Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Biochimica et Biophysica Acta 873 (1986) 73-78 Elsevier

73

BBA 32610

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy D e b r a S. C a s w e l l a n d T h o m a s G . S p i r o * Department of Chemistry, Princeton University, Princeton, NJ 08544 ( U. S.A.) (Received February 26th, 1986) (Revised manuscript received June 4th. 1986)

Key words: Ultraviolet resonance Raman spectroscopy; Tyrosine residue; Tryptophan residue: Chemical modification; Cytochrome c; Stellacyanin

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyi bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, and can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 c m - t tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface. Introduction

It has recently become possible to probe the chemical environment of aromatic residues in proteins with high sensitivity using ultraviolet resonance Raman spectroscopy [1-4]. Laser excitation in the deep ultraviolet region produces strong enhancement of vibrational Raman modes of aromatic molecules via resonance with their 7r-Tr* electronic transitions [3]. Excitation at 218 and 200 nm is conveniently obtained via the second and third anti-Stokes lines from an H 2 Raman

* To whom correspondence should be addressed. Abbreviation: HNBB, 2-hydroxy-5-nitrobenzyl bromide.

shifter pumped by the fourth harmonic (266 nm) of an N d : Y A G pulsed laser. Tryptophan (trp) modes are selectively enhanced at 218 nm, while with 200 nm excitation, one sees enhanced modes of tyrosine (Tyr) and phenylalanine (Phe) [3], as well as amide modes of the polypeptide backbone [2,5,6]. While the Phe and Tyr spectra overlap significantly, unique bands from Tyr can be observed, particularly the well-known 850/830 c m - t Tyr doublet. Environmental influences such as H-bonding and stacking interactions [7] can be monitored for Trp and Tyr at these two wavelengths. Since most proteins contain more than one Trp or Tyr residue, detailed structural interpretation is rendered problematic by their overlapping contri-

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

74 butions. In this study we explore the potential for increasing the discrimination of residue environments with selective chemical modification of Tyr and Trp. Tyr is selectively nitrated under mild conditions by tetranitromethane [8], while Trp can be reacted selectively with 2-hydroxy-5-nitrobenzyl bromide (HNBB) [9]. For a native protein, these reactions occur at surface residues, which are accessible to the reagents. The electronic absorption spectra of the modified Tyr and Trp residues are strongly redshifted [8,9], and their vibrational spectra and resonance Raman enhancement pattern are expected to be greatly altered. The possibility of obtaining nitrotyrosine resonance Raman spectra with near-ultraviolet laser excitation has already been demonstrated [10,11]. We were interested in the possibility that the contribution of the modified residues to the deep-ultraviolet resonance Raman spectra would be de-enhanced, thereby exposing the spectral contributions of buried residues. The present work demonstrates that de-enhancement does occur, and can be used to quantify the extent of the modification reaction. It points the way to selective studies of buried residues.

Experimental

Tyrosine modification Nitration of tyrosine was carried out by adding 125/~1 of tetranitromethane (Sigma) to 6 ml of 0.5 M phosphate buffer, pH 8.5, containing 1 mM tyrosine, and allowing the reaction to proceed for several hours. Horse heart cytochrome c (Sigma) and stellacyanin (isolated from Japanese lacquer acetone powder (Saito and Co.) according to the method of Reinhammer [12]) were modified by the general procedure for nitration of proteins given by Sokolovsky et al. [8]. An approximately 50-fold molar excess of tetranitromethane was added to a Tris-buffered (50 mM) solution, pH = 8.0-, approx. 4 m g / m l in protein and allowed to incubate (at 20°C for stellacyanin and 0°C for cytochrome c) with gentle stirring for several hours. The protein was dialyzed extensively against the Tris buffer. The extent of nitration was determined spectroscopically by employing e248 4100 M 1. cm 1 for quantitation of nitrotyrosine [8]. :

Tyrptophan modification HNBB modification of tryptophan residues was carried out according to the procedure outlined by Horten et al. [9]. A 0.2 molar stock solution of HNBB (Sigma) was prepared in dry acetone immediately prior to use. Tryptophan was modified by adding 4.5 ml of this solution to 10 ml tryptophan in 0.5 M phosphate buffer, pH 3.1, and allowing the reaction to proceed for 1 h. Stellacyanin was modified by adding 1.5 ml of the HNBB solution to 20 ml of 35/,M protein in 1 M phosphate buffer, pH 3.1, and incubating for several hours in the dark, then dialyzing against the phosphate buffer. For quantitation of the reaction, a value of e410 18000 M 1. cm 1 was employed at pH > 10 [9]. =

Raman spectra UV resonance Raman spectra were obtained by focusing the pulses of an H 2 Raman-shifted N d : Y A G laser into a free-following stream or wire-guided sheet of recirculating samples and collecting the Raman signal with a scanning single monochromator equipped with a solar blind photomultiplier and integrating electronics. The apparatus is described elsewhere [13]. Stellacyanin samples were replaced after every second scan (approx. 30 min), while cytochrome c samples were replaced after every scan (approx. 15 min).

