The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching

314

Biochimica et Biophysica Acta, 577 ( 1 9 7 9 ) 3 1 4 - - 3 2 3 © Elsevier/North-Holland Biomedical Press

BBA 38147

THE R E A C T I V I T Y OF T R Y P T O P H A N RESIDUES IN PRO T E IN S STOPPED-FLOW KINETICS OF F L U O R E S C E N C E QUENCHING

B.F. PETERMAN and K.J. L A I D L E R

Department of Chemistry, University of Ottawa, Ottawa, K I N 9B4 (Canada) (Received September l l t h , 1978)

Key words: Stopped-flow kinetics, Fluorescence quenching; Tryptophan residue reactivity Summary T h e quenching o f t r y p t o p h a n fluorescence by N-bromosuccinamide, studied by th e fluorescence stopped-flow technique, was used to compare t he reactivities o f t r y p t o p h a n residues in pr ot e i n molecules. The reaction o f N-bromosuccinamide with the indole group o f N - a c e t y l t r y p t o p h a n a m i d e , a model comp o u n d for b o u n d t r y p t o p h a n , followed second-order kinetics with a rate constant o f (7.8 + 0.8) • l 0 s dm 3 • mol -~ • s -1 at 23°C. The rate does n o t depend on t h e ionic strength or on the pH near neutrality. The non-fluorescent intermediate f o r med from N - a c e t y l t r y p t o p h a n a m i d e on t he reaction with N-bromosuccinamide appears to be a b r o m o h y d r i n c o m p o u n d . The second-order rate constant for fluorescence quenching of t r y p t o p h a n in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 -+ 0.8) • l 0 s dm 3 • mo1-1 • s -1. A p o c y t o c h r o m e c has t he c o n f o r m a t i o n o f a r a n d o m coil with t he single t r y p t o p h a n largely exposed to t he solvent. The rate constant for t he fluorescence quenching o f t he t r y p t o p h a n in a p o c y t o c h r o m e c by N-bromosuccinamide was (3.7 + 0.3) • l 0 s dm 3 • m ol - ' • s -1. The fluorescence quenching by N-bromosuccinamide o f t he t r y p t o p h a n residues incorporated in a - c h y m o t r y p sin at pH 7.0 showed three exponential terms from which t he following rate constants were derived: 1.74 • l 0 s, 0.56 • l 0 s and 0.11 • l 0 s dm 3 • m o l - ' • s -1. This protein is k n ow n to have eight t r y p t o p h a n residues in t he native state, six residues at th e surface, and two buried. Three o f t he surface t r y p t o p h a n s have the indole rings protruding o u t o f t he molecule and m a y account for the fastest kinetic phase o f the quenching process. The intermediate phase m ay be due to t h r ee surface t r y p t o p h a n s whose indole rings poi nt inwards, and the slowest to the two interior t r y p t o p h a n residues. Abbreviations: NBS, N-bromosuccinamide; N-AcTrpNH 2 , N-acetyl-L-tryptophanamide.

