The sulfhydryl groups of rhodopsin

BIOCHIMICA ET BIOPHYSICA ACTA

PRELIMINARY

409

NOTE

BBA 41 050

The sulfhydryl groups of rhodopsin* WALD AND BROWN1 demonstrated t h a t the photolysis of rhodopsin exposed several sulfhydryl groups and rendered them titratible b y AgNO 3 in ammonium buffer at p H 9. Although it has been conjectured that the exposure of these sulfhydryl groups accompanies the neural trigger reaction 2 the specific stage in the sequence of thermal reactions at which the exposure occurs has not been determined. ERHARDT, OSTROY AND ABRAHAMSON3 showed that illumination of rhodopsin at --29 °, where metarhodopsinaTs m~ is normally stable, does not uncover titratible sulfhydryl groups, while 3 sulfhydryl groups react with Ag(NHs)2 + (pH 9.5) after illumination at room temperature. On the basis of this evidence we concluded that the SH groups were uncovered after the completion of the lumirhodopsin497m~ ~ metarhodopsin47smt~ reactions. It would seem a simple problem to relate the exposure of SH groups to some stage in the decay of metarhodopsin b y merely stopping the reaction at the appropriate stage and titrating with a sulfhydryl reagent. We found 4, however, that on incubation of rhodopsin with the sulfhydryl-blocking reagent p-mercuribenzoate prior to illumination, neither metarhodopsinaTs m# nor metarhodopsin3s0 m/~ were detectable as intermediates in the process. Sulfhydryl analysis with Ag(NH3)2 + involves a preliminary titration of exposed sulfhydryl groups on rhodopsin prior to illumination at p H 9-9.5. This, therefore, raises the question of the extent to which the very alkaline p H ' s and altered SH groups affect the intermediates in the normal thermal decay sequence (pH 5-8). In this note we wish to describe some experiments which m a y clarify this point. We have employed the technique of BENESCH, LARDY AND BENESCH5 for the titration of sulfhydryl groups with AgNO 3 in Tris buffer (Ag(Tris)2+) under neutral p H conditions. Assuming only SH groups react with Ag(Tris)2+ we found 8 that 4 sulfhydryl groups per rhodopsin molecule m a y be titrated initially in the dark. In the presence of excess titrant, upon illumination, 4 additional sulfhydryl groups are titrated. Because this titration is performed at neutrality, it is possible to investigate directly the effect of this procedure on the normal sequence of thermal intermediates following illumination and to correlate the exposure of SH groups with a particular stage in this sequence. When the 4 " d a r k " sulfhydryl groups are titrated prior to illumination no change can be observed in the 498-m~ maximum. Upon illumination, however (Fig. I), metarhodopsin47sm~, the predominant intermediate under normal conditions, is no longer evident under the conditions of the experiment and metarhodopsin3somt~ is now the chief product. The subsequent thermal decay of metarhodopsin3som~ to metarhodopsin465m~ appears to proceed normally. If the metarhodopsin3somt,-~ metarhodopsina6~m~ reaction is allowed to go to completion and additional Ag(Tris)~+ * Taken in p a r t from a doctoral dissertation by S. E. OSTROY,Case I n s t i t u t e of Technology, April 1966.

Biochim. Biophys. dcta, 126 (1966) 4o9-412

410

Iq¢lClA M I N A R Y

NI)ll

a d d e d at the metarhodopsin46~mt, stage an a d d i t i o n a l I---2 SH groups can be titrat('d. Similarly, if rhodopsin solutions are i r r a d i a t e d w i t h o u t prior t i t r a t i o n ()f SH gr(~ut)s. 5-6 SH groups are t i t r a t i b l e at the completion of the metarhodopsinas0m/~ ~ metarhodopsin465 m/~ reaction. W h e n the d a r k t i t r a t i o n is carried out a n d excess t i t r a n t is a d d e d no significanl change in t h e 498-m/z p e a k is observed. Upon illumination, however, m e t a rhodopsin478 mt~ is c o m p l e t e l y absent. In addition, no f o r m a t i o n of metarhodopsin465 mt, is o b s e r v e d a n d t h e p e a k at 38o m/, decays v e r y slowly to p r o d u c t s w i t h o u t d e t e c t a b l e a b s o r p t i o n in the visible region of t h e s p e c t r u m . A t t h e same t i m e 4 a d d i t i o n a l moles of Ag(Tris) 2+ are t a k e n up per mole of r h o d o p s i n at a r a t e c o m p a r a b l e with the n o r m a l metarhodopsina80m# -> metarhodopsin46sm/~ process b u t much faster t h a n the disa p p e a r a n c e of t h e p e a k at 38o m/x. The r a t e of c o n s u m p t i o n of Ag(Tris),i is t e m p e r a t u r e d e p e n d e n t , requiring approx. I - 3 h at 3 ° a n d 5--3o min at 25'~" b u t the kinetics are too complex for a s t r a i g h t f o r w a r d analysis. T h e s p e c t r a which result from illumin a t i o n in t h e presence of excess t i t r a n t are shown in Fig. 2. Dark titration Rhodopsin4~s m/~ 4 SiJI groups per rhodopsin No excess titrant retinal387 m,~+ + opsin

