J. Phys. Chem. 1083, 87, 189-190
189
Direct Observation of Monophotonic Photoionization in Tryptophan Excited by 300-nm Radiation. A Laser Photolysls Study M. Bazln,+ L. K. Patterson," and R. Santust Radiation Laboratory, Universi?. of Notre Dame, Notre Dame, Indiana 46556, and Laboratoire de Physico-Chimie de I'Adaptation, ERA 951 du CNRS, Musbum National d'Histoire Netureile, 75231 Paris, Cedex 05, France (Received: September 10, 1982; In Final Form: November 22, 1962)
Direct observation of photoionization in aqueous tryptophan by laser photolysis at 280,292, and 300 nm gives a constant value for the monophotonic quantum yield of hydrated elecrons, CP = 0.075 f 0.02, over this range. These findings, along with previous studies at 265 nm, indicate uniform photoionization over the whole of the tryptophan near-UV band. Additionally, the yield of tryptophan triplet is found to be essentially constant over the same range.
Introduction A wide variety of photochemical transformations in protein systems may be associated with events occurring in tryptophan residues which function as chromophoric centers in interaction with UV components of the solar spectrum. Principal among these is photoionization to produce both hydrated electrons (ea;) and tryptophan cation.' Hence, an elucidation of the mechanism by which this process occurs is essential to a clear understanding of damage in the more complex systems. At present there is considerable disagreement about the quantum yield of photoionization and ita origin from tryptophan excited in the most accessible long wavelength absorption band. Photoionization data from various laboratories, taken together, fail to present a uniform picture of these primary events. In early papers Steen2proposed that photoionization over the whole of the So-S1 absorption band is uniform with CP = 0.015. This work was carried out by using protons as eaq- scavengers. More recently, with protonated imidazole as scavenger Pigault et al.3 have reported comparable results but with CP = 0.003. In experiments with N-acetyltryptophanamide Evans et d.4also reported a uniform yield of photoionization. Additionally, indication of photoionization over wavelengths up to 313 nm has been provided in a spin trapping study by Riesz an c o - ~ o r k e r s . ~ However, Bernas and co-workers,'using N20 to scavenge eaq-from photolyzed tryptophan, found a marked dependence of the quantum yield on the excitation wavelength.6 Their results indicate a quantum yield of CP = 0.08 at 242 nm (where there is some small overlap of the S,-,-S, band) which falls off to 0 at wavelengths greater than 278 nm. By the use of laser flash photolysis it is possible to (1) avoid complications which may occur with the use of scavenging techniques by direct observation of the hydrated electrons and (2) time resolve both the electron production and competing pathways, i.e., the triplet formation. However, the inherent high photodensity of laser photolysis systems introduces a major uncertainty concerning the origin-monophotonic or biphotonic-of photoionization intermediates. Additionally, laser studies of the problem have previously been limited by the inability to vary the excitation wavelength over the region of interest. Radiation Laboratory and Laboratoire de Physico-Chimie de 1'Adaptation. Address correspondence to the University of Notre Dame address. * Radiation Laboratory. Laboratorie de Physico-Chimie de l'hdaptation.
*
0022-3654/83/2087-0189$0 1.5010
Using a computerized dye laser system operated over
a low photon density range, we have examined both the wavelength and the excitation intensity dependences for the photoionization of tryptophan. We find that, in the first absorption band (265-300 nm), the yield of e, is both monophotonic and wavelength independent at low intensity. It is also shown that the ratio of the triplet formation yield to the ea; yield is constant under a given set of conditions.
Experimental Section The excitation source used in this study was the Quantaray YAG laser coupled to a dye laser system. The YAG second harmonic was used to pump the dye whose emission was then doubled in frequency and optically separated from the fundamental. By selection of the appropriate dye one may readily obtain the necessary output at the wavelengths employed here. Laser intensities were varied by changing the pumping energy. The laser pulse width was 6 ns. A conventional analyzing system with pulsed Xe flash lamp was used to monitor transient absorption. Computerized data gathering and storage facilities, using a Textronics R7912 transient recorder and labatory computer system, provide for averaging together many individual measurements to achieve the high signal-to-noise ratio indicated here. With this system it has been possible to resolve absorbances readily in the range 0.002-0.004 with a practical limit of about 0.0007. So that monophotonic quantum yields could be obtained, the requirements of this study dictated the use of very low laser intensities which, from tryptophan, generated ODs in the range stated. A plot of OD for ea; vs. exciting light intensity is given in Figure 1 to illustrate the range of this apparatus. At all wavelengths actinometry was carried out with anthracene triplet ,A( = 422 nm) as the primary standard? As a secondary standard a photodiode was employed in conjunction with a beam splitter and was calibrated against anthracene at each excitation wavelength. The (1) R. Santus, M. Bazin, and M. Aubailly, Reu. Chem. Int., 3, 231 (1980). (2) H. B. Steen, J. Phys. Chem., 61,3997 (1974). (3) C. Pigault, C. Hasselmann, and G. LaustHat, J. Phys. Chem., 86, 1755 (1982). (4)R. F.Evans, C. A. Ghiron, R. R. Kuntz, and W. A. Volkert, Chem. Phys. Lett., 42,415 (1976). ( 5 ) M. M. Mossoba, K.Makino, and P. Riesz, J.Phys. Chem., 86,3478 (1982). (6)E. Amouyal, A. Bernas, and D. Grand, Photochem. Photobiol., 29, 1071 (1979). (7) R. Bensasson and E. J. Land, Trans. Faraday. SOC.,67, 1904 (1971).