315 Introduction

Numerous studies of the structure-function relationship of proteins have suggested that residues of one kind do not all show the same reactivity. Those residues which are involved in important biological functions usually show higher reactivity toward ligands (substrates or DNA) as well as towards chemical reagents. In most cases the higher reactivity can be related to greater accessibility of the residue to the bulk aqueous environment. The reactivities of residues of the same kind can therefore be related to the distribution of the residues in the protein matrix. Tryptophan is a residue whose reactivity and distribution can be selectively studied by fluorescence methods. The exposure of tryptophan residues has been extensively studied by collisional fluorescence quenching techniques [ 1--3 ]. We employed the quenching of tryptophan fluorescence by N-bromosuccinamide (NBS) to study the relationship between the reactivities of tryptophan residues and their position in protein molecules. A similar, but significantly different, approach was used by Frazier et al. [4] to study the topography of tryptophan residues in mouse 2.5 S nerve growth factor. N-Bromosuccinamide has been shown to cleave tryptophyl bonds of peptides and proteins at low pH [5,6]. At higher pH values (near neutrality), NBS reacts preferentially with tryptophan residues of proteins, converting them into oxindole derivatives [ 7 - 9 ] . Equilibrium titrations of tryptophan residues in proteins by NBS indicate that, depending on the solvent accessibility, tryptophan residues show different reactivities towards NBS [8]. Furthermore, it was observed by Steiner [10] and Nagami and Senoh [11] that the addition of NBS to protein solutions diminishes the fluorescence emission on tryptophan residues. The modification of tryptophan residues by NBS has been reviewed by Spande et al. [12]. Our study of the quenching of indole fluorescence by NBS was undertaken in order to understand the kinetic mechanism of this process and the factors affecting it. With excitation at 280 nm the fluorescence of the indole ring is emitted with a maximum near 350 nm, and we followed the quenching of this fluorescence on reaction with NBS. This quenching reaction should discriminate between the protein-bound tryptophan residues of different reactivities, the exposed indole groups reacting more rapidly with NBS than the buried ones. Similar suggestions have been proposed for the NBS titration experiments [8,9]. However, in contrast to these titration experiments, where one studies the steady-state disappearance of the absorption band at 280 nm on addition of NBS, our approach focuses on the reaction kinetics of the fluorescence quenching of the indole group. The reaction of NBS with a protein containing two tryptophans of widely different reactivities is expected to show a different kinetic pattern from that containing a single tryptophan. Because of the high degree of complexity of protein structure, we first investigated the kinetic mechanism of the reaction between NBS and a model compound for bound tryptophan, N-acetyl-L-tryptophanamide (N-AcTrpNH2). The effects of pH and ionic strength on the reaction were examined. Since the reaction between the indole compounds and NBS occurs via a number of intermediates [8] it was important to characteriz~ the quenching process and the nonfluorescent intermediate. We continued our investigation by studying the reac-

316 tivity of t r y p t o p h a n residue in the tripeptide Gly-Trp-Gly. Finally, our work was extended to the study of the reactivities of t r y p t o p h a n residues in proteins.

Experimental procedure Materials. N-Bromosuccinamide was obtained from Fisher Chemical Co. N-Acetyl-L-tryptophanamide and cytochrome c (Type III), were purchased from Sigma Chemical Co. a-Chymotrypsin (3 times crystallized) was obtained from Worthington Biochemical Co. Indan, 1-indanol, 2-bromoindanol and oxindole were purchased from Aldrich Chemical Co. Tripeptide glycyltryptophylglycine was bought from Research Plus Laboratories. A p o c y t o c h r o m e c was prepared as described by Peterman and Laidler (submitted for publication). Two different preparations of a-chymotrypsin were used. a-Chymotrypsin was either used as obtained or chromatographed on a Sephadex G-25 column. In both cases, a-chymotrypsin was used immediately after dissolution or chromatography to prevent excessive autocatalysis. The concentrations of proteins were determined by measuring the absorbance of protein solutions near 280 nm using the following absorption coefficients: a-chymotrypsin, e(282) = 5.15 • 104 M -1 • cm -1 [13] and apocytochrome c, e(278) = 1.1 • 104 M -1 • cm -1 [14]. Phosphate and acetate buffers of defined pH and ionic strength were prepared according to Boyd [15] and Boyd [16], respectively. Spectroscopic measurements. Absorption measurements were carried out with a Pye-Unicam model SP 1800 spectrophotometer. Fluorescence measurements were made on a Perkin-Elmer 512 double-beam spectrofluorometer. Kinetic measurements. Kinetic measurements were carried out using a Durrum stopped-flow instrument equipped with a 75 W xenon-mercury lamp. The photomultiplier tube was positioned either parallel to the incident beam to follow the changes in absorbance, or perpendicularly to the incident beam to follow the changes in fluorescence. The signal-to-noise ratio of the instrument in the fluorescence mode was better than 300 with the time constant of 1 ms. Samples were excited at 280 nm for N-AcTrpNH2, Gly-Trp-Gly or at 296 nm for all proteins. In both cases, a Coming 0-54 filter was used on the emission side of the cuvette. A cuvette with a light path of 2 cm was used for all experiments. The transients were stored in the m e m o r y of a transient recorder (Biomarion 805) and displayed on an oscilloscope. The oscilloscope traces were photographed using a Polaroid camera, and the changes in fluorescence or absorbance vs. time were transferred to semilogarithmic paper. In order to avoid possible side-effects due to the presence of oxygen, all the samples were deaerated and placed uhder an atmosphere of nitrogen. This was later found to be very important for obtaining reproducible results. The measurement of the lifetime of the excited state of the t r y p t o p h a n residue in apocytochrome c was carried out with a single-photon fluorescence lifetime instrument described by Rayner et al. [17,18].

