observed in oxygen free solutions. The kinetic behaviour of the radical is also unclear. In one paper it was reported that in deoxygenated solutions the radical disappeared by three consecutive first order reactions (Grossweiner and Usui, 1971). Elsewhere (Santus and Grossweiner, 1972; Subramanyan and Tollin, 1972), the decay is said to be second order but the /+ values of different reports differ widely. In air saturated solutions, Pailthorpe et a/. (1973) mentioned a second order reaction between radical and oxygen but gave no details. In view of the relatively high concentration of dissolved oxygen in comparison to radical, a pseudo first order radical disappearance might have been expected. In the light of these and other difficulties, we have made a further investigation of the flash photolysis of trp. We have also been concerned with the possible differences in behaviour that might arise when trp is incorporated into simple peptides. Some flash photolysis studies of proteins have already been made but these have been on rather large molecules, such as lysozyme (Grossweiner and Usui, 1971; Subramanyan and Tollin, 1972; Kaluskar and Grossweiner, 1974). There has been little work on simple peptides until the recent study of Bent and Hayon (1975). In a steady photolysis study (R. B. Johns and P. Scheelings, personal communication) differences have been found in the product distribution for the photolysis of alanyl trp and tryptophyl alanine. We have thus been interested to investigate these molecules by flash photolysis with a view to comparing the yields of radical in the three cases. MATERIALS AND METHODS
The reagents L-tryptophan (trp), L-alanyl-L-tryptophan and L-tryptophyl-L-alanine were from Sigma and were used without further purification. All glassware was steam cleaned for 4 h before use. Solutions were made up with triple-distilled water. Solutions were deoxygenated by bubbling for more than 2 h with oxygen free N2 or Ar. In most cases no extra purification of Nz or Ar was attempted as they were sold as oxygen free. However, in some cases the gases were further purified by first passing them through a dry ice cold trap to condense traces of organic matter, and then through two Dreschel bottles each containing vanadous ion solution over amalgamated zinc. Two different flash apparatuses were used; one for the determination of transient spectra and the other for the study of kinetic behaviour. A double flash method was used to obtain transient spectra. The photolysis Aash was of 30 p s duration (at half-height) from an Ar-air filled flash lamp operated with a 3500 J input (120 pF, 7.6 kV).'The spectra were recorded on llford HP3 plates using a Hilger El spectrograph. The capillary spectroflash was fired at the maximum of the photolysis flash. Photographic plates were scanned with a Joyce-Loebl double beam recording microdensitometer and the photographic densities converted to absorbances using an emulsion calibration made on the same plate using the spectroflash lamp. The transient spectra were obtained by subtraction of the photographic densities of the short delay spectrum and a very long delay spectrum. To obtain suitable plate densities it
was necessary to use eight flashes and these wcrc applied to the one solution. As each flash causes appreciable permanent decomposition it is not possible to use such spectra to obtain quantum yields. However, the use of the abovementioned subtraction procedure eliminates the contri bution to the spectra by parent compound or permanent photolysis product. Thus the transient absorptions at different Wavelengths are relatively correct nnd can be used to obtain molar absorptivities provided only that thc spectrum is taken sufficiently soon after the photolysis flash so that different kinetic behaviours of the different species will have a negligible effect. All photolyses were done in a 27 cm long, doubled walled, silica cell, the outer jacket of which could be filled with a suitable filter solution. Kinetic data were obtained on a second apparatus. The photolysis flash was of 5 p s duration. It was produced by the discharge of 135 J (4.5 pF, 7.8 kV) through a 30 cm long ILC Xe flash lamp. The transient absorption was monitored using a Hanovia XBO 450 high pressure Xe source, a Bausch & Lomb monochromator and a Hamamdtsu R446 (red sensitive) PM tube feeding into a Tektronix 502A CRO. Decay curves were photographed with a Polaroid camera. Particular attention was given to the problem of minimising stray light by the use of baffles and suitable filter solutions in the photolysis cell jacket. A glass filter was used between the monitoring source and the absorption cell to prevent any decomposition of the cell contents by short wavelengths in the monitoring beam prior to flashing. When monitoring ea; at 670nm a red filter was used between the absorption cell and the monochromator to remove the possibility of second order light passing through the monochromator. Second order light may account for some results previously reported on L,,; decay. The linearity of the photomultiplier over the transient absorption range likely to be covered was checked using solutions of known absorbance.
RESULTS AND DISCUSSION
Radical spectra and uhsorptivities The transient spectrum obtained on flashing a 160 pA4, deoxygenated, N,-saturated solution of trp at pH 11 was similar to that reported earlier (Grossweiner and Usui, 1971) with a maximum at 510nm attributable to the neutral tryptophyl radical and an absorption at the red end of the spectrum due to ea;. The cell jacket was filled with glacial acetic acid in order to remove the possibility of ea; generation by photolysis of carboxyl groups. To determine the molar absorptivity of the radical the assumption is made that the absorbance above 630 nm is essentially due entirely to ea;. The ea; absorbance at 510 nm can then be determined from its published spectrum (Fielden and Hart, 1967). The absorbance of the radical at 510 nm is obtained by subtracting the ea; absorbance from the total absorbance. By comparison of the absorbance of radical at 510nm and that of e.+; at 630 nm and knowing the molar absorptivity of ed; at 630 nm (Fielden and Hart, 1967) the molar absorptivity of the radical at 510nm is found to be 8.8 1.0 x lo3 M - l cm-'. This procedure assumes that at the time of the spectroflash the radical and ea; are present in equal concentrations. By the same procedure the absorptivities of the radicals from alanyl trp and tryptophyl alanine were found to be
Flash photolysis of tryptophan
81
time region between 200 and 9 0 0 p , i.e. up to apspectively. The result for tryptophyl radical is in quite proximately two half-lives, the decay follows skcond good agreement with the earlier determination of order kinetics with the li,k being approximately Grossweiner and Usui (1971). However, Redpath et 4.6 x lo5 cm s-'. The k/e values found for a number ul. (1975) have recently re-determined the absorptivity of deoxygenated, N2- or Ar-saturated solutions of the neutral tryptophyl radical to be 1.75 x lo3 ranged from 3.5 x 10' to 5.7 x 105cm S C ' , with M--' cm-'. They claimed that errors can occur in there being a trend towards the rate constant being the two flash method if the electron has decayed sig- higher when the radical yield was lower. This would nificantly more than the radical at the time of record- suggest that a second minor reaction is contributing ing the spectrum. An alternative procedure is to use to the decay in some cases. We have been unable kinetic data to determine the extrapolated absor- to confirm this. In a number of subsequent experbances of ea; and radical at zero time and thus deter- iments covering a greater time range we were unable mine the absorptivity. There are two difficulties with to detect any deviation from second ordcr kinetics. this procedure in our case. The conditions for best In several experiments with deoxygenated, N20- or extrapolation of radical absorbance are