Biochimica et Biophysica Acta, 439 (19761 426 431

~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 37380 R E I N T E R P R E T A T I O N OF LUMINISCENCE PROPERTIES OF NEUROTOXINS F R O M T H E VENOM OF MIDDLE-ASIAN COBRA N A J A O X I A N A EICHW.

BUKOLOVA-ORLOVA~, E. A. PERMYAKOV", E. A. BURSTEIN ~ and L. YA. YUKELSON ~ T. G.

~lnstitute t~f Biological Physics, Academy of Sciences of the U.S.S.R., 142292 Pushchino, Moscow Region and "Institute of Biochemistry, Academy t~fSciences of the Uzbek. S.S.R., Tashkent ( U.S.S.R. )

(Received October 30th, 1975) (Revised manuscript received March 9th, 1976)

SUMMARY A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya. (1974) Biochim. Biophys. Acta 342, 275-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantum yield value, the shortened phosphorescence lifetime ( 8 0 ~ of the emission has zp < 0.5 s, 2 0 ~ has zp ~ 4.8 s, comparing with usual zp = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I - (0.455) is much higher than tha t for the positively charged Cs + (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive'quenchers is even more pronounced in the case~of the neurotoxin'l'(1.04~and'0"~0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together*with the-analysis-of theramino'acid sequence, suggest that the space folding'=of this' polypeptide-results in'~the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).

INTRODUCTION Knowledge of protein amino acid sequence gives a basis for-more"adequate and precise interpretation of spectroscopic data. Therefore, the publication of the

427 primary structures of neurotoxins I [1] and I1 [2, 3] (see Fig. 1) from middle-Asian cobra venom enabled us to better characterise the localization and environment of tyrosine and tryptophan residues in these proteins, using their fluorescence properties. In our previous work [4], we based the interpretation of the fluorescence features of these properties on the known primary structures of related cobra neurotoxins assuming their close homologies. However, it was found later [2, 3] that middle-Asian cobra neurotoxins have rather unusual content and positions of fluorescent aromatic residues. It turned out that both neurotoxins have two tryptophans per molecule. In the "short" neurotoxin II (61 residues) tryptophan residues are positioned side by side in positions 27 and 28, and in the "long" neurotoxin I (73 residues) the tryptophan residues are in positions 27 and 31 on both sides of the disulfide bridge Cys(28)-Cys(32). While the neurotoxin II, like most of the related toxins, has only an invariant Tyr-24, the neurotoxin I apart from the invariant Tyr-23 contains two more tyrosyls (Tyr-4 and Tyr-53). Tyr-4 in toxin I, like the invariant residue in both toxins, is adjoined to the disulfide bond Cys(3)-Cys(22)

TI

.

.

.

.

.

TII

.

.

.

.

.

~~gs~S ~ C ~-., u~

~-

*', ~

.

,-. . . . . . . (CuslSerP~

d, ~ - , ~ . . . . ~(Cus

Fig. 1. The covalent structures of neurotoxins 1 (A) and II (B) from the middle-Asian cobra venom. Owing to discovery of these new facts, we have attempted to give a more adequate interpretation of the data published earlier [4] and some new results on the luminescence of the Naja oxiana neurotoxins. MATERIALS AND METHODS The characterization of the preparations, the measurements and the treatment of fluorescence spectra were presented in detail in our previous paper [4]. The low temperature luminescence spectra (fluorescence and phosphorescence) were measured with a specialized recording instrument made in the Institute of Biophysics (Puschino, U.S.S.R.).

