J. Phys. Chem. lQ83, 87, 1559-1565
the phenyl radical, and the methyl radical. The observation that the phenyl radical does not react with COz when excited into its %, state precludes the formation of methyl benzoate via this route. In essence, the experiments only support formation of methyl benzoate by combination of the longer lived benzoyloxy radical with the methyl radical. Furthermore, since phenyl acetate could not be detected in the matrix experiments, it was concluded that the acetoxy radical must have a much shorter lifetime. Ab initio calculations were reported on the benzoyloxy radical in order to provide an explanation for the much shorter lifetime of the acetoxy radical vs. the benzoyloxy system. The calculations indicate that the facile decar-
1559
boxylation for both systems, and acyloxy radicals in general, is due to a curve crossing very close to the ground state of the acyloxy radical. The curve crossing moves closer to the minimum on the ground state as the AH for the decarboxylation becomes more exothermic. The shorter lifetime for the acetoxy radical is attributed to a more negative AH for loss of C 0 2 and to its lower symmetry because, in this case, the barrier for decarboxylation is lowered by an avoided crossing between two surfaces with the same symmetry. Registry No. Acetyl benzoyl peroxide, 644-31-5; benzoyloxy, 1854-28-0; phenyl, 2396-01-2.
Photophysics of Tryptophan in H20, D20, and in Nonaqueous Solvents'
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Eva Gudgln, Ricardo Lopez-Deigado,* and William R. Ware' Photochemistry Unit, Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 587 (Received: July 2, 1982; I n Final Form: December 6, 1982)
The fluorescence properties of tryptophan in water and deuterated water have been examined. Tryptophan molecules exhibit three distinct fluorescence lifetimes in water which become longer in deuterated water; the two shorter lifetimes are present below the pK of the amino group and the long lifetime appears as the pH is raised through this pK. The steady-state quenching of tryptophan fluorescence by hydrogen ion in the region of pH less than 3 shows a definite wavelength effect, consistent with less-pronounced quenching of the subnanosecond component whose emission maximum is at 330 nm. The Stern-Volmer plots show a marked curvature in the direction of decreasing Stern-Volmer constant as [H30+]increases. Deuterium ion also quenches tryptophan fluorescence at low pD. A kinetic scheme is proposed which reproduces both the steady-state and lifetime quenching results. Tryptophan in methanol or ethanol exhibits three fluorescence lifetimes; the relative percentage of the long component vs. the intermediate component can be varied by the additon of triethylamine or acid. In dimethyl sulfoxide, tryptophan and tryptophan deuterated at the amino and ring nitrogen positions show identical behavior, both having the same decay parameters. These results are discussed in light of the theories which have recently been proposed to account for the several components in tryptophan fluorescence decay. Solvent interaction is suggested to play a critical role.
Introduction The fluorescence properties of tryptophan and its derivatives in solution have been a subject of interest for a number of years, and they have been used to attempt to explain the fluorescenceof tryptophan residues in proteins. Recently there has been particular interest in the time dependence of the fluorescence of tryptophan since it has been established that the molecule does not follow a single exponential decay law at neutral pH.3-5 Rather, it fluoresces with two lifetimes, of about 0.6 and 3.2 ns. Further, when the anionic form is present a t higher pH a third lifetime of about 9.2 ns is o b ~ e r v e d .These ~ lifetimes are pH independent in the range of pH 4-10.5 where the concentrations of H30+ and OH- are kinetically insignificant. Some of the reasons which have been proposed for tryptophan's complex decay behavior are the existence of two uncoupled, close-lyingexcited states: the existence ~
(1)Publication No. 294 from the Photochemistry Unit, Department of Chemistry, The University of Western Ontario, London, Canada. (2) Permanent address: CNRS, Laboratoire de Photophysique MolBculaire, Bltiment 213,UniversiG de Paris Sud, 91405,Orsay, France. (3)E.Gudgin, R. Lopez-Delgado, and W. R. Ware, Can. J.Chem., 59, 1037 (1981). (4)A.G. Szabo and D. M. Rayner, J. Am. Chem. Soc., 102,554(1980). (5)R. J. Robbins, G. R. Fleming, G. S. Beddard, G. W. Robinson, P. J. Thistlewaite, and G. J. Woolfe, J . Am. Chem. Soc., 102,6271 (1980). 0022-365418312087-1559$01.50/0
of different ground-state conformations which are not rapidly interconvertible in the excited state," and a proton transfer from the side chain to the ring which would result in a diffusional transient component in the fluorescence decay.5 The presence of two pK's in the molecule, that of the carboxylate and the amino groups, is also a complicating factor, as there are thus three possible groundstate species, the cation, the zwitterion, and the anion, the relative concentrations of which depend on the conditions of the experiment. In this paper, we describe several aspects of the fluorescence behavior of tryptophan in an attempt to determine the extent of solvent involvement: its fluorescence in nonaqueous solvents, deuterated solvents, and its fluorescence in water and deuterated water at low pH. An attempt has been made to show that solvent interaction is an important parameter in the photophysics of tryptophan and perhaps is a critical factor in the determination of its excited-state behavior.