those of high Ar-saturated solutions, a value of approximately flash energy and large radical-electron yield. As will 3.5 x lo5 cm s f l was found. be explained later, the decay of the e& under these At times less than 150ps, there was in all cases conditions is very rapid and the kinetics complex. a negative deviation from the second order kinetics This being so it is difficult to extrapolate the e, yield holding beyond 200 ps. This we attribute to an initial back to zero time. A further difficulty is that under rapid removal of radical by back reaction with elecconditions of high ea; yield the lifetime of the ea; tron. To confirm this conclusion we have performed decay is short and the flash duration becomes signifi- experiments.with deoxygenated, N,O-saturated solucant in comparison. In this case extrapolation back tions. N 2 0 should scavenge e,; without any effect on to zero time is likely to be inaccurate. Extrapolation the radical. In N,O-saturated cases we find that the of the radical yield is more reliable due to its much decay remains second order down to quite short slower decay. In this case the flash may be regarded times, the rate constant being approximately the same as instantmeous. I t is for this reason that we have as found in the previous cases. The yield at zero times generally preferred to extrapolate radical rather than was also found to be approximately the same in both ea; absorbance. cases. This observation argues against an alternative explanation for the more rapid decay of absorbance Kinetic behuviour of the tryptophyt radicul in deoxy- seen below 1 5 0 ~ sThis . might have been attributed genated and air-saturated solutions to a contribution by eai to the 510nm absorbance. Particular attention was given to the decay kinetics The more rapid decay of ea; would then cause a rapid of the radical produced by flashing trp. Decays were decay of part of the 510nm absorbance. If this were monitored at 510 nm using the kinetic apparatus de- the case, then for an N2-saturated solution the absorscribed earlier. A filter solution consisting of an eth- bance should extrapolate back to a higher value than anol- HCI solution of CoCI, and NiCI, (Vladimirov, would be the case with N 2 0 present. In separate ex1966) was used in the outer jacket. This filter is highly periments to be described later it was shown that the transmitting in the region 260 to 390nm and fairly ea; absorbance at 670 nm has mostly disappeared by opaque (transmission generally less than 20%) else- 5 0 p s from the time of the flash. Thus the plotted where. The purpose of this filter was to minimise the points of Fig. 1 should at most contain a small contrieffect of stray light. In fact, stray light was found to bution from edg absorption. Our observations may be insignificant after 15ps at all wavelengths moni- be contrasted with those of Grossweiner and Usui tored. The filter's transmission properties were found to change after exposure to light and with age. In 24r some cases where it was desired to compare radical yields, special precautions were taken to ensure that the filter's properties did not change during the course of a series of experiments. Thus, in general, the results presented in any one figure apply for a given filter solution. However, the radical yields cannot be compared from one figure to another due to changes in filter transmission in the photolysis region. Fig. 1 shows the decay of tryptophyl radical plotted as a second order process in deoxygenated and deoxyI I I I I 200 400 600 800 genated, N20-saturated solutions. In both cases the t /ps trp concentration was M and the pH was 11. The plot for the deoxygenated case is typical of a Figure 1. Second ordcr decay of tryptophyl radical in (a) large number of decays observed for different trp con- deoxygcnated and (b) deoxygcnated, N,O-saturated centrations and flash energies. In all cases, for the solution. 7.5 & 1.0 x 10' and 9.3 & 1.0 x lo3 M - ' cm-' , re-
,
H. TEMPLER and P.J. THISTLETHWAITE
82
(1971) who reported that the tryptophyl radical underwent three consecutive first order decays. They too found an initial rapid decay of absorbance followed by B slower decay and then a still slower decay at times greater than 1 ms. Grossweiner and Usui also studied the decay of 510 nm tryptophyl absorption in lysozyme and attributed the intermediate time first order decay to a pseudo first order reaction of tryptophyl radical groups with hydroxide ion. However, an unsatisfactory aspect of their interpretation was the fact that the observed first order rate constant varied only slowly with pH. In further experiments using a slower time base we have monitored the radical decay out to approximately 2ms (more than 3 halflives) without observing any change in kinetics. Our conclusion is that, in oxygen free solutions, except for the initial back reaction with e&, radicals are removed by second order reaction with themselves. M trp solution at On flashing a deoxygenated pH 6.3, we again observed second order decay in the region 200 to lOOOps, this time with a kjc value of 8.4 x lo5 cm s-'. We consider the change in rate constant to be most likely due to the switch from the net negative charged radical existing at a pH of eleven to the zwitterion form occurring at pH 6.3. The yield of the radical was also found to be reduced in line with the results of Bent and Hayon (1975). In air-saturated solutions, the radical decay kinetics become complex although the initial steep section is no longer present as would be expected in view of the very efficient scavenging of e& by oxygen. Fig. 2 shows a decay typical of those found in several experiments with different flash energies. The results have been plotted both as first and second order proM and cesses. Again the trp concentration was the pH was 11. If a reaction occurs between radicals and oxygen then this would be expected to be pseudo
first order, as the dissolved oxygen concentration (approximately 3 x 10-4M) is very much greater than that of radical. If such a reaction was in competition with the second order radical radical reaction, complex kinetics would result. The differential rate law for such a situation is
k [ R I 2 + k'[R] dt where k and k' are the second order and pseudo first order rate constants, respectively. On integration, we obtain [R] = k / ( K exp k'l - k) _ drR1 _-
where K = (k' + k[R],,)/[R],,([R10= radical concentration at t = 0). A non-linear least squares computer program was used to fit the observed [ R ] vs time data, for a number of experiments with different flash energies, to the above function. In all cases the data could be well fitted (computed and experimental points within 5'%,, standard errors in k and k' of approximately 4%). The value of k required for best fit lay in the range 4.9-5.3 x lo9 M - ' s- (k,k in range 5.5-6.0 x lo5cm s-'). The values of k' required for a fit lay in the range 1.2--1.8 x 103s-'. The values of the second order constant, it will be noted, lie at the upper end of the range found earlier for deoxygenated solutions. This is in line with the earlier statement that the apparent second order rate constants seem slightly dependent on radical yield. Attempts were made to verify the pseudo first order reaction with oxygen by doing runs with solutions equilibrated with different pressures of air. Lowering the pressure of air did in fact lower the pseudo first order constant whilc leaving the second order constant unchanged. For an air pressure of 33 cm Hg, a pseudo-first-order constant of 1.0 x 103s-' was found. Taking this value and combining it with the solubility of oxygen in solution equilibrated with 33 cm Hg of air, we obtain a value of X.2 x lo6 M ' s-' for thc second order rate constant for the reaction between radical and oxygen. Eflkct of oxygen on radicul yield
t,
ps
Figure 2. Decay of tryptophyl radical in air equilibrated solution: filled circles, plotted as second order process; open circles, plottcd as first order process.