Results and Discussion

Tyrosine nitration Tetranitromethane reacts readily with tyrosine under mildly alkaline conditions to give 3-nitrotyrosine, which has a strong absorption band (~ = 4100 M 1.cm 1 ) a t 4 2 8 n m , shifting to 360 ( e = 2790 M 1.cm l) upon acidification [8]. These absorption bands can be used to quantitate the modification reaction, although the protein sample must first be dialyzed (or undergo gel filtration or tributylphosphate extraction) [14] to remove nitroformate formed during the reaction, which has a strong absorption band (e = 14400 M 1. cm i) at 350 nm. Fig. 1 shows the 200 nm-excited resonance Raman spectrum of 1 mM tyrosine before and after treatment with tetranitromethane. The 988 cm 1 band of the phosphate buffer (0.5 M, pH 8.5) serves as an intensity standard. Clearly

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Fig. 1. 200 nm-excited resonance Raman spectra of 1 mM tyrosine before and after nitration with tetranitromethane (TNM). The 988 cm-1 phosphate band (P) is due to the 0.5 M, pH 8.5, phosphate buffer, present as an internal intensity standard.

the tyrosine spectrum is eliminated by the nitration reaction, and the spectrum of the reacted solution shows only very weak contributions at approx. 1350 and approx. 1610 cm -~. These might be due to nitrotyrosine, or to the nitroformate side-product, which was not removed in this case. Fig. 2 shows absorption spectra for stellacyanin before and after tetranitromethane treatment. This is a 'blue' Cu protein with a characteristic 604 nm absorption band due to a thiolate ~ Cu(II) charge transfer transition [15]. It also exhibits a weak

(e = 950 M -~ • cm ~) absorption band at 442 nm, which has been suggested to arise from imidazole ---, Cu(II) charge transfer [15]. After tetranitromethane treatment and dialysis to remove nitroformate, the spectrum shows a strong 422 nm nitrotyrosine band. Using e = 4100, we calculate that approx. 4.7 of the seven tyrosine residues of the stellacyanin have been modified. The 604 nm absorbance is unaffected by the nitration reaction, indicating that the modified protein remains in the native conformation. Fig. 3 shows 200 nm-excited resonance Raman spectra for stellacyanin before and after tetranitromethane treatment. There is a clear intensity loss for tyrosine bands at 1612, 1595, 1265, 1207 and 851 cm-~. Most of the bands are overlapped by phenylalanine bands, or by the polypeptide amide III band ( = 1250 cm-1). The 851 cm 1 band is an isolated tyrosine mode, however, and can be used for quantitation. Using the intensity

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Fig. 2. Absorption spectra for stellacyanin before and after treatment with tetranitromethane (TNM). Conditions: 4.8 m g / m l stellacyanin in 50 mM, pH 8.3, Tris buffer.

Fig. 3. 200 nm-excited resonance Raman spectra of stellacyanin before and afer nitration with tetranitromethane (TNM). The approx. 1660 cm -1 amide I band serves as the intensity standard. The tyrosine band monitored is marked with an asterisk. Conditions: see Fig. 2. Data were collected at 0.05 A increments for 6 (unmodified) and 10 (modified) s per point.

76

ratio of this b a n d to that of the a m i d e I b a n d at 1660 cm , we calculate that 4.3 _+ 0.2 tyrosines can be modified. (The error limit is based on the estimate of c o m b i n e d reading errors in the intensity measurements.) This calculation is rendered s o m e w h a t uncertain by the d e p e n d e n c e of the 8 3 0 / 8 5 0 cm ~ T y r d o u b l e t intensity ratio on the H - b o n d i n g status of Tyr. W h e n Tyr is an H - b o n d d o n o r to a strong acceptor, the 8 3 0 / 8 5 0 cm intensity ratio increases [16]. This seems to occur in the t e t r a n i t r o m e t h a n e - t r e a t e d stellacyanin, suggesting that the T y r residues inaccessible to tetran i t r o m e t h a n e are strongly H - b o n d e d . I m p r o v e d signal-to-noise will be needed to confirm this indication. Despite this uncertainty, the a g r e e m e n t between the a b s o r p t i o n a n d R a m a n m e a s u r e m e n t s regarding the n u m b e r of modified tyrosines is encouraging. In the absence of a crystal structure for this protein, these results indicate that 4 - 5 of the tyrosine residues are on the surface of the protein, while 2 - 3 are buried. Fig. 4 shows a b s o r p t i o n and 200 nm-excited R a m a n spectra for horseheart c y t o c h r o m e c before and after t e t r a n i t r o m e t h a n e t r e a t m e n t and dialysis. The a b s o r p t i o n s p e c t r u m is d o m i n a t e d by the heme group whose intense 408 nm (Soret) b a n d and quite strong 360 nm b a n d obscure b a n d s of nitrotyrosine (360 nm) or n i t r o t y r o s i n a t e (428 nm), and make q u a n t i t a t i o n of the nitration reaction problematic. The 200 nm-excited R a m a n spectrum, however, contains no d e t e c t a b l e heme contribution. All of the b a n d s are a t t r i b u t a b l e to amide, p h e n y l a l a n i n e and tyrosine. A m a r k e d loss in the tyrosine b a n d intensities is seen after tetranitromethane treatment. The 860 c m l ( t y r o s i n e ) / 1 6 6 0 cm , (amide I) intensity ratio indicates that 2.1 _+ 0.2 of the four tyrosines in c y t o c h r o m e c have been nitrated. The crystal structure [17] shows that two of the tyrosines are buried and two are at the surface, so this result is exactly as expected. Both buried tyrosines are H - b o n d e d , one to a t h r e o n i n e - O H group, the other to a heme p r o p i o n a t e side-chain. The latter should form a strong H - b o n d and is expected to show an increased 8 3 0 / 9 5 0 cm -t intensity ratio. I m p r o v e d s i g n a l / n o i s e will be needed to evaluate this point.