Experimental Section L-Tryptophan (Trp) (Aldrich, 99%) was recrystallized once from an alcohol-water solution. Deuterated L-tryp(6) D. M. Rayner and A. G. Szabo, Can. J . Chem., 56, 743 (1978).
0 1983 American Chemical Society
1560
TABLE I: Effect of Solvent on Tryptophan Lifetimes (A,, solvent MeOH EtOH (95%) EtOH (abs) MelSO
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Gudgin et ai.
The Journal of Physical Chemistry, Vol. 87, No. 9, 1983
7 , Ins
0.2 f 0.1 0.2 f 0.1 0.2 f 0.1 2.6
f
0.4
= 280
I, 0.05
rl/ns 1.90 f 0.05
0.07
2.46 f 0.1 2.0 0.1 8.0 t 0.06
0.05 0.06
*
tophan (Trp-d,) was prepared by recrystallizing L-tryptophan three times from deuterated water (Merck Sharpe and Dohme, Canada, 99.7 atom % D). A large majority of the Trp molecules were deuterated, as was confirmed by an infrared spectrum of a Nujol mull, taken on a Beckman IR4250 spectrophotometer. Aqueous solutions were prepared from triply distilled water, and the pH was altered by the addition of HC1 or NaOH. The pD of deuterated water solutions was altered by the addition of deuterium chloride (99 atom % D, 35% (w/w) in D20)or sodium hydroxide-d (98 atom % D, 40% (w/w) in D20) obtained from Merck Sharp and Dohme, Canada. The pH was measured on a Fisher Accumet Model 600 pH meter. For D20 solutions, the pH was calculated with the formula pD = pHo + 0.40 where pHo is the observed reading on the pH meter.' Nonfluorescent methanol and 95% ethanol were prepared by distillation from a solution with triply distilled water and Triton X-100 detergent (Fisher) which removed fluorescent impurities at all wavelengths examined. Dimethyl sulfoxide (Me2S0, Fisher, spectranalyzed) contained 0.05 mol/L of water, measured with a Karl Fischer water titrator (Photovolt Aquatest IV). In order to examine the effect of this trace amount of water on tryptophan fluorescence in Me2S0, we prepared some Me2S0 in which this trace amount consisted of deuterated water. This was done by adding 0.5 mol/L of deuterated water to the M e a 0 and vacuum distilling at 6 mmHg pressure, from dried alumina, into molecular sieves. The last fraction of Me2S0 collected contained 0.02 mol/L deuterated water. Me2S0 samples containing trace amounts of water and deuterated water are referred to as Me2SO(H20)and Me2SO(D20),respectively. Solutions of Trp and Trp-d, were prepared in both Me2SO(H20)and Me2SO(D20)by using syringes for transferring of solvent and for nitrogen bubbling in the cuvette, in order to prevent any addition of atmospheric water to the samples. The water content of solutions was checked after lifetime measurements were completed and did not increase appreciably from the solvent water content value. The very weak solvent fluorescence was subtracted during lifetime measurements. Oxygen-freesolutions were prepared by bubbling pure N2gas through the sample cuvette for 20 min immediately before the measurement. All lifetime measurements were performed on oxygen-free solutions except for H 2 0 and D20at low pH where oxygen removal had no effect on the results. AU measurements were performed at 20 OC on IO4 M solutions. Absorption spectra were recorded on a Cary 219 spectrophotometer;fluorescence spectra were recorded on a Perkin-Elmer MPF-4 spectrofluorometer. Fluorescence lifetime measurements were performed on a PRA3000 single-proton counting nanosecond spectrofluorimeter which has been previously de~cribed.~ For the analysis of the fluorescence decay curves, we have used a procedure, also previously described? the experimental decay I ( t ) is divided into time windows; each portion of the decay, limited by the time window edges, is analyzed separately (7) R. Lumry, E.L. Smith, and R. R. Glantz, J. Am. Chem. SOC.,73, 4334 (1951).