To determine the effect of oxygen on the initial radical yield an air-saturated and a carefully deoxygenated solution were flashed, taking particular care that the filter solution was precisely the same in both cases. The tryptophan concentration was lo-" M and the pH was 11. Fig. 3 shows that a reduction of more than 50% in initial yield occurs in air-saturated solutions compared to oxygen free solutions. In the deoxygenated case, the extrapolation back to zero time might be criticised on the ground that until all electron has been removed the absorbances at 510 nm contain a contribution from ea; as well as from radical. However, as the points used in the extrapolation are for times longer than 5 0 p , by which time most of the electron has disappeared, the error will be small and the basic conclusion that the initial radical yield is reduced by oxygen remains valid. The reduction
Flash photolysis of tryptophan
83
by the filter for various flash energies were determined by ferrioxalate actinometry. Fig. 4 shows a plot of extrapolated initial radical absorbance versus the relative number of photons in the flash for air-saturated, 10-4M trp solutions at pH 11. In fact a slight fall in radical quantum yield seems to occur at higher flash energies. In an earlier study, Pailthorpe er ul. (1973) found no variation in permanent destruction quantum yield for an air-saturated solution, as the flash energy was varied. However, if most of the permanent destruction observed by them occurred by a reaction between oxygen and triplet formed independently of the formation of the radical, then their experiment would not be expected to reveal any biphotonic pathway to radical formation. It should be recalled that Pailthorpe et al. observed a permanent 200 400 600 800 destruction quantum yield in the presence of oxygen several times higher than the radical yield observed t , /Ls in oxygen free solutions. Although our results indicatc Figure 3. Effect of oxygen on the yield of tryptophyl radino biphotonic photoionization in our case, they do cal : (a) deoxygenated solution (b) air equilibrated solution. not rule out the possibility of some contribution by a biphotonic pathway if a flash containing the tripletin radical yield observed here contradicts the earlier triplet absorption wavelength were used. Our results observations of Grossweher and Usui (1971) and should be contrasted with some recent results of Bent and Hayon (1975) and suggests some involve- Kaluskar and Grossweiner (1974) which they ment of the triplet state in our case. One possibility explained on the basis of a biphotonic photoionizafor reconciling the discrepancy is to assume that at tion of trp, via an intermediate triplet state, becoming short times the 510 nm absorbance contains a contri- significant at higher flash energies. bution from the triplet state. As reported by Bent Our results point to the triplet state as a precursor and Hayon, the absorptivity of this species at 510 nm of the radical but not as the absorber of a second is not negligible. As the triplet is strongly quenched photon. The partial quenching of the radical yield by oxygen this could account for an apparent drop in the presence of oxygen could mean either that in radical yield in the presence of oxygen. However, photoionization from the triplet state competes with there are difficulties with this explanation. The triplet quenching of the triplet by oxygen, or that photoionistate lifetime in oxygen free solutions is reported zation is occurring from both the singlet and the trip(Bent and Hayon, 1975) to be approximately 2 0 p , let and that the partial quenching in the presence of while the plotted points of Fig. 3 are all for times oxygen reflects the blocking of the triplet pathway. greater than 5 0 p s by which time the triplet should If the latter was the case, then there would be no have disappeared. Secondly, if the triplet state was possibility of observing a biphotonic process involvmaking a contribution to the 510 nm absorbance the ing the triplet when oxygen was present. For this rearapid decay of this absorbance could account for the son an experiment similar to the one depicted m Fig. initial steep section of the plot of Fig. la. However, 4 was done with deoxygenated solutions. The result if thk were so then the N 2 0 should not be effective was similar to before. in removing this initial steep section. Our results point to ionization being possible from Our observation that oxygen reduces the radical more than one excited state, one of which is sensitive yield, suggesting an involvement of the triplet state, 0.31 raises the question of whether the triplet state is involved as the absorber of a second photon, as is known to be the case in low temperature glasses. We have found no evidence that biphotonic processes are playing any appreciable part in the radical production we observe. A biphotonic process involving the absorption of a second photon by the triplet state would, a priori, seem unlikely in our case, due to our use of a flash filter with a low transmission at 450 nm, the wavelength reported by Bent and Hayon (1975) for the triplet-triplet absorption. If any consecutive biphotonic process were involved, the quantum yield No. photons, orb u n i t s of radicals would be expected to rise at higher flash Figure 4. Initial radical absorbance versus the relative energies. The relative numbers of photons transmitted number of photons in the photolysis Ilash.
H TEMPLER and P. J. TIII$II
84
I I IWAI
I
cal production is low, The pseudo first order reaction is also favoured by higher parent concentrations. Both the above sets of experiments involved initial electron-radical concentrations of approximately 6 x 10- ' M , this being dependent mainly on the flash energy rather than the parent concentration. In the case of the series in the 10-5-10-4 M concentration range, there is sufficient second order electron-radical reaction to increase the apparent rate constant of the Kinetic hehaviour of the hydrated electron pseudo-first-order reaction without producing plots A puzzling feature of earlier studies has been the with a very noticeable deviation from first order kindisagreement on the fate of e&. As mentioned earlier, etics. To check these ideas, further experiments were at least three possibilities have been reported. The done with a high transmission filter which gave a results to be described have to be seen in the light much higher electron-radical yield. In these cases the of our earlier conclusion that at least some of the ea&decay was considerably faster and the kinetics now electron undergoes a back reaction with tryptophyl showed appreciable deviation from first order. The radical. We have performed two series of experiments. ea&+ e& + H, + 2 OH- reaction could also be In the first instance, a series of deoxygenated trp solu- expected to make a contribution to a deviation from tions of pH 11, ranging in concentration from M first order kinetics. A rough calculation serves to to 7 x M, was flashed and the eLq decays moni- further emphasize the difference between the situation tored. These experiments were done with a filter solu- depicted in Fig. 1 and that in the experiments where tion somewhat less transmitting in the photolysis strictly first order decay of ea; is observed. In Fig. region than that used for the experiments depicted 1, the initial yield of e; and radical in the oxygen in Fig. 1. In each case the decay appeared to be free case is approximately 1.5 x lO-'PJ, while the M. In one of the strictly first order, and the rate constant varied syste- parent trp concentration was matically with trp concentration, indicating a pseudo experiments of Fig. 4, the initial yields were approxifirst order reaction between ea; and trp. A plot of mately 6 x lO-'M and the trp concentration pseudo first order constant versus trp concentration 3 x 10-4M. Thus in terms of initial rates, there is led to a straight line passing through the origin and a change in the ratio of first to second order reaction the slope gave a figure of 1.2 x 1 0 8 M - ' s - ' for by a factor of approximately 19 in going from one the second order rate constant (Fig. 5). This result case to the other. agrees with the pulse radiolysis work of Braams (1966) in which eai was generated quite independently of the trp. In a second series of experiments in the Relative radical yields for tryptophan, uhnyl tryptolo-' M-8 x M trp concentration range, appar- phan and tryptophyl alanine at naturul p H ently pseudo first order decay was again found. This To compare the yields of radicals, deoxygenated, time a plot of pseudo first order constant versus trp M solutions of the three compounds, at pH 6.3, concentration yielded a second order constant of were flashed with exactly the same flash energy. The 4 x lo8 M - ' s- Our conclusion is that in general decay curves for the radicals from all three species e& undergoes at least two reactions simultaneously. were found to be of approximately the same form These are a second order reaction with tryptophyl as found previously for trp in the high pH region. radicals and a pseudo first order reaction with un- There were, however, some differences in the second photolysed trp. These can occur in competition, or order rate constants from those found earlier for trypin some circumstances, the pseudo first order reaction tophyl radical. The initial yield of radical from trp may dominate the kinetics. This will be the case at was somewhat reduced (approximately 25%) in comlow flash energies where the initial electron and radi- parison to the yield at pH 11. This observation is in agreement with the earlier work of Pailthorpe el al. (1973) and with the recent work of Bent and Hayon (1975). The initial absorbance found for the radical from tryptophyl alanine was somewhat lower, and that for the case of alanyl trp was much lower again. Using the molar absorptivities previously found, the relative yields were calculated to be 1.0, 0.54 and 0.27, for trp, tryptophyl alanine and alanyl tryptophan respectively. Similar results have been reported by Bent and Hayon (1975) who found that the photoionization yields of glycyi and tyrosyl pep( [ t r v l /M ) x lo4 tides of trp were lower than trp itself. In an earlier steady photolysis study (Johns and Figure 5. Variation of pseudo-first-order rate constant for e,Yu decay with tryptophan concentration. Scheelings, private communication), the quantum
to oxygen. The excited singlet state is known to be unaffected by oxygen at the concentration found in atmosphere equilibrated solutions (Lakowicz and Weber, 1973). However, in the light of the results of Bent and Hayon (1975) obtained with apparatus better able to determine concentrations at very short times, our conclusions on the effect of oxygen must be subject to some doubt.