Tryptophan modification 2-Hydroxy-5-nitrobenzyl

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Fig. 4. Absorption and 200 rim-excited resonance Raman spectra of horse heart cytochrome c before and after treatment with tetranitromethane (TNM). The approx. 1660 cm i amide I band serves as the intensity standard. The tyrosine band monitored is marked with an asterisk. Conditions: 3.4 mg/ml horse heart cytochrome c in 50 mM, pH 7.7, Tris buffer. The unmodified cytochrome c resonance Raman spectrum is from Ref. 5. Data were collected at 0.05 ,~ increments for 10 (unmodified) and 20 (modified) s per point.

forms a highly reactive i n t e r m e d i a t e due to reson a n c e stabilization of its incipient c a r b o n i u m ion [18]. A m o n g protein residues, t r y p t o p h a n is attacked most readily while cysteine reacts less than one-fifth as r a p i d l y as t r y p t o p h a n [18]. In alkaline solution tyrosine is also attacked, but in mildly acid solution this reaction is inhibited [9]. The m o d i f i e d t r y p t o p h a n has a red-shifted a b s o r p t i o n spectrum. A t p H a b o v e 10, an intense a b s o r p t i o n

b a n d (e = 18000 M -~ • c m -~) develops at 410 nm, a n d can be used for q u a n t i t a t i o n after removal of h y d r o l y z e d reagent, which will also a b s o r b strongly at this wavelength [9]. Fig. 5 shows 218 rim-excited resonance R a m a n spectra of t r y p t o p h a n before a n d after t r e a t m e n t with H N B B . The strong t r y p t o p h a n resonance R a m a n s p e c t r u m is c o m p l e t e l y eliminated, and r e p l a c e d with strong b a n d s at 1596 and 1344 cm ~, and weaker ones at 1291 and 1243 cm -~. These are due to h y d r o l y z e d H N B B , as shown in the top s p e c t r u m of the figure. In p r o t e i n - m o d i f i c a t i o n studies, this material is readily removed by dialysis or gel filtration [9]. Fig. 6 shows 218 nm-excited resonance R a m a n s p e c t r a for stellacynin, before a n d after H N B B

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Fig. 6. Absorption spectrum of stellacyanin after HNBB treatment, dialysis, and pH adjustment ( > 10) and the 218 nm-excited resonance Raman spectra of stellacyanin before and after treatment with HNBB (pH 3.1). The 1178 cm ] phenylalanine plus tyrosine band serves as the intensity standard. The tryptophan band monitored is marked with an asterisk. Conditions: 0.7 mg/ml stellacyanin in 1 M, pH 3.1, phosphate buffer. Data were collected at 0.05 ,~ increments for 6 (unmodified) and 10 (modified) s per point.

treatment, followed by dialysis (pH 3.1), a n d the a b s o r p t i o n s p e c t r u m for the modified, dialyzed, p H - a d j u s t e d ( > 10) protein. The 604 n m a b s o r p tion b a n d is unaltered by the modification, indicating that the p r o t e i n remains in its native conformation. The 408 nm a b s o r p t i o n b a n d a p p e a r s at p H 10, and its intensity indicates that one of the three stellacyanin t r y p t o p h a n residues has been modified. T h e 218 nm-excited resonance R a m a n s p e c t r u m shows the t r y p t o p h a n b a n d s at 759, 877, 1008 and 1550 cm L but also strong c o n t r i b u t i o n s from Tyr a n d Phe (1605, 1267, 1207, 1178 a n d 1000 cm 1), since stellacyanin c o n t a i n s 7 T y r a n d 5 Phe residues. H N B B t r e a t m e n t reduces the t r y p t o p h a n b a n d intensities relative to the rem a i n i n g bands. T h e ratio of the 759 cm I trypt o p h a n b a n d to the 1178 c m - t p h e n y l a l a n i n e plus