0 1983 American Chemical Society
190
The Journal of Physical Chemistty, Vol. 87,
r----
TABLE
1
I
/
/
/
L
I
1
l,xlO9
Einstein
x
1
2.02 2.40 2.48 2.58 3.22 1.55 3.02
5560
cm-2
Flgure 1. Optical density of the hydrated electron in a 2.0-mm photolysis -11 as a function of laser excitation intensity. All measurements of absorption were made at 650 nm.
ea.-
presence of eaq-as reflected by 650-nm absorption was confirmed with NzO quenching. In both actinometry and the tryptophan measurements, the data from up to 50 individual shots were averaged together with the laser intensity from each individual shot being measured. Solution was changed after 5 shots. Tryptophan from Calbiochem was used and solutions were prepared in 20 mM phosphate buffer at pH 7.1. All experiments were carried out at ambient temperature (20 "C).
Results and Discussion The results of this study for each wavelength are given in Table I. These include excitation intensities, yields of hydrated electrons and triplets, and the relevant groundstate parameters. It may be clearly seen from the small variation in the 4(ea;) obtained even when Io is varied by a factor of 5 that the process obtained is essentially monophotonic. In agreement with the steady-state results of Pigault et al.3 it may be seen that tryptophan can be photoionized by radiation at 300 nm, the practical absorptiuon limit for this residue in proteins. However, the results of this study suggest a quantum yield for e,; of 0.075 i 0.02 which is somewhat at variance with the scavenging measurements of Pigault3 recently presented. When compared to the monophotonic yield of ea; reported by Grossweiner et at 265 nm with the fourth harmonic from a Nd:glass laser system, these results are in excellent agreement. All these results taken together are consistent with the interpretation that tryptophan photoionization can proceed by a monophotonic mechanism under the conditions of normal solar irradiation. These results also represent a direct observation of hydrated electron production in the long wavelength end of the first absorption band well beyond the 275-run limit proposed by Bernas and co-worker@from the N 2 0 scavenging experiments. From Table I, one may also see that, within the uncertainty of individual measurements, the ratio @(ea;)/@T is also constant for the wavelengths considered. From this finding, it appears that, for monophotonic ionization, both eaq-and tryptophan triplet are produced from a common ~~~~
P
10-~1,, einstein/ E , M-' cm' cm-'
E 13
~~
Letters
No. 2, 1983
~~
(8) R. Klein, I. Tatischeff, M. Bazin, and R. Santus, J.Phys. Chem.,
85,670 (1981). (9) L. I. Grossweiner. A. M. Brendzel. and A. Blum, Chem. Phvs.. 57, 147 (1981).
@(e,;)
h = 280 nm 0.068 0.075 0.067 0.059 0.071
av = 0.068 0.75 1.54 2.59 3.35 0.75 1.77 2.88
2730
510
0.254 0.277 av = 0 . 2 6 5
h = 292 nm 0.061 0.096 0.097 0.092
av = 0.086 4.5 3.45 2.7 4.6
@(T)
0.325 0.197 0.292 av = 0.27
h = 300 n m 0.050 0.092
av = 0.071
0.27 0.20 av = 0.24
lo2? 2.59 3.07 3.19 3.31 4.1 1.99 3.88 0.26 0.47 1.00 1.62 2.11 0.47 1.11 1.80 0.32 0.53 0.41 0.32 0.54 0.30
a The parameters in the Table are defined as follows: I , is the photon density of the incident exciting light; E is the ground-state extinction coefficient for tryptophan a t the wavelengths under study; @(eaq-)is the quantum yield of hydrated electron measured at 650 n m ; @ ( T )is the quant u m yield of tryptophan triplet obtained by using the extinction coefficient a t 460 nm from indole;' q is the fraction of ground-state depopulation by absorption of exciting light.
excited state. While these data are not inconsistent with the scheme suggested by Grossweiner and co-workersg which involves a prefluorescent state, S1', whose energy is significantly higher than the S1 state, such a scheme is not required for an understanding of our results. In light of the present study any such S i state must be characterized by an energy level very close to that of the fluorescent state, S1. When one considers both the constant fluorescence quantum yield throughout the first absorption bandlo as well as the uniformity of the ratio, @(eaq-)/@p, it is sufficient to evoke SIalone as the precursor for the monophotonic photoionization. This behavior was proposed earlier from the steady-state studies for tryptophan and deri~atives.~Jl It has been demonstrated with laser photolysis for indole.8
Acknowledgment. We thank Professor R. W. Fessenden for his advice and assistance during the use of the laser system. The research described herein was supported by the office of Basic Energy Sciences of the Department of Energy. This is Document No. NDRL 2378 from the Notre Dame Radiation Laboratory. Registry No. Tryptophan, 73-22-3. (10)I. Tatischeff and R. Klein, Photochem. Photobiol., 22,221 (1975). (11)R. Klein and I. Tatischeff, Chem. Phys. Lett., 51, 333 (1977).