'.
Flash photolysis of tryptophan
yields for permanent destruction and NH3 production, respectively, were: trp 0.01 5, 0.005; alanyl tryptophan 0.036, 0.030; and tryptophyl alanine 0.017, 0.014. These figures provide an interesting comparison with our own data. The order of permanent destruction quantum yields is the reverse of our order of radical yields. Both tryptophyl alanine and alanyl tryptophan show higher quantum yields for NH3 production than does trp. The high quantum yield for NH3 may be connected with the reduced radical yield we observe for alanyl trp. A rapid rearrangement of the molecule at the time of, or just after, the ejection of the electron could bypass the formation of the radical. If this is so, it appears that the structure of the alanyl tryptophan is particularly favourable to such a rearrangement. A check was made on the eai yield
85
in the alanyl tryptophan case and this was found to be correspondingly reduced. Very rapid electron migrations are thought to occur in both lysozyme and trypsin where the formation of the disulphide bridge electron adduct has been shown not to occur by trapping of ea; from solution. The electron apparently makes an intramolecular migration to the cystine (Kaluskar and Grossweiner, 1974; Grossweiner and Usui, 1971; Adams et d.,1969). The present results emphasize the importance of avoiding the assumption of constant photoionization quantum yield for trp regardless of the environment. They indicate that significant differences can occur, even in quite simple compounds. Further work at higher time resolution is indicated in order to elucidate the reasons for the observed differences.
REFERENCES
Adams, G. E., R. L. Willson, J. E. Aldrich and R. B. Cundall (1969) Intern. J . Rudiution B i d . 16, 33 3-342. Bent, D. V. and E. Hayon (1975) J . Am. Chem. Soc. 97, 2612-2619. Braams, R . (1966) Radiation Res. 27, 319--329. Fielden, E. M. and E. J. Hart (1967) Trans. Faraday Soc. 63, 2975-2982. Finnstrom, B. (1971) Photochem. Photohiol. 13, 375-377. Grossweiner, L. I., G. Swenson and E. Zwicker (1963) Science 141, 805-806. Grossweiner, L. 1. and Y. Usui (1971) Photocheni. Photobiol. 13, 195-214. Guermonprez, R., C. Helene and M. Ptak (1967) J . Chim. Phys. 64, 13761384. Helene, C., M. Ptak and R. Santus (1968) J . Chim. Phys. 65, 160-166. Joschek. H. I. and L. 1. Grossweiner (1966) J . Am. Chem. Soc. 88. 3261-3268. Kaluskar, A. G. and L. I. Grossweiner (1974) Photochem. Photobiol. 20, 329-338. Lakowicz, J. R. and G. Weber (1973) Biochemistry 12, 41614170. Pailthorpe, M. T. and C . H. Nicholls (1972) Photochem. Phofohiol. 15, 465-477. Pailthorpe, M. T., J. P. Bonjour and C. H. Nicholls (1973) Photochem. Photobiol. 17, 209- 223. Redpath, J. L.. R. Santus, J. Ovadia and L. I. Grossweiner (1975) Inttrn. J . Radiation Biol. 27, 201-204. Santus, R. and L. I. Grossweiner (1972) Photochem. Photobiol. 15, 101-105. Santus, R., C. Helene and M. Ptak (1968) Photochem..f!&qFiol. 7, 341-360. Santus, R., A. Helene, C. HClene and M. Ptak (1970) J . Phys. a e m . 74, 550-561. Santus, R., M. Bazin, M. Aubailly and R. Guermonprez (1972) Photochi.m:"Photobiol. 15, 61 69. Subramanyan, V. and G. Tollin (1972) Photochrm. Photobiol. 15, 449456. Vladimirov, Y. (1966) Photochem. Photobiol. 5, 243-250. Vladimirov, Y. and E. Fesenko (1968) Photochem. Photobiol. 8, 209-212. Viadimirov, Y., D. I. Roschupkin and E. Fesenko (1970) Photochent. Photubiol. 11, 227-246.
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FLASH PHOTOLYSTS OF AQUEOUS TRYPTOPHAN, ALANYL TRYPTOPHAN AND TRYPTOPHYL ALANINE H. TEMPLER and P. J. THISTLETHWAITE* Chemistry School, Melbourne University, Parkville, Victoria, 3052, Australia (Receiwd 3 July 1975; accepted 1 October 1975)
Abstract-Tryptophan has been flash photolysed in deoxygenated and air equilibrated solulions. Thc kinetics of the subsequent reactions of the tryptophyl radical and the hydrated electron havc bccn elucidated. Oxygen has been found to reduce the primary photoionization yield, indicating a triplet state precursor, but no evidence has been found for a biphotonic process. Appreciable differences in the photoionization quantum yields have been found for tryptophan, alanyl tryptophan and tryptophyl alanine.
