Fast Relaxation Processes in a Protein Revealed by the Decay Kinetics of Tryptophan Fluorescence+ A . Grinvaldl and I . Z. Steinberg*
A H S T R A C r:
The fluorescence decay of chicken pepsinogen is not monoexponential throughout the emission spectrum. For light emitted a t the long wavelength region of the fluorescence spectrum, the decay can be described by two exponential terms, one of them exhibiting a negative amplitude. l'his behavior shows that in this region of the spectrum the fluorescence builds up before it decays, indicating that the electronically excited species involved has been created during the fluorescence lifetime. For a variety of reasons it seems very unlikely that this emitting species as formed by energy transfer from other entities in the protein. The buildup of the fluorescence at the red edge of the spectrum prior to emission thus reflects a genuine relaxation process i n the protein molecule i n the nanosecond time scale. the exc,i:ed chromophore shifting its emission spectrum to the red i n the course of the relaxation process. The reaction in-
volved i n the relaxation may be a nonspecific orientation of various groups around the excited chromophore or a forniation of a more specific excited state complex, i.e.. an exciplex. This nanosecond relaxation process is Conformation dependent and disappears upon denaturation. Similarly, chicken pepsin a t neutral pH fails to show i t . This sce1m to be the first case in which a relaxation process i n the iianosecond time range has been demonstrated with a native chromophore in a protein and complements the observa. tions of Brand and Gohlke and of Lakowicz and Weber of other nanosecond relaxation processes in proteins. Fast relaxation reactions demonstrated in the present study for tryptophans are not necessarily limited to indole side chains, except that these residues permit the study of the re. laxation processes by perturbation of interactions with the environment through electronic excitation.
T f i e fluorescence emitted by molecules in condensed phases is invariably shifted to longer wavelengths relative to the absorption spectrum. The larger the interaction between the excited chroniophore with its environment the greater the shift of the fluorescence band to the red. This behavior of fluorescence spectra is due to relaxation processes which the excited molecule undergoes prior to the emission process, the rate of relaxation being usuallj. much faster than the rate of light emission. The processes which take place in the excited state may be of a variety of kinds: dissipation of vibrational energy or change of conformation of the electronicall4 excited molecules, interactions with surrounding solvent molecules, association or dissociation reactions in the excited state, or processes of fast energy transfer from one chrornophoric group to another in the system studied. Part of the energy of the excited molecules is lost in these processes and the photon emitted subsequently is left N i t h less energ) t o carry; the emitted light is thus of longer wavelerig t h . I n all cases in which the reactions i n the excited state are much faster than the rate of light emission, the fluorescence collected at the various wavelengths of the spectrum deca) s ni ono t on I)us1y with ti iri e. IJ nder certain circumstances son1e of t h e relaxation processes are not fast compared t o the lifetime of the excited state. Such a situation is disclosed by buildup of the fluorescence intensity prior to the decay at some region of the emission spectrum. Examples of this kind were reported when the electronically excited molecule undergoes association reactions (Speed and Selinger. 1969)
or ionization reactions (Loken et ill., 1972) or when if is embedded in a highly viscous medium (Ware et al., 1971; Brand and Gohlke, 1971). It may be recalled that the lifetime of the first singlet excited state, which is the one that is usually involved in the fluorescence process, is of thz order of 1-100 nsec. The study of the decay kinetics at different wavelengths or of the behavior of the emission spectrum at various times after excitation may thus serve as a powerful tool in the investigation of excited-state processes in the nanosecond time range. While relaxation processes may follow very complicateit kinetics depending on the exact details of the reactions involved, it is useful to assume a simple scheme for the chair] of reactions which follows the electronic excitation in order to gain qualitative understanding of the events that take place. The following scheme (eq 1) has been adopted. where
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From t h e Department of Chemical Phqsics. The h e i 7 i n a n n Insrit u l e of Science. Rehovot, Israel. Rrc.ri\,ed Ju/>' 10, 1974. t Part of a ph,D. Thesis t o be submitted to t h e Feinhere (;raduatz S c h o i of t h e U e i z m a n n lnatitute of Sciznce
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BIOCHEMISTRY.