428 The low temperature luminescence was excited by light of mercury line at 289.7 nm, isolated by the grating monochromator from the emission of the superhigh pressure lamp SWD-120. The sample was contained in an open brass cell that was plunged into a transparent quartz Dewar flask tilled with liquid nitrogen. In order to prepare the sample, the solution was poured out into the cell (precooled at 77 ' K ) and covered with a brass lid. After freezing, the lid was removed. The sample thickness was 0.2 cm. The luminescence light was focused on the entrance slit of the grating monochromator SPM-2 (Karl Zeiss, Jena, G.D.R.) from the [¥ont surface of the sample at a right angle to the exciting beam direction. Spectral slit width of the monochromator was ~ 1 nm. Photomultiplier FEU-39 (Sb-Cs cathode) was used as a detector of fluorescence light. Photocurrent was registered on a recording potentiometer EPP-09M3. All the luminescence spectra were corrected for the spectral sensitivity of the instrument. The correction was made point by point with 2-nm steps. Intensities in the corrected spectra are proportional to the number of photons per unit wavelength interval. To measure phosphorescence lifetime values %, the phosphorescence decay curves were registered at 460 nm after rapidly closing the exciting beam. The phosphorescence lifetime value was estimated from the slope of a straight line that was obtained from the plot of In(P~/P°Oversus t (P~ and P~, phosphorescence intensities at initial and arbitrary moments t). Besides the main long-living component of phosphorescence ( ~ ~ 4-6 s). the short living one is also present in protein phosphorescence spectra (~v < 0.5 s). The contributions of the long living (P~) and short living (P~) emission in the total spectra of protein phosphorescence were estimated from the decay plots after their extrapolation to the zero time poivt. RESULTS AND DISCUSSION

First of all, knowledge of tyrosine and tryptophan content in the neurotoxins enabled a more accurate estimating of fluorescence quantum yield values of tyrosine residues in aqueous protein solutions at 20 °C. Such an estimation required the calculation of correction coefficients for screening effects of tryptophan residues on the excitation of tyrosyls. These coefficients W~, at the excitation wavelength 2 were calculated according to equation [5]: W,~ --

1 -- TTr~ 1 - - TTyr'TTrI~

ATyr -~- ATr~ ATy r

where A is absorbance, T = 10 -A is the transparency of the solution containing tyrosine (index Tyr) or tryptophan (index Trp) in concentrations equal to those in the protein solution under study. Molar extinction coefficients at 2 = 280 nm were 1340. M - 1. cm - 1 for tyrosine and 5600. M - 1. cm - 1 for tryptophan. The tyrosine component of the total emission spectrum excited at 280 nm was isolated by means of subtraction of the pure tryptophan component (measurements at 296.7 nm excitation where only tryptophanyls absorb) from the total fluorescence spectrum obtained at 280 nm excitation following their intensity equalization at 370 nm at which wavelength the emission of tyrosyls is practically absent.