nm) 12 0.85 0.83 0.79 0.94
?,Ins 7.0
I3
0.2
0.10
8.4 f 0.3 6.9 ?: 0.4
0.10
f
0.16
remarks A,, -...> 305 nm A,, = 330 nm A,, = 330 nm A,, = 355 nm
by weighted least-squares interative convolution of the instrumental function g(t) with a double-exponential decay function until the best fit, defined by requiring a minimum x2 and randomly distributed residuals, is obtained. Particular attention is paid to both the beginning and end of the time distribution, where short and long decay components, respectively, are likely to be more important. If a double exponential fit is not adequate, the best set of decay parameters obtained are then fed into a triple-exponential interative convolution program. x2 and randomness of residuals of both sets of results are then compared. The intensity of fluorescence of each component of the decay is calculated with the equation AiTj
1, = CAjTj i
where Ai and iiare, respectively, the preexponential factor and lifetime of the ith component. The experiments to observe the steady-state quenching of tryptophan by hydrogen or deuterium ions were performed by preparing individual solutions immediately before measurement of fluorescence; the solutions consisted of 5.0 X M tryptophan with known volumes of standardized HCl or DC1 solutions. All solutions were nonfluorescent in the absence of tryptophan, and there was no scattered light at the observation wavelengths. The fluorescence was observed at different wavelengths with a 4-nmbandwidth, and the excitation wavelength was kept constant at 280 nm (4-nm bandwidth). The absorption spectra were then measured immediately, in order to confirm that solutions had not degraded; solutions left standing showed a significant increase in absorption at 280 nm and a decrease in fluorescence intensity. Computer modeling of steady-state quenching was performed with a Digital MINC-11 computer.
Results We have measured the fluorescence lifetimes of tryptophan in methanol, ethanol, 95% ethanol, and dimethyl sulfoxide (Me2SO). These results are listed in Table I. For the measurements in methanol, either an emission monochromator or cutoff filter (305 nm) were used on the emission side; other measurements were performed with an emission monochromator. Three decay times are observed in methanol, ethanol, and 95% ethanol while only two are observed in Me2S0. Measurements showing the effect of triethylamine and acetic acid on Trp lifetimes in methanol are reported in Table 11. Addition of increasing quantities of triethylamine increases the relative intensity while , the addition of acetic acid removes the conof i3 altogether. tribution from i3 We have examined the fluorescence lifetimes of tryptophan in D20at a variety of pD's. In Table I11 are given the results for pD 7.0 and 9.9. In both cases only two components could be resolved although a total of four decay times were observed, two of which were identical. These values are compared with values in H20and in methanol and methanol-d4in Table 111. As with water, the lifetime values obtained in D20 were independent of pD over the range where [D30+]or [OD-] were kinetically
The Journal of Physlcal Chemistry, Vol. 87, No. 9, 1983
Photophysics of Tryptophan
1561
TABLE 11: Effect of Triethylamine and Acetic Acid on Tryptophan Lifetimes in Methanol (hex, = 280 n m ) solvent MeOH MeOH MeOH MeOH MeOH MeOH a
7, still
t t t t t
r Ins
0.0004 M TEAb 0.0014 M TEA 0.0058 M TEA 0.014 M TEA 0.0007 M HOAcC
1.90 f 1.88 1.79 f 1.84 f 1.94 f 1.91 f
0.05
0.85 0.33 0.23 0.11 0.05 0.95
* 0.1
0.1 0.2 0.2 0.05
I,
r ,Ins
I3
remarks
0.05
7.0 f 0.2 7.46 f 0.05 7.39 f 0.03 7.33 0.03 7.27 0.03
0.10 0.67 0.77 0.89 0.95
hem> 3 0 5 n m hem = 345 nm hem = 345 nm hem = 345 nm hem = 345 nm hem = 345 nm
r ,ins
I1
0.2
0.1
f
a a a a
a
a a
a
0.2
0.1
f
* *
0.05
TEA = triethylamine.
present, but only long time portion of decay analyzed.
HOAc = acetic acid.