Results Reaction between NBS and N-AcTrpNH2 In order to examine the fundamental aspects o f the reaction between NBS

317 and a protein-bound t r y p t o p h a n residue, we first investigated the factors affecting the reaction b e t w e e n NBS and a model c o m p o u n d , N-acetyl-L-tryptophanamide. The reaction was studied b y the steady-state and transient-fluorescence techniques. Titration o f N-AcTrpNH2 fluorescence b y NBS shows that 1.1 + 0.1 mol of NB8 react with 1 mol of N-AcTrpNH2 in sodium phosphate buffer, at T = 23°C, pH 7.0, and an ionic strength of 0.05 M (these conditions were used througho u t this investigation, unless otherwise stated). This is in good agreement with the results reported b y Green and Witkop [8]• When N-AcTrpNH2 was rapidly mixed with NBS in large excess (at least ten times) in the stopped-flow spectrofluorometer, the fluorescence of N-AcTrpNH2 decreased exponentially to the equilibrium level which was identical to the fluorescence of the buffer (i.e. the fluorescence was completely abolished; see also Fig. 4). Increasing the concentration of NBS resulted in an increase of the rate of the disappearance of the N-AcTrpNH2 fluorescence. Fig. 1 shows that there exists a linear relationship between the first-order rate coefficient and the concentration of NBS. From the slope the second-order rate constant for the reaction b e t w e e n NBS and N-AcTrpNH2 was found to be (7.8 + 0.8) • l 0 s dm 3 • mo1-1 • s -1. The rate did not depend on the ionic strength in the range from 0.01 to 0.2 M. Near neutral pH the rate of the reaction is not very sensitive to the pH, b u t it dramatically increases when the pH is lowered below 5 (Fig. 2). At pH 2.0 the reaction becomes immeasurably fast (i.e. it is over in less than 4 ms, which is the measured dead time o f the fluorescence stopped-flow instrument).

Characterization o f the non-fluorescent intermediate and the quenching process As reported b y Green and Witkop [8], the reaction of indole derivatives with NBS occurs b y a mechanism which can be simplified as follows: OH

NBS H

N H Br

oxindole or brominated oxindole

In order to characterize the non-fluorescent intermediate we followed the reaction between N-AcTrpNH2 and NBS in the fluorescence and absorption modes• In contrast to the disappearance of N-AcTrpNH2 fluorescence, where only one relaxation of half-time of approx. 4 ms was observed, when N-AcTrpNH2 reacts with NBS the absorption change occurs first b y a very rapid process (half-time of approx. 4 ms), followed b y a much slower relaxation of approx. 200 s which was measured by the stopped-flow instrument in its absorption mode• By measuring the time course o f t h e reaction at various wavelengths (240--350 nm) we were able to obtain the absorption spectrum of the intermediate (Fig. 3); it has a broad band with a maximum b e t w e e n 290 and 300 nm, characteristic o f a b r o m o h y d r i n c o m p o u n d [8]. We found t h a t ' b o t h indan and 1-indanol fluoresce, b u t that 2-bromo-l-indanol does not. This suggests that the insertion of bromide into the fluorescent

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Fig. 1. T h e f i r s t - o r d e r r a t e c o e f f i c i e n t s f o r t h e N - A c T r p N H 2 f l u o r e s c e n c e q u e n c h i n g as a f u n c t i o n o f NBS c o n c e n t r a t i o n . T h e c o n c e n t r a t i o n of N - A c T r p N H 2 w a s k e p t c o n s t a n t a t 5 • 10 -6 M ( a f t e r m i x i n g ) . Condit i o n s o f t h e e x p e r i m e n t : b u f f e r , s o d i u m p h o s p h a t e , ionic s t r e n g t h I = 0 . 0 5 M, p H 6.0, T = 23°C. E x c i t a t i o n a t 2 8 0 n m was e m p l o y e d a n d t h e e m i t t e d light s e l e c t e d b y a C o m i n g 0 - 5 4 filter. Fig. 2. E f f e c t o f p H o n t h e f i r s t - o r d e r r a t e c o e f f i c i e n t o f t h e r e a c t i o n b e t w e e n NBS a n d N - A c T r p N H 2. f o l l o w i n g s o l u t i o n s w e r e u s e d : p H 2.6 + 0 . 0 5 , 0.1 M suecinic acid; p H 3 . 0 + 0 . 0 5 , p H 4.0, p H 4 . 9 5 , a n d p H 5.8, a c e t a t e b u f f e r s o f ionic s t r e n g t h 0 . 0 5 M; p H 7.0, s o d i u m p h o s p h a t e b u f f e r ; p H 8.7, b o r i c a c i d ] b o r a x b u f f e r . C o n c e n t r a t i o n s o f NBS a n d N - A c T r p N H 2 w e r e k e p t c o n s t a n t a t 1 0 • 1 0 - s M a n d 5 • 10 -6 M, r e s p e c t i v e l y . E x c i t a t i o n a t 2 8 0 n m , e m i s s i o n s e l e c t e d b y a C o m i n g 0-54 filter. The