photoionization is a single photon process involving a singlet state as precursor to the radical. Our own results support the idea of a single photon process. However, we have observed a decrease in radical yield in the presence of oxygen which points to triplet state involvement and is in contradiction to Bent and Hayon (1975). Work prior to that of Bent and Hayon on the effect of oxygen on the radical yield was in some cases rather ambiguous in that the possible contribution by hydrated electron to the absorbance at 510 nm, the absorption maximum of the neutral radical,was overlooked. Removal of ea; in the presence of oxygen could thus have accounted for the drop in 510nm absorbance observed by Paikhorpe et uI. (1973). As will be described, we have attempted to overcome this source of confusion. The observation of Pailthorpe et al. that the overall destruction yield increased in the presence of oxygen indicated that the triplet may be involved in the photochemical change but not necessarily as the electron ejecting species. The fate of the electron and radical after their formation is also uncertain. In oxygen free solutions the electron has been reported to undergo a back reaction with radical (Grossweiner and Usui, 1971) and elsewhere to decay by second order reaction with itself (Pailthorpe er al., 1973). Other data suggest that the electron should predominantly react with unphotolysed parent molecule (Braams, 1966; Finnstromm, 1971). Attempts have been made to correlate the quantum yield for permanent destruction with the quantum yields for radical and electron production (Pailthorpe et al., 1973). Such correlations require answers to the questions raised above. Grossweiner and Usui (1971) have sought to explain the low permanent photolysis yields in oxygen free solutions on the basis of a back reaction of electrons and radicals. An increased permanent photolysis yield found in the presence of oxygen could then in part result from interference with this back reaction due to e& scavenging by oxygen. It should, however, be pointed
INTRODUCTION
The photochemistry of tryptophan (trp) has been the subject of a number of investigations. The interest in trp arises because, as a photochemically labile amino acid, it is thought to play a crucial role in the light sensitivity of a number of peptides and proteins. Previous studies have utilized steady photolysis and EPR at low temperatures (Guermonprez et ul., 1967; Santus et a!., 1968, 1970, 1972; Vladimirov et d., 1968, 1970; Hklkne et ul., 1968; Pailthorpe et ul., 1972),flash photolysis (Grossweiner et al., 1963, 1971; Santus and Grossweiner, 1972; Joschek and Grossweher, 1966; Finnstromm, 1971; Subramanyan and Tollin, 1972; Pailthorpe et ul., 1973), and most recently, laser flash photolysis (Bent and Hayon, 1975). Despite the wide attention it has received, a number of aspects of the behaviour of trp remain unclear. There is general agreement that absorption of light in the 280 nm band initially leads to the ejection of an electron from the indole ring and the formation of a radical cation. In aqueous solutions at pH greater than zero this cation is thought to lose the proton from the indole nitrogen to form the neutral radical (Santus and Grossweiner, 1972; Bent and Hayon, 1975). Overall, the radical, like the trp molecule, may still have a net positive or negative charge according to the state of ionization of the amino and carboxyl groups. For pH between 4 and 9 the predominant species will be that of zero net charge. In low temperature glasses there is some evidence that the electron ejection is a consecutive biphotonic process with the probability of the triplet state acting as an intermediate (Santus et al., 1968, 1970; Vladimirov and Fesenko, 1968).The triplet has been detected by EPR in such glasses. A recent laser flash photolysis study by Bent and Hayon (1975), which appeared while the present work was being prepared for publication, has indicated that in room temperature solution the *To whom correspondence should be directed. 19
80
H. TEMPLER and P. J. THISTLETHWAITE
out that the permanent destruction yield observed by Pailthorpe et al. in the presence of oxygen was several times the radical yield observed in oxygen free solutions. The kinetic behaviour of the radical is also unclear. In one paper it was reported that in deoxygenated solutions the radical disappeared by three consecutive first order reactions (Grossweiner and Usui, 1971). Elsewhere (Santus and Grossweiner, 1972; Subramanyan and Tollin, 1972), the decay is said to be second order but the /+ values of different reports differ widely. In air saturated solutions, Pailthorpe et a/. (1973) mentioned a second order reaction between radical and oxygen but gave no details. In view of the relatively high concentration of dissolved oxygen in comparison to radical, a pseudo first order radical disappearance might have been expected. In the light of these and other difficulties, we have made a further investigation of the flash photolysis of trp. We have also been concerned with the possible differences in behaviour that might arise when trp is incorporated into simple peptides. Some flash photolysis studies of proteins have already been made but these have been on rather large molecules, such as lysozyme (Grossweiner and Usui, 1971; Subramanyan and Tollin, 1972; Kaluskar and Grossweiner, 1974). There has been little work on simple peptides until the recent study of Bent and Hayon (1975). In a steady photolysis study (R. B. Johns and P. Scheelings, personal communication) differences have been found in the product distribution for the photolysis of alanyl trp and tryptophyl alanine. We have thus been interested to investigate these molecules by flash photolysis with a view to comparing the yields of radical in the three cases. MATERIALS AND METHODS
The reagents L-tryptophan (trp), L-alanyl-L-tryptophan and L-tryptophyl-L-alanine were from Sigma and were used without further purification. All glassware was steam cleaned for 4 h before use. Solutions were made up with triple-distilled water. Solutions were deoxygenated by bubbling for more than 2 h with oxygen free N2 or Ar. In most cases no extra purification of Nz or Ar was attempted as they were sold as oxygen free. However, in some cases the gases were further purified by first passing them through a dry ice cold trap to condense traces of organic matter, and then through two Dreschel bottles each containing vanadous ion solution over amalgamated zinc. Two different flash apparatuses were used; one for the determination of transient spectra and the other for the study of kinetic behaviour. A double flash method was used to obtain transient spectra. The photolysis Aash was of 30 p s duration (at half-height) from an Ar-air filled flash lamp operated with a 3500 J input (120 pF, 7.6 kV).'The spectra were recorded on llford HP3 plates using a Hilger El spectrograph. The capillary spectroflash was fired at the maximum of the photolysis flash. Photographic plates were scanned with a Joyce-Loebl double beam recording microdensitometer and the photographic densities converted to absorbances using an emulsion calibration made on the same plate using the spectroflash lamp. The transient spectra were obtained by subtraction of the photographic densities of the short delay spectrum and a very long delay spectrum. To obtain suitable plate densities it
was necessary to use eight flashes and these wcrc applied to the one solution. As each flash causes appreciable permanent decomposition it is not possible to use such spectra to obtain quantum yields. However, the use of the abovementioned subtraction procedure eliminates the contri bution to the spectra by parent compound or permanent photolysis product. Thus the transient absorptions at different Wavelengths are relatively correct nnd can be used to obtain molar absorptivities provided only that thc spectrum is taken sufficiently soon after the photolysis flash so that different kinetic behaviours of the different species will have a negligible effect. All photolyses were done in a 27 cm long, doubled walled, silica cell, the outer jacket of which could be filled with a suitable filter solution. Kinetic data were obtained on a second apparatus. The photolysis flash was of 5 p s duration. It was produced by the discharge of 135 J (4.5 pF, 7.8 kV) through a 30 cm long ILC Xe flash lamp. The transient absorption was monitored using a Hanovia XBO 450 high pressure Xe source, a Bausch & Lomb monochromator and a Hamamdtsu R446 (red sensitive) PM tube feeding into a Tektronix 502A CRO. Decay curves were photographed with a Polaroid camera. Particular attention was given to the problem of minimising stray light by the use of baffles and suitable filter solutions in the photolysis cell jacket. A glass filter was used between the monitoring source and the absorption cell to prevent any decomposition of the cell contents by short wavelengths in the monitoring beam prior to flashing. When monitoring ea; at 670nm a red filter was used between the absorption cell and the monochromator to remove the possibility of second order light passing through the monochromator. Second order light may account for some results previously reported on L,,; decay. The linearity of the photomultiplier over the transient absorption range likely to be covered was checked using solutions of known absorbance.