TABLE 111: Effect of Deuterated Solvents o n Tryptophan Lifetimes (Aexc = 280 n m ) solvent H,O, pH 7.0 D,O, pD 7.0 H,O, pH 10.0 D,O, pD 9.9 MeOH MeOH-d,
I1
71. ns 3.19 f 0.1 6.05 f 0.1 3.15 f 0.2 6.2 f 0.1 1.90 f 0.05 2.50 f 0.05
I,
7 2 , ns 0.60 f 0.15 1.3 i 0.2
0.94 0.97 0.10 0.48 0.85 0.62
73,ns
I3
9.18 f 0.1 11.5 f 0.1 7.0 f 0.2 8.0 f 0.1
0.89 0.52 0.10 0.35
0.06 0.03
> k,'[H30+] and k6 >> kg. However, if this were true, one would expect a linear or upwardcurving Stern-Volmer plot; the reverse is osberved. In fact, there is residual fluorescence long after the Trp molecule is completely converted to the cation form in the ground state. Thus a more realistic model involves a fluorescent cation and/or an excited-state pK shift which will give some zwitterion fluorescence. We have modeled both the steady-state and lifetime rate equations for the proposed kinetic scheme and searched for the best fit of the computer-simulated curves simultaneously to the fit of the steady-state quenching curves at 410 nm and the lifetime quenching of the 3 4 s component. Calculations were based on eq 1, where [Z,,*] is defined by eq 2, and
The Journal of Physical Chemism, Vol. 87, No. 9, 1983 1563
TABLE VI: Results of Computer Modeling of H,O+ and D,O+Quenching
kl k, k5 Keq
D,O+
H,O+
quencher
Assumed Rate Constants 4 x 107 4 x 107 2.9 X 10' 1.25 X 10' 4 x 107 4 x 107 245 630 Fitted Rate Constants
k,' kc.' k3
GI
x 107
k6
$1 x 10' (2.3 k 0.1)x 10'' (1.5 k 0.1)x 109 ( 2 . 6 k 0.1)x 109
Keg* rc/ns
15 0.24
k4
$1 x 1 0 7 61 x 107 ( 1 . 7 * 0.1)x 1Olo (3.7 ?: 0.1)x 109 ( 1 . 4 i: 0.1) x 109
Resulting Values 46 0.55
Values of K,, (which determines 6, the fraction of light absorbed by the Z form) k,, k2, and k5 were assumed. Values were based on the known pK, radiative lifetimes calculated from the quantum yield of 0.14 for the Z form, and the value of T ~ The . extinction coefficients of the Z and C forms were equal. Since excitation was at the isosbestic point,1° this is a valid assumption. (Also, the absorption spectra were checked for each solution in the quenching experiments.) The best fit to the steady-state quenching data in H 2 0 is illustrated in Figure 3. Some variability in the parameters used to fit the steady-state curve alone was possible. However, with the simultaneous requirement of a fit to lifetime quenching data, a unique best fit was obtained for the rate parameters. The fit to the lifetime quenching data, obtained by using the same rate parameters as were used for fitting the data in Figure 3, is illustrated in Figure 4. Calculated curves, based on different assumed values of K,,, kl, k 2 , and k5, for quenching by D30+are also illustrated in Figures 3 and
4. The value of K,, for the C-Z equilibrium in D 2 0 is based on an assumed value for the pK of 2.8; this value has not been exactly determined but is likely not in error by more than fO.l pK unit. This introduces a small error in the calculation but has little effect on the magnitudes of the rate constants obtained. The values we have assumed for KW,k,,k2, and k5 and the values of k;, k6') k,, k4, KW*,and T~ which result from the computer fitting are summarized in Table VI. The rate constants k2' and kg/ which were introduced into the scheme to allow for some unknown mechanism of quenching by H30+were neither necessary nor desirable in order to obtain a good fit. Thus, in the final analysis we have varied only three rate constants. Tabulated also are the values of the excited-state equilibrium constant KW*= k3/k4, and the cation lifetime at zero hydrogen ion concentration TC = ( k 4 k5 kc)-'. These values of Keq* and TC imply that residual fluorescence at high hydrogen ion concentration is due to both a small amount of cation fluorescence and a shift in the excited state pK. The values of k3 obtained, namely, 2.3 X 1O'O in H 2 0 and 1.7 X 1O'O in D20, are consistent with an approximately diffusion-controlled reaction," and there is a decrease in the rate in D 2 0 as expected. The decay time of the cation is difficult to confirm experimentally owing to difficulties caused by sample decomposition and interference by the T~ component which is of a similar magnitude when quenched in acid.3 We plan to attempt this measurement using a synchronously-pumped, modelocked dye laser as the excitation source, which will decrease the data collection time and increase the time resolution. The prediction of an increase in the dissociation of the
(10)J. W. Donovan in 'Physical Principles and Techniques of Protein Chemistry", Part A, S. J. Leach, Ed., Academic Press, New York, 1969, p 101.