429 Fluorescence quantum yield values of tyrosine component obtained were equal to 1.3 ± 0.3 ~o for neurotoxin II and 0.45 ~ 0.3 ~o for neurotoxin I. Such a considerable difference of tyrosyl fluorescence yields may be due to the possibility that at least one tyrosine residue in neurotoxin I is practically nonfluorescent, and serves as a trap for the energy absorbed by the other chromophores and transferred by the F6rster resonance mechanism. Tyr-4 may play the role of that trap because of its position near the Cys(3)-Cys(22) disulfide bridge which is an effective quencher of tyrosine fluorescence [6]. This interpretation suggests rather short mutual distances between tyrosine phenolic moieties in the macromolecule. It seems highly probable if one takes into consideration that disulfide Cys(3)-Cys(22) draws together Tyr-4 and Tyr-23 (Fig. 1). Moreover, investigations of iodination of neurotoxin I demonstrated that a product of the modification is an iodotyronine structure which is possible only if these moieties are close together [7]. The effective energy transfer to tryptophan residues may be an additional cause of the low fluorescence quantum yield of tyrosine residues in toxin I. As noted earlier [4], the maximum position (2m 345--347 rim) and the half width (A2 -- 57-58 nm) of tryptophan fluorescence spectra of both toxins suggest the localization of the emitting centers on the protein globule surface in contact with rather fast relaxing water molecules [8]. However, in spite of the presence of two tryptophan residues per molecule in both proteins, their fluorescence parameters 2,, and A2 strictly correspond to one-component spectra [8]. It may be a result of the fact that, due to the covalent structures, both indole chromophores in each of the two toxins are close to one another, at distances which enable a highly effective resonance energy exchange between them. While the neurotoxin II fluorescence quantum yield value (q, 21 ~k l~o) at room temperature strictly corresponds to the surface tryptophan residues of class III [8], the fluorescence of toxin I is less than one third as much (q = 6.5 ± 0.7 ~). Taking into consideration the data on the primary structure of the latter, it is highly probable that such strong additional quenching is brought about by the contact of the indole moieties of Trp-27 and Trp-33 with the disulfide bond Cys(28)-Cys(32). For an examination of this assumption we made use of the fact that the contact with heavy sulphur atoms must result in an increase of the probability of electron spin reversal and hence of the rates of singlet-triplet and triplet-singlet interconversions. In turn, it has to induce the fluorescence quenching, the phosphorescence lifetime shortening and, in many cases, the phosphorescence quenching. Therefore, the luminescence spectra of the neurotoxins at low temperature (77 °K) were measured in comparison with spectra of some other proteins. These spectra normalized in fluorescence maxima are seen in the Fig. 2. The phosphorescence lifetime of proteins devoid of disulfides in contact with emitting indole moieties (chymotrypsinogen, human and bovine serum albumin, papain) is 5.5-5.9 s and the ratio of phosphorescence intensity at 460 nm to fluorescence intensity at 355 nm (P/F) is about 0.4-0.7 (Table 1"). On the other hand, the lifetime of the long-living phosphorescence component of neurotoxin l, like that of lysozyme (which contains 4 tryptophan residues in contact with disulfides [9], is lowered to 4.8 s (3.8 s for lysozyme), and the P/Fratios drop as low as 0.042 (compare 0.037 for lysozyme). Moreover, about 8 0 ~ of neurotoxin I phosphorescence has a lifetime shorter than 0.5 s, although usually the protein phosphorescence at 460 nm

430

~0

~- 0.5

U','.7.q'i" -

E

•

300

%.,7

;

"

" ~ -

c ~

400

"-c

""~ -

500

~

Fig. 2. Luminescence spectra of neurotoxins I G . . . . . ) and 11 ( -) at low temperatures (77 "K), ,.~ ~--, in comparison with that for lysozyme (,~--., ~) and chymotrypsinogen ( . . . . ). All proteins are in snowy frozen aqueous solutions. The spectra are equalized at maxima[ fluorescence intensities. c o n t a i n s n o t m o r e t h a n 50 ~,,i o f s u c h s h o r t - l i v i n g e m i s s i o n . T h e s e f a c t s s u g g e s t t h a t i n t h e n e u r o t o x i n I m o l e c u l e t h e t r y p t o p h a n r e s i d u e s a r e a c t u a l l y in c o n t a c t w i t h t h e disulfide. F o r n e u r o t o x i n II, it w a s d e m o n s t r a t e d t h a t t h e P / F r a t i o (0.21), p h o s p h o r e s c e n c e l i f e t i m e (5.5 s) a n d t h e c o n t r i b u t i o n o f s h o r t - l i v i n g e m i s s i o n a t 460 n m (0.64) have values much closer to those obtained for unperturbed tryptophan residues. TABLE I THE CHARACTERISTICS OF LOW T E M P E R A T U R E L U M I N E S C E N C E OF N E U R O TOXINS I A N D II IN COMPARISON WITH THAT OF SOME OTHER PROTEINS ).~x, the excitation wavelength; P . P~, the intensities of, respectively long and short living components in the emission at 460 nm; F, the fluorescence intensity at 356 nm; rp, the lifetime of long living phosphorescence ; ;~0-o,the wavelength of 0-0 electron transition in the phosphorescence spectra ; Ps/(P~ ~ Ps), the relative contribution of short living emission in the total phosphorescence intensity at 460 nm. Protein

,)rex (nm)

P1/F

Ps

zp (s)

2o-o (nm)