c o m p o u n d quenches the fluorescence. The effect of bromination on the fluorescence of organic compounds was studied b y Ermolaev and Svitashev [19] (see also Ref. 20). They observed that the addition of a halogen atom to a fluorescent organic c o m p o u n d (an aromatic hydrocarbon, for example) increases the probability of the radiationless energy transfer from the lowest singlet-excited state to the triplet state (intersystem crossing). We also found that oxindole, the final product in the above scheme, does not fluoresce; this explains why fluorescence does n o t reappear after bromination has occurred. It is apparent that the substitution of bromine into the indole ring increases the intersystem crossing, which competes with the fluorescence emission to the extent that the fluorescence emission is completely quenched. We therefore conclude that the quenching of N-AcTrpNH2 fluorescence is essentially the insertion of a bromine atom into the indole ring with rearrangement of the ring.

Reaction o f NBS with the tryptophan residue in Gly-Trp-Gly NBS reacts with the indole ring of the tripeptide Gly-Trp-Gly in a similar manner as with N-AcTrpNH2. The fluorescence of Gly-Trp-Gly decreased exponentially on reaction with NBS. As observed with N-AcTrpNH2, the first-order rate coefficient varied linearly with the concentration of NBS. The secondorder rate constant was (8.8 -+ 0.8) • l 0 s M -I • s -~, which is approximately 10%

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Fig. 3. A b s o r p t i o n a n d f l u o r e s c e n c e spectra o f N - A c T r p N H 2 , N B S - N - A o T r p N H 2 i n t e r m e d i a t e a n d f i n a l p r o d u c t . C o n c e n t r a t i o n o f N - A c T r p N H 2 w a s 2 . 5 • 1 0 -5 M ( a f t e r m i x i n g ) , c o n c e n t r a t i o n o f N B S w a s 2 5 • 1 0 -5 M ( a f t e r m i x i n g ) i n s o d i u m p h o s p h a t e b u f f e r ; p H 7 . 0 , i o n i c s t r e n g t h 0 . 0 5 M; - - , absorption spectrum of N-AcTrpNH2; • • , a b s o r p t i o n s p e c t r u m o f t h e i n t e r m e d i a t e ; z~ ~, absorption spectrum of the final product; o o, f l u o r e s c e n c e s p e c t r u m o f N - A c T z p N H 2 ( e x c i t a t i o n 2 9 6 r i m , not corrected); • -', f l u o r e s c e n c e s p e c t r u m o f t h e i n t e r m e d i a t e . The absorbance d u e t o N B S w a s s u b tracted.

higher than that found for N-AcTrpNH2. Subtle differences in the neighboring groups probably contribute to a slightly increased reactivity of the indole group in the tripeptide.

Reaction o f N B S with tryptophan residues in proteins We also studied two proteins ( a p o c y t o c h r o m e c and ~-chymotrypsin) with largely different numbers of t r y p t o p h a n residues. A molecule of horse heart c y t o c h r o m e c contains only one tryptophan residue. Once the heine group of c y t o c h r o m e c is removed, the polypeptide chain is unable to assume a globular conformation similar to that of the native prorein. Thus, a p o c y t o c h r o m e c has the conformation of a random coil, with the t r y p t o p h a n largely exposed to the solvent [14]. The reaction between NBS and the single t r y p t o p h a n residue in a p o c y t o c h r o m e c followed a single exponential decay as was observed for the model compounds. A linear relationship was obtained b e t w e e n the rate of decrease of fluorescence and the concentration of NBS, with a second-order rate constant of (3.7 _+0.3) • 105 dm 3 • tool -1 • s -1. This value is only a b o u t one-half of that for N-AcTrpNH2. Presumably this is partially due to the shielding of the t r y p t o p h a n residue b y the protein matrix, and partially due to the side-reactions. We used acrylamide as a collisional fluorescence quencher [21] to t e s t the exposure of the single t r y p t o p h a n in a p o c y t o c h r o m e c. According to the Stern-