(11)M. Eigen and L. de Maeyer in 'Investigation of Rates and Mechanisms of Reactions", Part 11, S. L. Friess, E. S. Lews, and H. Weissberger, Ed., Wiley-Interscience, New York, 1963,p 1031.
where
+ +
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Gudgin et al.
The Journal of Physical Chemistry, Vol. 87, No. 9, 1983
cation into the zwitterion and hydrogen ion by a factor of about 15 in the excited state is surprising since one would not predict that excitation of a chromophore would influence the dissociation of an isolated substituent. In order to interpret this result, one must postulate that the side chain is not, in fact, truly isolated from the chromophore, something which has been often suggested. The computer modeling of Trp photophysics has not explicitly involved solvent interaction; however, its importance in the excited state of Trp has been previously emphasized by studies indicating that the large red shift in the emission of the indoles in solution with respect to the absorption is due to either solvent reorientation after excitation, a solvent indole exciplex, or emission from a solvated Rydberg state (see ref 4 for a discussion of these proposed explanations). This large value of the dissociation rate constant k4,as well as the large nonradiative rate constant k6,could be attributed to some specific solvent shell configurations adopted in the excited state, differing from the ground state configuration, which would facilitate loss of the carboxylic proton to the solvent and possibly assist proton transfer to the chromophore or facilitate some other mechanism which would increase the nonradiative relaxation rate. In our discussion we have emphasized the importance of solvent interaction to explain the photophysical behavior of tryptophan. We would also like to discuss some of the other mechanisms which have been proposed. In the model proposed by Robbins et al.? it is suggested that the nonradiative pathway responsible for the decrease in lifetime from 9 to 3 ns may be attributed to intramolecular proton transfer. Thus, in the absence of this process, the zwitterion should have the same lifetime as the anion if one assumes the same radiative lifetime. They suggest that the proton is transferred to the 2-position of the indole ring, a position which, according to calculations, should be a suitable acceptor site. They propose that the 0.5-11s component in this model may arise from a transient effect in the intramolecular quenching process, Le., an intramolecular diffusion transient involving motion of the side chain relative to the indole ring. We suggest that this model has the following weaknesses: (a) A transient effect due to side-chain diffusion would probably not produce two exponentials with a nearly exact fit. (b) There is no transient term4 in the behavior of the compound where indole is substituted at the 3-position with -CH2CH2COOCH2CH3.But this compound has a lifetime of 3 ns which is due to some nonradiative process not present in 3-methylindole. Inductive effects are highly unlikely at these chain lengths. (c) The results obtained in Me,SO suggest that the deuterium effect is a solvent effect and thus cannot be used to support a proton transfer model. (d) The H30+ quenching data, both steady state and via lifetime changes, suggest that the 3- and 0.5-11s components belong to separate species. (e) A transient effect would not have associated with it wavelength effects such as are seen with H30+ quenching and in the timeresolved spectral measurements of Szabo and Rayner which have been confirmed in this laboratory. (f) In Nacetyltryptophan ethyl ester proton transfer is quite unlikely from the -CH3COONHz group to the ring. Nevertheless, a short component has been r e p ~ r t e d . ~ Another of the models proposed to explain the multiplicity of Trp lifetimes is that there are several groundstate conformations around the C,-C, bond, which is illustrated in Figure 5. Two rotamers around the C,-C, bond are also possible; in both conformations the indole ring is parallel to the plane formed by the amino, carboxylate, and Hagroups, but the ring nitrogen can point
Flgure 5. to c,.