RESULTS AND DISCUSSION
Radical spectra and uhsorptivities The transient spectrum obtained on flashing a 160 pA4, deoxygenated, N,-saturated solution of trp at pH 11 was similar to that reported earlier (Grossweiner and Usui, 1971) with a maximum at 510nm attributable to the neutral tryptophyl radical and an absorption at the red end of the spectrum due to ea;. The cell jacket was filled with glacial acetic acid in order to remove the possibility of ea; generation by photolysis of carboxyl groups. To determine the molar absorptivity of the radical the assumption is made that the absorbance above 630 nm is essentially due entirely to ea;. The ea; absorbance at 510 nm can then be determined from its published spectrum (Fielden and Hart, 1967). The absorbance of the radical at 510 nm is obtained by subtracting the ea; absorbance from the total absorbance. By comparison of the absorbance of radical at 510nm and that of e.+; at 630 nm and knowing the molar absorptivity of ed; at 630 nm (Fielden and Hart, 1967) the molar absorptivity of the radical at 510nm is found to be 8.8 1.0 x lo3 M - l cm-'. This procedure assumes that at the time of the spectroflash the radical and ea; are present in equal concentrations. By the same procedure the absorptivities of the radicals from alanyl trp and tryptophyl alanine were found to be
Flash photolysis of tryptophan
81
time region between 200 and 9 0 0 p , i.e. up to apspectively. The result for tryptophyl radical is in quite proximately two half-lives, the decay follows skcond good agreement with the earlier determination of order kinetics with the li,k being approximately Grossweiner and Usui (1971). However, Redpath et 4.6 x lo5 cm s-'. The k/e values found for a number ul. (1975) have recently re-determined the absorptivity of deoxygenated, N2- or Ar-saturated solutions of the neutral tryptophyl radical to be 1.75 x lo3 ranged from 3.5 x 10' to 5.7 x 105cm S C ' , with M--' cm-'. They claimed that errors can occur in there being a trend towards the rate constant being the two flash method if the electron has decayed sig- higher when the radical yield was lower. This would nificantly more than the radical at the time of record- suggest that a second minor reaction is contributing ing the spectrum. An alternative procedure is to use to the decay in some cases. We have been unable kinetic data to determine the extrapolated absor- to confirm this. In a number of subsequent experbances of ea; and radical at zero time and thus deter- iments covering a greater time range we were unable mine the absorptivity. There are two difficulties with to detect any deviation from second ordcr kinetics. this procedure in our case. The conditions for best In several experiments with deoxygenated, N20- or extrapolation of radical absorbance are those of high Ar-saturated solutions, a value of approximately flash energy and large radical-electron yield. As will 3.5 x lo5 cm s f l was found. be explained later, the decay of the e& under these At times less than 150ps, there was in all cases conditions is very rapid and the kinetics complex. a negative deviation from the second order kinetics This being so it is difficult to extrapolate the e, yield holding beyond 200 ps. This we attribute to an initial back to zero time. A further difficulty is that under rapid removal of radical by back reaction with elecconditions of high ea; yield the lifetime of the ea; tron. To confirm this conclusion we have performed decay is short and the flash duration becomes signifi- experiments.with deoxygenated, N,O-saturated solucant in comparison. In this case extrapolation back tions. N 2 0 should scavenge e,; without any effect on to zero time is likely to be inaccurate. Extrapolation the radical. In N,O-saturated cases we find that the of the radical yield is more reliable due to its much decay remains second order down to quite short slower decay. In this case the flash may be regarded times, the rate constant being approximately the same as instantmeous. I t is for this reason that we have as found in the previous cases. The yield at zero times generally preferred to extrapolate radical rather than was also found to be approximately the same in both ea; absorbance. cases. This observation argues against an alternative explanation for the more rapid decay of absorbance Kinetic behuviour of the tryptophyt radicul in deoxy- seen below 1 5 0 ~ sThis . might have been attributed genated and air-saturated solutions to a contribution by eai to the 510nm absorbance. Particular attention was given to the decay kinetics The more rapid decay of ea; would then cause a rapid of the radical produced by flashing trp. Decays were decay of part of the 510nm absorbance. If this were monitored at 510 nm using the kinetic apparatus de- the case, then for an N2-saturated solution the absorscribed earlier. A filter solution consisting of an eth- bance should extrapolate back to a higher value than anol- HCI solution of CoCI, and NiCI, (Vladimirov, would be the case with N 2 0 present. In separate ex1966) was used in the outer jacket. This filter is highly periments to be described later it was shown that the transmitting in the region 260 to 390nm and fairly ea; absorbance at 670 nm has mostly disappeared by opaque (transmission generally less than 20%) else- 5 0 p s from the time of the flash. Thus the plotted where. The purpose of this filter was to minimise the points of Fig. 1 should at most contain a small contrieffect of stray light. In fact, stray light was found to bution from edg absorption. Our observations may be insignificant after 15ps at all wavelengths moni- be contrasted with those of Grossweiner and Usui tored. The filter's transmission properties were found to change after exposure to light and with age. In 24r some cases where it was desired to compare radical yields, special precautions were taken to ensure that the filter's properties did not change during the course of a series of experiments. Thus, in general, the results presented in any one figure apply for a given filter solution. However, the radical yields cannot be compared from one figure to another due to changes in filter transmission in the photolysis region. Fig. 1 shows the decay of tryptophyl radical plotted as a second order process in deoxygenated and deoxyI I I I I 200 400 600 800 genated, N20-saturated solutions. In both cases the t /ps trp concentration was M and the pH was 11. The plot for the deoxygenated case is typical of a Figure 1. Second ordcr decay of tryptophyl radical in (a) large number of decays observed for different trp con- deoxygcnated and (b) deoxygcnated, N,O-saturated centrations and flash energies. In all cases, for the solution. 7.5 & 1.0 x 10' and 9.3 & 1.0 x lo3 M - ' cm-' , re-
,
H. TEMPLER and P.J. THISTLETHWAITE
82
(1971) who reported that the tryptophyl radical underwent three consecutive first order decays. They too found an initial rapid decay of absorbance followed by B slower decay and then a still slower decay at times greater than 1 ms. Grossweiner and Usui also studied the decay of 510 nm tryptophyl absorption in lysozyme and attributed the intermediate time first order decay to a pseudo first order reaction of tryptophyl radical groups with hydroxide ion. However, an unsatisfactory aspect of their interpretation was the fact that the observed first order rate constant varied only slowly with pH. In further experiments using a slower time base we have monitored the radical decay out to approximately 2ms (more than 3 halflives) without observing any change in kinetics. Our conclusion is that, in oxygen free solutions, except for the initial back reaction with e&, radicals are removed by second order reaction with themselves. M trp solution at On flashing a deoxygenated pH 6.3, we again observed second order decay in the region 200 to lOOOps, this time with a kjc value of 8.4 x lo5 cm s-'. We consider the change in rate constant to be most likely due to the switch from the net negative charged radical existing at a pH of eleven to the zwitterion form occurring at pH 6.3. The yield of the radical was also found to be reduced in line with the results of Bent and Hayon (1975). In air-saturated solutions, the radical decay kinetics become complex although the initial steep section is no longer present as would be expected in view of the very efficient scavenging of e& by oxygen. Fig. 2 shows a decay typical of those found in several experiments with different flash energies. The results have been plotted both as first and second order proM and cesses. Again the trp concentration was the pH was 11. If a reaction occurs between radicals and oxygen then this would be expected to be pseudo
first order, as the dissolved oxygen concentration (approximately 3 x 10-4M) is very much greater than that of radical. If such a reaction was in competition with the second order radical radical reaction, complex kinetics would result. The differential rate law for such a situation is
k [ R I 2 + k'[R] dt where k and k' are the second order and pseudo first order rate constants, respectively. On integration, we obtain [R] = k / ( K exp k'l - k) _ drR1 _-
where K = (k' + k[R],,)/[R],,([R10= radical concentration at t = 0). A non-linear least squares computer program was used to fit the observed [ R ] vs time data, for a number of experiments with different flash energies, to the above function. In all cases the data could be well fitted (computed and experimental points within 5'%,, standard errors in k and k' of approximately 4%). The value of k required for best fit lay in the range 4.9-5.3 x lo9 M - ' s- (k,k in range 5.5-6.0 x lo5cm s-'). The values of k' required for a fit lay in the range 1.2--1.8 x 103s-'. The values of the second order constant, it will be noted, lie at the upper end of the range found earlier for deoxygenated solutions. This is in line with the earlier statement that the apparent second order rate constants seem slightly dependent on radical yield. Attempts were made to verify the pseudo first order reaction with oxygen by doing runs with solutions equilibrated with different pressures of air. Lowering the pressure of air did in fact lower the pseudo first order constant whilc leaving the second order constant unchanged. For an air pressure of 33 cm Hg, a pseudo-first-order constant of 1.0 x 103s-' was found. Taking this value and combining it with the solubility of oxygen in solution equilibrated with 33 cm Hg of air, we obtain a value of X.2 x lo6 M ' s-' for thc second order rate constant for the reaction between radical and oxygen. Eflkct of oxygen on radicul yield
t,
ps
Figure 2. Decay of tryptophyl radical in air equilibrated solution: filled circles, plotted as second order process; open circles, plottcd as first order process.