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25. 1974
A* is the excited species foriiied immediately upon light a h sorption by the absorbing molecule. I t is then comerted with a rate constant k , into a second excited speLies H*. A * can also fluoresce or decay nonradiatibelq with rate constants k t z and k / , respectivelq, B* tnay undergo similar reaction with respective rate constant k l " and X,,". Sinzc energy is dissipated on the conversion of A * into B*. the light emitted by B* will be red-shifted relative to t h a t einiLted by A * . The fluorescence at the red-edge of the emission spectrum, where the emission frorn B* is dominant, shmld thus show a buildup prior to decay. For thr simplc h c h c n i e presented in eq 1 the analytical expression for thc d c c s j ot' the concentration of A * and B* I S giLen b\
NANOSECOND RELAXATION
PROCESSES IN
A
where [A*]o is the concentration of A* a t t = 0, and 7 1 = k P k q A k , and 72 = k p kqB. Equation 2 is of the same form as the equations given by Loken et al. (1972) for ionization reactions in the excited state when the proton transfer is not reversible. Under real circumstances, eq 2 cannot be expected to hold exactly since the scheme presented in eq 1 is probably a n over-simplification, and it is experimentally difficult to isolate the emission due to B* alone. However, the appearance of an exponent with a negative amplitude in the expression obtained for the fluorescence decay a t the red-edge of the emission spectrum shows that a relaxation process of one kind or another has taken place in the system studied. The above approach has been applied in the study to be reported below to the detection and examination of nanosecond processes which take place in protein molecules. The intrinsic fluorescence of proteins due to their tryptophan residues was measured a t different spectral ranges and analyzed for its time course after excitation. In the case of the protein chicken pepsinogen a buildup of the fluorescence intensity a t the red edge of the emission spectrum was demonstrated, indicating that relaxation processes d o indeed take place i, this protein with a lifetime of less than a nanosecond.
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Experimental Section Materials. Highly purified chicken pepsinogen and chicken pepsin have been kindly donated by Dr. Zvi Bohak of the Department of Biophysics, the Weizmann Institute of Science. The procedures for purification and characterization of these proteins were described elsewhere (Bohak, 1969). Most of the measurements were carried out a t p H 7.1 (0.1 M phosphate buffer in doubly distilled water) using freshly prepared solutions. For measurements a t different p H values the solution was titrated in the cuvet by addition of either 1 N HC1 or 1 N NaCI, the p H being monitored by a p H meter (Radiometer, Copenhagen). Purified N- acetylL-tryptophanamide and N-methylpyridinium perchlorate were a generous gift from Dr. M. Shinitzky. Solvents used were of Spectrograde quality; lack of fluorescent impurities was tested in every case by using the very sensitive decay fluorimeter. Methods. Protein concentrations were determined by the absorbance a t 280 nm using absorption coefficients of 1.26 and 1.46 mg-' cm2 for the pepsinogen and pepsin, respectively (Bohak, 1969). Protein concentrations used were alM. Unless otherwise stated all meaways less than 3 X surements were carried out a t a temperature of 23 f 2'. Absorption spectra were measured with a Zeiss Model P M Q I1 or with a Cary 15 spectrophotometer. Corrected fluorescence spectra were obtained with a Turner 210 spectrofluorimeter using cells of 1-cm optical path, the optical density of the solution being less than 0.1 a t the excitation wavelength. Fluorescence polarization spectra' were obtained with an instrument of the type described by Weber and Bablouzian (1966), built by Dr. M . Shinitzky, the
' The
fluorescence polarization p is defined as (111 - I l ) / ( / 1 ~
+
II), where 11,and 1 1 are the fluorescence intensities observed through
a polarizer oriented parallel and perpendicular, respectively, to the direction of polarization of the exciting light.
PROTEIN
Weizmann Institute of Science. The fluorescence was excited by linearly polarized light. Special filters were used to isolate either the blue or the red part of the emission spectrum, Kodak 18A filter for the blue region, and a Schott KV-380 filter for the red region (see Figure 3). For the fluorescence po!lvaiii:i 191 18. Rrcriwrl F