P~ !-P~ Neurotoxin I Neurotoxin I1 Lysozyme ct-Lactalbumin

289.7 289.7 289.7 289.7

0.042 0.212 0.037 0.187

0.787 0.636 0.546 0.575

4.76 5.50 3.77 3.77

407.0 412.5 416.0 411.5

Chymotrypsinogen A Bovine serum albumin Papain Human serum albumin

289.7 289.7 289.7 289.7

0.406 0.576 0.684 0.588

0.043 0.208 0.316 0.446

5.89 5.78 5.50 5.56

413.0 408.5 410.0 410.0

431 However they still show 1he tendency characteristic of the presence o f a weak heavy atom. Therefore, it ~eems possible that the polypeptide chain folding in this toxin enables also some closing o f Trp-27 and Trp-28 side moieties with one of four disulfide bonds in the molecule. The tryptophan fluorescence quenching by the neighbouring disulfide in neurotoxin I is in accordance with the relatively small increase of fluorescence yield during protein denaturation by heat, urea or extreme pH values. I m p o r t a n t information about the three-dimensional structural features o f the neurotoxins can be obtained from the data on fluorescence quenching by anions ( I - ) and cations (Cs+). The main results of the quenching experiments have been communicated earlier [4]. It has been shown that the tryptophan fluorescence o f neurotoxin II is effectively quenched by iodide (accessibility in relation to that o f the free tryptophan is 0.455) and much less effectively by cesium (relative accessibility is 0.08). These facts suggest a perfect exposure o f tryptophans to the solvent and the localization of a small positive charge besides. The contributions in this charge may belong to Lys-25, Lys-26 and His-31. However, neurotoxin I fluorescence quenching by the ions demonstrates a stronger positive charge: the relative accessibility for I - is 1.04 (approx. 0.5-0.6 for denatured proteins [8]) and for Cs + is 0 ~ 0.02. At the same time, the n u m b e r of cationic residues in the neighbourhood of tryptophyl residues in the primary structure o f the toxin I is less than that of the toxin II (only Lys-25). The explanation o f this fact therefore seems to be in some peculiarities o f the polypeptide chain folding. In this connection, the most significant peculiarity o f neurotoxin I is the C-terminal peptide region which is absent in toxin II and contains four cationic amino acid residues (the residues 67-73: - H i s ( + ) - P r o - L y s ( + ) - G l n L y s ( + ) - A r g ( + ) - P r o ( - - ) ) . Thus, one can assume that the peptide chain in neurotoxins is folded in such a way that C-terminal region o f toxin I is located in the vicinity of Trp-27 and Trp-31. Some kind o f the two-dimensional representation of such a situation is given on Fig. 1. ACKNOWLEDGMENTS The authors are grateful to Mrs. A. S. Beskodarova for her productive technical assistance. REFERENCES 1 Grishin, E. V., Sukhikh, A. P., Slobodyan, L. N., Ovchinnikov, Yu. A. and Sorokin, V. M. (1974) FEBS Lett. 45, 118-121 2 Grishin, E. V., Sukhikh, A. P., Lukyanchuk, N. N., Slobodyan, L. N., Lipkin, V. M., Ovchinnikov, Yu. A. and Sorokin, V. M. (1973) FEBS Lett. 36, 77-78 3 Ryden, L., Gabel, D. and Eaker, D. (1973) Int. J. Pept. Protein Res. 5, 261-272 4 Bukolova-Orlova, T. G., Burstein, E. A. and Yukelson, L. Ya. (1974) Biochim. Biophys. Acta 342, 275-280 5 Burstein, E. A. (1968) Biofizika 13, 433~42 6 Cowgill, R. W. (1967) Biochim. Biophys. Acta 140, 37 44 7 Gussakovsky, E. E. (1975) Thesis, Tashkent 8 Burstein, E. A., Vedenkina, N. S. and Ivkova, M. N. (1973) Photochem. and Photobiol. 18, 263 .. 279 9 Longworth, J. W. (1971) in: Excited States of Protein and Nucleic Acids (Steiner, R. F. and Weinryb, I., eds.), pp. 319-484, MacMillan, London