320 TABLE I COLLISIONAL QUENCHING CONSTANTS, APPARENT RATE CONSTANTS FOR QUENCHING AND LIFETIMES OF THE EXCITED STATE FOR N-AcTrpNH 2 AND APOCYTOCHROME c

N-AcTrpNH2 Apocytochrome c

K q (M -z)

70 (ns)

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Fig. 4. Analysis o f t h e oscKloscope t r a c e r e p r e s e n t i n g t h e d e c r e a s e o f ~ - c h y m o t r y p s i n f l u o r e s c e n c e o n r e a c t i o n w i t h NBS (inset: o r d i n a t e , 0 . 2 V / d i v i s i o n ; abscissa, 0.1 s/division). T h e d i f f e r e n c e b e t w e e n t h e f l u o r e s c e n c e i n t e n s i t y at t i m e t a n d t h e e q u i l i b r i u m [ F ( t ) - F ( o o ) ] ( i n s e t ) w a s t r a n s f e r r e d to s e m i l o g a r i t h m i c p a p e r (o). T h e s l o w e s t r a t e c o n s t a n t w a s e s t i m a t e d f r o m t h e l i n e a r p o r t i o n o f t h e e x p e r i m e n t a l c u r v e . T h e i n t e r m e d i a t e r a t e c o n s t a n t was d e t e r m i n e d f r o m t h e l i n e a r p o r t i o n of t h e c u r v e r e p r e s e n t i n g t h e diff e r e n c e b e t w e e n t h e e x p e r i m e n t a l c u r v e a n d t h e s l o w e s t r e l a x a t i o n (~). T h e r e m a i n i n g d i f f e r e n c e , w h i c h a p p e a r e d in all t r a c e s a n a l y z e d , y i e l d e d t h e fastest r a t e c o n s t a n t . T h e c o n d i t i o n s o f t h e e x p e r i m e n t : conc e n t r a t i o n of ~ - c h y m o t r y p s l n , 6 • 1 0 - 7 M; c o n c e n t r a t i o n o f NBS, 20 • 1 0 -5 M; b u f f e r , s o d i u m p h o s p h a t e , 0 . 0 5 M, p H 7.0; T , 23°C; e x c i t a t i o n 296 n m , e m i s s i o n , C o m i n g filter 0-54. T h e f i r s t - o r d e r r a t e coeffic i e n t s are: 31 s-1, 9.8 s-1 a n d 2.4 s -z.

321

Volmer equation [ 1--3 ] the decrease in the fluorescence intensity is related to the concentration of quencher [X] by the equation F o / F = 1 + Kq

[X]

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= k'T0

where k' is apparent rate constant for the collisional quenching and To the lifetime of the excited state in the absence of quencher. As can be seen from Table I, the apparent quenching rate constant, which represents the exposure of the indole ring, for t r y p t o p h a n in a p o c y t o c h r o m e c is approximately one-half o f that for N-AcTrpNH2. A molecule o f ~-chymotrypsin has a heterogeneous population o f eight trypt o p h a n residues [3]. When these t r y p t o p h a n residues reacted with NBS the fluorescence decreased with time (see inset in Fig. 4) and, as shown in Fig. 4, required three exponential terms. Purification of ~-chymotrypsin on a Sephadex G-25 column did not remove any of the three relaxations. The first~rder rate coefficients o f all three relaxations increase linearly with NBS concentration (Fig. 5), and the slopes give the following second-order rate constants: 1.74 • 10 s dm 3 • mo1-1 • s-', 0.56 • l 0 s dm 3 • mo1-1 • s -1 and 0.11 • l 0 s dm ~ • mo1:1 • s -1, with an estimated error of from 10% to 15%. The amplitudes o f the three relaxations were, within the error of the experiment, independent o f the NBS concentration. After correction for the dead-time of the stopped-flow instru-