H,,
H
H
I
I7
In
a-/3 rotamers of tryptophan. The indole ring is attached
in either direction. If these rotamers are not interconvertible in the excited state, they could give rise to different lifetimes due to differing side-chain interactions. This would depend on the population distribution. NMR experiments on several uncharged Trp derivatives, such as N-acetyltryptophan ethyl ester, show a definite dependence on solvent dielectric constant of the ground-state a-/3 rotamer populations.12 Rotamer I becomes predominant as the dielectric constant of the solvent increases, for most derivatives, while at low dielectric constant in non-hydrogen-bondingsolvents, the predominant rotamer depends on the derivative. In polar hydrogen-bonding solvents, deviations from population trends occur, indicating that there are specific solvent interactions influencing rotamer populations, particularly in charged derivatives such as the cationic form of tryptophan-methylamide. NMR studied3 on the Trp zwitterion itself in water at pH 5 indicate that rotamer I predominates (70%) in the P-7 configuration with the ring nitrogen nearest Ha. There is a small amount of I11 (-15%) in the 6-y configuration with the ring nitrogen nearest the amino group, and small percentages of other remaining rotamers. Szabo and Rayner4 suggest that the answer to the multiplicity of lifetimes lies in this multiplicity of conformers. In this model lifetime changes from the anion value of 9 ns are attributed to three ground-state conformers I, 11, and 111. Because of the particular configuration of 11, there is postulated a strong intramolecular quenching and 11* decays with a lifetime of 0.5 ns. I* and III* also are subject to weaker intramolecular quenching which is responsible for the 3-ns lifetime. Deprotonation of the -NH3+ group is thus postulated to so vastly alter these interactions as to produce a single emitting species with a lifetime of 9 ns. They suggest that these intramolecular interactions involve the -NH3+ and -COO- groups of the side chain. The idea that the side-chain conformation alone can account for a multiplicity of lifetimes has the following weaknesses: (a) The zwitterion of glycine is a poor quencher of 3-methylindoleand indole in neutral aqueous solution with no static ~0mponent.l~ (b) The anion fluoresces with a single lifetime. (c) When the indole is substituted with the side chain -CH2CH2COO- there appears to be minimal intramolecular q ~ e n c h i n g . ~ The conclusion that a solvent effect is involved in the lifetime and quantum yield changes when one replaces HzO with D20 (with concomitant deuteration of Trp) might appear to be in conflict with the results of Nakanishi et a1.16 They observe a transient growth over tens of (12) J. Kobayashi, T. Higashijima, S.Sekido, and T. Miyazawa, Int. J. Peptide Protein Res., 17,486 (1981).
(13) B. Dezube, C. M. Dobson, and C. E. Teague, J . Chem. Soc., Perkin Trans. 2,4, 730 (1981). (14) E. Gudgin, R. Lopez-Delgado, and W. R. Ware, unpublished re-
sults.
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Photophysics of Tryptophan
milliseconds in the fluorescence intensity of Trp in a stop-flow experiment where HzO and DzO are mixed. However, the possibility exists in the case of Trp that this transient, which they observe, is related to changes in solvation plus the final completion of mixing which might take place on this time scale and be picked up only with a system which was sensitive to solvation, i.e., Trp. Thus there may be no real conflict between our results and those of Nakanishi et al. It is probably premature to postulate a model to account for what is clearly a complex photophysical system. However, the following hypothesis may stimulate additional fruitful studies. The above results along with those in the literature point to solvent involvement in the behavior of the excited state of tryptophan and tryptophan-like compounds. Indole is thought to form exciplexes or excited-state complexes with definite stoichiometry.16 It is suggested that the effects being observed with Trp have to do with solvation of both the indole ring and the side chain, hence the influence of the side chain on the photophysics. In particular, it is postuated that one particular configuration involving the side chain and solvation shell may perturb the indole chromophore and make a very rapid nonradiative process possible. Other configurations of the solvent-excited molecule system also (15) N. Nakanishi, M. Kobayashi, M. Tsuboi, C. Takasaki, and N. Tamiya, Biochemistry, 19, 3204 (1980). (16) N. Lasser, J. Feitelson, and R. Lumry, Isr. J. Chem., 16, 330 (1977), and references contained therein.
The Journal of Physical Chemistry, Voi. 87, No. 9, 1983
1505
perturb the indole ring and this averages out to give a lifetime of 3 ns. Elimination of the zwitterion is postulated to drastically alter the solvation configurations and thus the photophysics. The influence of the side chain is thus postulated to operate through its influence on overall solvation, which in turn also influences the acid-base reactions in both the ground and excited state. The population of ground-state conformers appears to be solvent dependent12 and, in addition, mechanisms of interaction between the side chain and the ring will of course depend upon conformation. Thus, we are not rejecting the notion that conformers play a role in the photophysics of Trp; rather we are suggesting that solvation and specific solvent-Trp interactions may be involved and these effects in turn may be intimately related to the various conformations available to Trp. This is clearly a complex photophysical and photochemical problem. The details of the nonradiative pathways are not entirely clear although considerable progress in this regard w8s made by Bent and Hayon" in their laser flash photolysis studies. The ultimate resolution of the numerous questions raised by this work and the work of others will no doubt involve studies which go beyond simply the measurement of lifetime and quantum yields of fluorescence. Registry No. L-Tryptophan, 73-22-3. (17) D.V. Bent and E. Hayon, J. Am. Chem. SOC.,97, 2612 (1975).