~[{()4P~m/~ "~,, +: NI~Ov;5 ,n,,

metarhodopsin4~5 m/~ (Maximum 1-2 additional SH groups titratible) (Total 5--6 SH groups if no dark titration)

lihodopsin498 m1~ [)ark titration 4 SH groups per rhodopsin + excess titrant

Scheme ~.

hv (l.ittle or no) -37; + lnetarhodopsin47,~ m,.

nletarhodopsin3,~t,

,~

h*, -~ metarhodopsin;,,,, llltt 3' 4 additional i SH groups Product not absorbing in visible region of spectrunl

On t h e basis of t h e Ag(Tris) 2 ~ e x p e r i m e n t s which are s u m m a r i z e d in Scheme I it w o u l d a p p e a r t h a t t h e t i t r a t i o n of S H groups in t h e presence of excess Ag(Tris) 2 has e l i m i n a t e d t h e i n t e r m e d i a t e s metarhodopsin478 m# a n d metarhodopsin4~5 mp in the t h e r m a l sequence of reactions in t h e photolysis of rhodopsin. This suggests t h a t the p a t h w a y s to t h e p r o t e i n c o n f o r m a t i o n s r e p r e s e n t e d b y these i n t e r m e d i a t e s are in some w a y a l t e r e d b y t h e u p t a k e of Ag(Tris)~+. A l t h o u g h the m e c h a n i s m of this a l t e r a t i o n is u n k n o w n a t this time, it m a y b e significant t h a t t h e i n t e r m e d i a t e s which are affected, metarhodopsin4v8m/~ a n d metarhodopsin465m~, result from t h e r m a l reactions which have large n e g a t i v e entropies of a c t i v a t i o n a,4 i n d i c a t i v e of a refolding or charge reordering process. There is t h e possibility, however, t h a t t h e failure to observe a p r o d u c t a b s o r b i n g in t h e visible region of t h e s p e c t r u m (Fig. 2) resulting from the t h e r m a l d e c a y of metarhodopsinasomt, m a y arise from a complexing reaction of Ag(Tris) 2+ w i t h t h e exposed c h r o m o p h o r e at t h e b i n d i n g site in a d d i t i o n to the normal reaction of Ag(Tris)2+ with the exposed S H groups. T h e complexing reaction m a y Biochim. Biophys. Acta, a20 (i966) 4o9-412

PRELIMINARY NOTE

4II

lead to a much slower thermal process b y which the 38o-m~ peak disappears. Ag(Tris)~+ consumed at the binding site could lead to an overestimate of the number of SH groups uncovered b y illumination, but would not affect the conclusion about the position in the thermal decay sequence where the SH groups are exposed, i.e., the thermal decay of metarhodopsin3s0m~. 0.5