78 tyrosine peak gives an estimate of 1.1 _+ 0.2 out of the three t r y p t o p h a n s modified, consistent with the results from the a b s o r p t i o n spectrum. (It had been hoped that the 1 M phosphate buffer could provide an internal intensity standard, but its 871 cm 1 band overlaps with the 877 c m - 1 Trp band, while its 1076 cm ~ b a n d is too weak for reliable intensity measurement.) Evidently only one of the stallacyanin t r y p t o p h a n residues is at the surface of the molecule. The t r y p t o p h a n b a n d s of the HNBB-treated protein do not differ significantly from those of the untreated protein, suggesting no special e n v i r o n m e n t a l effect of the buried tryptophans, although the signal-to-noise needs improvement to confirm this. U n f o r t u n a t e l y the most e n v i r o n m e n t a l l y sensitive bands, the 1 3 5 0 / 1 3 3 0 cm 1 t r y p t o p h a n doublet [2], are weak, and are obscured in the treated protein sample by residual HNBB hydrolysis product.

Conclusion The present results d e m o n s t r a t e that UV reson a n c e R a m a n s i g n a l s from t y r o s i n e a n d t r y p t o p h a n can be eliminated by treatment with t e t r a n i t r o m e t h a n e and HNBB, respectively. The R a m a n intensities for these residues can be used to q u a n t i t a t e the extent of modification. The results for cytochrome c is entirely consistent with the two buried tyrosine residues being inaccessible to nitrating reagent. In the case of stellacyanin, 4 5 tyrosines and one t r y p t o p h a n are modified, and are p r e s u m a b l y at the surface of the protein. The ' b l u e ' Cu site is unaffected by the modification reactions. These reactions can be used to eliminate the c o n t r i b u t i o n of surface tyrosine and t r y p t o p h a n residues, exposing the buried residues to a detailed study of UV resonance R a m a n spectroscopy. The

presently available signal-to-noise will need to be improved somewhat to make such studies reliable.

Acknowledgements This work was supported by N S F grant C H E 79-09433 a n d N I H grant GM25158.

References 1 Rava, R.P. and Spiro, T.G. (1984) J. Am. Chem. Soc. 106, 4062 2 Rava, R.P. and Spiro, T.G. (1985) Biochemistry 24, 1861 3 Rava, R.P. and Spiro, T.G. (1985) J. Phys. Chem. 89, 1856 4 Johnson, C.R., Ludwig. M., O'Donnell, S. and Asher, S.A. (1984) J. Am. Chem. Soc. 106, 5008 5 Copeland, R.A. and Spiro, T.G, (1985) Biochemistry 24, 4960 6 Copeland, R.A. and Spiro, T.G. (1986) J. Am. Chem. Soc. 108. 1281-1285 7 Copeland, R.A., Dasgupta, S. and Spiro, T.G. (1985) J. Am, Chem. Soc. 107, 3370 8 Sokolovsky, M., Riordan, J.F. and Vallee, B.L. (1966) Biochemistry 5, 3582 9 Horlen, H.R. and Koshland, D.E,, Jr. (1965) J. Am. Chem. Soc. 87, 1126 10 Kumar, K. and Carey, P.R. (1975) J. Chem. Phys. 63. 3697 11 lzzo, G.E., Jordan, F. and Mendelsohn, R. (1982) J. Am. Chem. Soc. 103, 3178 12 Reinhammar, B. (1970) Biochim. Biophys. Acta 205, 35 13 Fodor. S.P.A., Rava, R.P., Copeland, R.A. and Spiro, T.G. (1986) J. Raman Spectrosc., in the press 14 Beaven, G.H. and Grazen, W.B. (1968) Biochim. Biophys. Acta 168, 456 15 Gray, H.B. and Solomon, E.I. (1981) in Copper Proteins (Spiro, T.G., ed.), Vol. 3, pp. 1-39, John Wiley & Sons, New York 16 Siamwiza, M.N., Lord, R.C., Chert, M.C., Takamatsu, T., Harada, I., Matsuura, H. and ShimanouchLT. (1975) Biochemistry 14, 4870 17 Dickerson, R.E., Takano, T., Eisenberg, D., Kallai, O.B., Samson, L., Copper, A. and Margoliash, E. (1971) J. Biol. Chem. 246, 1511 18 Means, G.E. and Feeney, R.E. (1971) Chemical Modification of Proteins, pp. 123 125, Holden-Day, San Francisco