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322 ment (4 ms), the following relative values for the relaxation amplitudes were obtained: aF = 0 . 3 5 + 0 . 0 4 , aM = 0.37 + 0.04 and as = 0.28 +- 0.02, where aF, aM and as are the relaxation amplitudes for the fastest, the intermediate and the slowest component. Discussion

Our results suggest that the quenching of indole fluorescence b y NBS is essentially the insertion o f a bromine atom, which is highly electrophflic, into the indole ring. Bromination is accompanied b y the splitting of the doublebond and hydroxylation producing a bromohydrin c o m p o u n d as judged from the ultraviolet spectrum. That a bromohydrin is an early intermediate of the oxidation of indole by NBS was first proposed b y Green and Witkop [8]. The quenching process was found to follow second-order kinetics and is very sensitive to pH below 5. The inherent problem of the reaction between NBS and the indole rings of b o u n d tryptophan residues is that side-reactions can occur. NBS, being a potent oxidizing reagent, reacts also with the sulfhydryl group, and with tyrosine, histidine and methionine residues [22]. Except with the sulfhydryl group, NBS reacts with tyrosine, histidine and methionine much more slowly than with the indole group [22]. Preliminary results that we have obtained on the effect of the reactive groups on the rate of the reaction between NBS and the indole group indicate that if the ratio of the reactive groups to tryptophan does not exceed approx. 5 the rate is not decreased b y more than 20%. The side-reactions do somewhat limit the usefulness of the comparison of the rate constants between various proteins. The s t u d y o f the reactivity of the single tryptophan residue in apocytochrome c shows that the tryptophan is only approximately half as reactive towards NBS as N-AcTrpNH:. We attribute the drop in the reactivity to both the steric shielding of the t r y p t o p h a n residue b y the protein matrix and the possible competing side-reactions. Significant steric shielding of the tryptophan residue b y the protein matrix was shown b y quenching experiments with acrylamide. Eftink and Ghiron [3], using acrylamide as a collisional fluorescence quencher, observed that the tryptophan residues in a-chymotrypsin vary in the extent of their exposure to the solvent. The simplest interpretation of our results is that there exist three groups of tryptophan residues having significantly different reactivities. The X-ray analysis [23] shows that a molecule of ~-chymotrypsin has six t r y p t o p h a n residues located on the surface and two buried in the interior of the molecule. Three of the six surface residues have the indole rings protruding o u t of the molecule, and the remaining three are pointed inward. It is tempting to conclude that the fastest c o m p o n e n t is due to the reaction between NBS and the surface residues having exterior indole groups. The intermediate phase is probably the bromination o f the three surface tryptophans having interior indole groups and the slowest phase the bromination of the t w o interior tryptophan residues. The values of the relaxation amplitudes indicate that 2.8 + 0.3, 2.96 + 0.3 and 2.24 + 0.2 tryptophans contribute to the fastest, the intermediate and the slowest relaxation, respectively. These values agree, within the errors of the experiment, with what one would

323

expect if each o f the eight tryptophans emits with the same intensity. Although this assumption should be made with considerable caution, since it was observed that the quantum yield of the tryptophan residue depends on its location in the protein matrix [24], the values of the relaxation amplitudes seem to support our interpretation. The present work has shown that the fluorescence quenching of tryptophan residues in proteins by NBS can be used to study the reactivity o f tryptophan residues and has suggested that one may be able to relate the reactivity of tryptophan residues to their position in the protein matrix. An advantage o f this technique is that an exposure of protein to NBS of less than approx. 100 ms is necessary for the observation to be made. It is believed that during this short exposure, drastic conformation changes (unfolding) are unlikely. Steady-state titrations of tryptophan residues by NBS take much longer and therefore some unfolding and partial cleavage is more likely. The advantage o f the reaction quenching technique over the coUisional quenching technique [2,3] is that it can provide information on the number o f classes of tryptophan residues of different reactivities (i.e. of different solvent accessibility). However, if the number of tryptophan residues is large the kinetic pattern becomes complicated and less discernible. In order to obtain a more complete picture o f the reactivities o f tryptophan residues in proteins, a study o f a number of peptides and proteins is currently under way in our laboratory. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

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