To determine the effect of oxygen on the initial radical yield an air-saturated and a carefully deoxygenated solution were flashed, taking particular care that the filter solution was precisely the same in both cases. The tryptophan concentration was lo-" M and the pH was 11. Fig. 3 shows that a reduction of more than 50% in initial yield occurs in air-saturated solutions compared to oxygen free solutions. In the deoxygenated case, the extrapolation back to zero time might be criticised on the ground that until all electron has been removed the absorbances at 510 nm contain a contribution from ea; as well as from radical. However, as the points used in the extrapolation are for times longer than 5 0 p , by which time most of the electron has disappeared, the error will be small and the basic conclusion that the initial radical yield is reduced by oxygen remains valid. The reduction
Flash photolysis of tryptophan
83
by the filter for various flash energies were determined by ferrioxalate actinometry. Fig. 4 shows a plot of extrapolated initial radical absorbance versus the relative number of photons in the flash for air-saturated, 10-4M trp solutions at pH 11. In fact a slight fall in radical quantum yield seems to occur at higher flash energies. In an earlier study, Pailthorpe er ul. (1973) found no variation in permanent destruction quantum yield for an air-saturated solution, as the flash energy was varied. However, if most of the permanent destruction observed by them occurred by a reaction between oxygen and triplet formed independently of the formation of the radical, then their experiment would not be expected to reveal any biphotonic pathway to radical formation. It should be recalled that Pailthorpe et al. observed a permanent 200 400 600 800 destruction quantum yield in the presence of oxygen several times higher than the radical yield observed t , /Ls in oxygen free solutions. Although our results indicatc Figure 3. Effect of oxygen on the yield of tryptophyl radino biphotonic photoionization in our case, they do cal : (a) deoxygenated solution (b) air equilibrated solution. not rule out the possibility of some contribution by a biphotonic pathway if a flash containing the tripletin radical yield observed here contradicts the earlier triplet absorption wavelength were used. Our results observations of Grossweher and Usui (1971) and should be contrasted with some recent results of Bent and Hayon (1975) and suggests some involve- Kaluskar and Grossweiner (1974) which they ment of the triplet state in our case. One possibility explained on the basis of a biphotonic photoionizafor reconciling the discrepancy is to assume that at tion of trp, via an intermediate triplet state, becoming short times the 510 nm absorbance contains a contri- significant at higher flash energies. bution from the triplet state. As reported by Bent Our results point to the triplet state as a precursor and Hayon, the absorptivity of this species at 510 nm of the radical but not as the absorber of a second is not negligible. As the triplet is strongly quenched photon. The partial quenching of the radical yield by oxygen this could account for an apparent drop in the presence of oxygen could mean either that in radical yield in the presence of oxygen. However, photoionization from the triplet state competes with there are difficulties with this explanation. The triplet quenching of the triplet by oxygen, or that photoionistate lifetime in oxygen free solutions is reported zation is occurring from both the singlet and the trip(Bent and Hayon, 1975) to be approximately 2 0 p , let and that the partial quenching in the presence of while the plotted points of Fig. 3 are all for times oxygen reflects the blocking of the triplet pathway. greater than 5 0 p s by which time the triplet should If the latter was the case, then there would be no have disappeared. Secondly, if the triplet state was possibility of observing a biphotonic process involvmaking a contribution to the 510 nm absorbance the ing the triplet when oxygen was present. For this rearapid decay of this absorbance could account for the son an experiment similar to the one depicted m Fig. initial steep section of the plot of Fig. la. However, 4 was done with deoxygenated solutions. The result if thk were so then the N 2 0 should not be effective was similar to before. in removing this initial steep section. Our results point to ionization being possible from Our observation that oxygen reduces the radical more than one excited state, one of which is sensitive yield, suggesting an involvement of the triplet state, 0.31 raises the question of whether the triplet state is involved as the absorber of a second photon, as is known to be the case in low temperature glasses. We have found no evidence that biphotonic processes are playing any appreciable part in the radical production we observe. A biphotonic process involving the absorption of a second photon by the triplet state would, a priori, seem unlikely in our case, due to our use of a flash filter with a low transmission at 450 nm, the wavelength reported by Bent and Hayon (1975) for the triplet-triplet absorption. If any consecutive biphotonic process were involved, the quantum yield No. photons, orb u n i t s of radicals would be expected to rise at higher flash Figure 4. Initial radical absorbance versus the relative energies. The relative numbers of photons transmitted number of photons in the photolysis Ilash.
H TEMPLER and P. J. TIII$II
84
I I IWAI
I
cal production is low, The pseudo first order reaction is also favoured by higher parent concentrations. Both the above sets of experiments involved initial electron-radical concentrations of approximately 6 x 10- ' M , this being dependent mainly on the flash energy rather than the parent concentration. In the case of the series in the 10-5-10-4 M concentration range, there is sufficient second order electron-radical reaction to increase the apparent rate constant of the Kinetic hehaviour of the hydrated electron pseudo-first-order reaction without producing plots A puzzling feature of earlier studies has been the with a very noticeable deviation from first order kindisagreement on the fate of e&. As mentioned earlier, etics. To check these ideas, further experiments were at least three possibilities have been reported. The done with a high transmission filter which gave a results to be described have to be seen in the light much higher electron-radical yield. In these cases the of our earlier conclusion that at least some of the ea&decay was considerably faster and the kinetics now electron undergoes a back reaction with tryptophyl showed appreciable deviation from first order. The radical. We have performed two series of experiments. ea&+ e& + H, + 2 OH- reaction could also be In the first instance, a series of deoxygenated trp solu- expected to make a contribution to a deviation from tions of pH 11, ranging in concentration from M first order kinetics. A rough calculation serves to to 7 x M, was flashed and the eLq decays moni- further emphasize the difference between the situation tored. These experiments were done with a filter solu- depicted in Fig. 1 and that in the experiments where tion somewhat less transmitting in the photolysis strictly first order decay of ea; is observed. In Fig. region than that used for the experiments depicted 1, the initial yield of e; and radical in the oxygen in Fig. 1. In each case the decay appeared to be free case is approximately 1.5 x lO-'PJ, while the M. In one of the strictly first order, and the rate constant varied syste- parent trp concentration was matically with trp concentration, indicating a pseudo experiments of Fig. 4, the initial yields were approxifirst order reaction between ea; and trp. A plot of mately 6 x lO-'M and the trp concentration pseudo first order constant versus trp concentration 3 x 10-4M. Thus in terms of initial rates, there is led to a straight line passing through the origin and a change in the ratio of first to second order reaction the slope gave a figure of 1.2 x 1 0 8 M - ' s - ' for by a factor of approximately 19 in going from one the second order rate constant (Fig. 5). This result case to the other. agrees with the pulse radiolysis work of Braams (1966) in which eai was generated quite independently of the trp. In a second series of experiments in the Relative radical yields for tryptophan, uhnyl tryptolo-' M-8 x M trp concentration range, appar- phan and tryptophyl alanine at naturul p H ently pseudo first order decay was again found. This To compare the yields of radicals, deoxygenated, time a plot of pseudo first order constant versus trp M solutions of the three compounds, at pH 6.3, concentration yielded a second order constant of were flashed with exactly the same flash energy. The 4 x lo8 M - ' s- Our conclusion is that in general decay curves for the radicals from all three species e& undergoes at least two reactions simultaneously. were found to be of approximately the same form These are a second order reaction with tryptophyl as found previously for trp in the high pH region. radicals and a pseudo first order reaction with un- There were, however, some differences in the second photolysed trp. These can occur in competition, or order rate constants from those found earlier for trypin some circumstances, the pseudo first order reaction tophyl radical. The initial yield of radical from trp may dominate the kinetics. This will be the case at was somewhat reduced (approximately 25%) in comlow flash energies where the initial electron and radi- parison to the yield at pH 11. This observation is in agreement with the earlier work of Pailthorpe el al. (1973) and with the recent work of Bent and Hayon (1975). The initial absorbance found for the radical from tryptophyl alanine was somewhat lower, and that for the case of alanyl trp was much lower again. Using the molar absorptivities previously found, the relative yields were calculated to be 1.0, 0.54 and 0.27, for trp, tryptophyl alanine and alanyl tryptophan respectively. Similar results have been reported by Bent and Hayon (1975) who found that the photoionization yields of glycyi and tyrosyl pep( [ t r v l /M ) x lo4 tides of trp were lower than trp itself. In an earlier steady photolysis study (Johns and Figure 5. Variation of pseudo-first-order rate constant for e,Yu decay with tryptophan concentration. Scheelings, private communication), the quantum
to oxygen. The excited singlet state is known to be unaffected by oxygen at the concentration found in atmosphere equilibrated solutions (Lakowicz and Weber, 1973). However, in the light of the results of Bent and Hayon (1975) obtained with apparatus better able to determine concentrations at very short times, our conclusions on the effect of oxygen must be subject to some doubt.
'.
Flash photolysis of tryptophan
yields for permanent destruction and NH3 production, respectively, were: trp 0.01 5, 0.005; alanyl tryptophan 0.036, 0.030; and tryptophyl alanine 0.017, 0.014. These figures provide an interesting comparison with our own data. The order of permanent destruction quantum yields is the reverse of our order of radical yields. Both tryptophyl alanine and alanyl tryptophan show higher quantum yields for NH3 production than does trp. The high quantum yield for NH3 may be connected with the reduced radical yield we observe for alanyl trp. A rapid rearrangement of the molecule at the time of, or just after, the ejection of the electron could bypass the formation of the radical. If this is so, it appears that the structure of the alanyl tryptophan is particularly favourable to such a rearrangement. A check was made on the eai yield
85
in the alanyl tryptophan case and this was found to be correspondingly reduced. Very rapid electron migrations are thought to occur in both lysozyme and trypsin where the formation of the disulphide bridge electron adduct has been shown not to occur by trapping of ea; from solution. The electron apparently makes an intramolecular migration to the cystine (Kaluskar and Grossweiner, 1974; Grossweiner and Usui, 1971; Adams et d.,1969). The present results emphasize the importance of avoiding the assumption of constant photoionization quantum yield for trp regardless of the environment. They indicate that significant differences can occur, even in quite simple compounds. Further work at higher time resolution is indicated in order to elucidate the reasons for the observed differences.
REFERENCES
Adams, G. E., R. L. Willson, J. E. Aldrich and R. B. Cundall (1969) Intern. J . Rudiution B i d . 16, 33 3-342. Bent, D. V. and E. Hayon (1975) J . Am. Chem. Soc. 97, 2612-2619. Braams, R . (1966) Radiation Res. 27, 319--329. Fielden, E. M. and E. J. Hart (1967) Trans. Faraday Soc. 63, 2975-2982. Finnstrom, B. (1971) Photochem. Photohiol. 13, 375-377. Grossweiner, L. I., G. Swenson and E. Zwicker (1963) Science 141, 805-806. Grossweiner, L. 1. and Y. Usui (1971) Photocheni. Photobiol. 13, 195-214. Guermonprez, R., C. Helene and M. Ptak (1967) J . Chim. Phys. 64, 13761384. Helene, C., M. Ptak and R. Santus (1968) J . Chim. Phys. 65, 160-166. Joschek. H. I. and L. 1. Grossweiner (1966) J . Am. Chem. Soc. 88. 3261-3268. Kaluskar, A. G. and L. I. Grossweiner (1974) Photochem. Photobiol. 20, 329-338. Lakowicz, J. R. and G. Weber (1973) Biochemistry 12, 41614170. Pailthorpe, M. T. and C . H. Nicholls (1972) Photochem. Phofohiol. 15, 465-477. Pailthorpe, M. T., J. P. Bonjour and C. H. Nicholls (1973) Photochem. Photobiol. 17, 209- 223. Redpath, J. L.. R. Santus, J. Ovadia and L. I. Grossweiner (1975) Inttrn. J . Radiation Biol. 27, 201-204. Santus, R. and L. I. Grossweiner (1972) Photochem. Photobiol. 15, 101-105. Santus, R., C. Helene and M. Ptak (1968) Photochem..f!&qFiol. 7, 341-360. Santus, R., A. Helene, C. HClene and M. Ptak (1970) J . Phys. a e m . 74, 550-561. Santus, R., M. Bazin, M. Aubailly and R. Guermonprez (1972) Photochi.m:"Photobiol. 15, 61 69. Subramanyan, V. and G. Tollin (1972) Photochrm. Photobiol. 15, 449456. Vladimirov, Y. (1966) Photochem. Photobiol. 5, 243-250. Vladimirov, Y. and E. Fesenko (1968) Photochem. Photobiol. 8, 209-212. Viadimirov, Y., D. I. Roschupkin and E. Fesenko (1970) Photochent. Photubiol. 11, 227-246.