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400 WAVELENGTH

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0.1

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300

400

500

6 0i 0

,

,

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WAVELENGTH

Fig. I. P h o t o l y s i s of c a t t l e r h o d o p s i n a f t e r t i t r a t i o n of i n i t i a l 4 s u l f h y d r y l g r o u p s w i t h Ag(Tris)2 +. O r i g i n a l r h o d o p s i n A49s m# = 0.542 (I cm). Ten ml of r h o d o p s i n (1.29- lO -5 M) w a s a d d e d t o 20 ml of Tris buffer 5. A g N O s (I. IO -s M) was a d d e d (at r o o m t e m p e r a t u r e ) u n t i l t h e first t r a c e of add i t i o n a l c u r r e n t w a s noted. The s i l v e r n i t r a t e a d d e d a m o u n t e d t o 5.4" lO-7 mol e s or 4.19 m o l e s / m o l e r h o d o p s i n . Six ml of t h e t i t r a t e d r h o d o p s i n was t h e n p l a c e d i n t o a 2-cm cell t h e r m o s t a t e d in t h e Cary I I a t a p p r o x . 3 °. S p e c t r u m i w a s t a k e n before t h e i r r a d i a t i o n of t h e r h o d o p s i n . S p e c t r u m 2 was s t a r t e d a t 700 m # (scan t i m e 5 m#/sec), 0. 3 mi l l a f t e r t h e e nd of t h e 4 - m i n i r r a d i a t i o n 4. S p e c t r u m 3, 3 .o rain; s p e c t r u m 4, 9.2 rain; a n d s p e c t r u m 5, 20 rain a f t e r t h e e n d of t h e i r r a d i a t i o n . F i n a l s o l u t i o n p H was 7.50. S p e c t r u m 2' w o u l d h a v e be e n o b s e r v e d i m m e d i a t e l y a f t e r i r r a d i a t i o n if no t i t r a t i o n h a d b e e n performed. Fig. 2. P h o t o l y s i s of c a t t l e r h o d o p s i n in t h e p r e s e n c e of excess Ag(Tris)2 + a f t e r t i t r a t i o n of t h e i n i t i a l 4 " d a r k ' s u l f h y d r y l groups. O r i g i n a l r h o d o p s i n A 498 ma = o.48 (I cm). F i v e ml of r h o d o p s i n (1.14. IO 5 M) was a d d e d to 25 m l of Tris buffer 5. A g N O 3 w a s a d d e d a m o u n t i n g to 6. lO -7 mol e s or lO. 5 m o l e s / m o l e r h o d o p s i n r e s u l t i n g in a n excess of 6. 5 m o l e s of Ag(Tris) 2+ pe r mol e of r h o d o p s i n . F i f t e e n ml of t h e s o l u t i o n w a s t h e n a d d e d to a 5-cm cell a n d t h e r m o s t a t e d in t h e C a ry i i a t IO °. S p e c t r u m I wa s s t a r t e d a t 700 m F (scan speed 5 m/~/sec) 0. 4 m i n a f t e r t h e e nd of t h e 4-rain irr a d i a t i o n 4. S p e c t r u m 2, 16.1 m i n ; s p e c t r u m 3, 32 m i n ; s p e c t r u m 4, 81 rain; s p e c t r u m 5, 3 h 36 m i n ; a n d s p e c t r u m 6, 18 h 18 m i n a f t e r t h e end of t h e i r r a d i a t i o n . F i n a l s o l u t i o n p H w a s 7-47. A b s o r b a n c e s are o n l y r e l a t i v e as b l a n k d i g i t o n i n s o l u t i o n w a s f o u n d t o be more t u r b i d t h a n t h e .solution of p i g m e n t .

Currently, we are applying other sulfhydryl reagents to this study. Of particular interest is the behavior of rhodopsin on incubation with ELLMAN'S reagent 6 (5,5'dithiobis-(2-nitrobenzoic acid)) which reacts readily with available SH groups. At p H 7.0 it was found that only 1-2 of the S H groups are immediately reactive in the dark and 2-3 further groups are always readily exposed upon illumination. Upon prolonged dark incubation in a closed cell, depending on the temperature, as m a n y as 8-1o SH groups have been detected. In summary, the major inferences to be drawn from this work are: (I) The titration of the SH groups on the rhodopsin molecule with Ag(Tris)~+ largely destroys the integrity of the intermediate metarhodopsin47sm/~. (2) The exposure of additional Biochim. Biophys. Acta, 126 (I966) 4o9-412

412

PRELIMINARY NOTE

SH groups s u b s e q u e n t to the i l l u m i n a t i o n of rhodopsin occurs after the formation of metarhodopsin380m/~ a n d therefore in p o i n t of time c a n n o t be associated with a neural trigger reaction. This i n v e s t i g a t i o n was supported b y a g r a n t from the N a t i o n a l I n s t i t u t e of Neurological Diseases a n d Blindness, No. Nb-234o.

Department of Chemistry, Case Institute of Technology, Cleveland, Ohio (U.S.A.)

SANFORD E. OSTROY* HARRY RUDNEY** E. W. ABRAHAMSON

I G. V~'ALD AND P. BROWN, dr. Gen. Physiol., 35 (1952) 797. 2 G. ~rALD, P. BROWNAND I. GIBBONS,J. opt. Soc. Am., 53 (I963) 20. 3 F. ERHARDT, S. E. OSTROY AND E. W. ABRAHAMSON, Biochim. Biophys. Acta, It2 (1960) 256. 4 S. E. OSTROY, F. ERFIARDT AND E. W. ABRAHAMSON, Biochim. Biophys. ,4cta, 112 (1966) 265. 5 R. E. BENESCH, H. A. LARDY AND R. BBNESCH, J. Biol. Chem., 216 (1955) 663.

6 G. L. ELLMAN,.4rch. Bioehem. Biophys., 82 (1959) 7o. Received J u l y 7th, 1966 * Present address: Department of Chemistry, Cornell University Ithaca, N.Y. (U.S.A.) ** Research Career Awardee U.S. Public Health Service on leave of absence from Western Reserve University, Cleveland, Ohio (U.S.A.).

Biochim. Biophys. dcla, 126 (1966) 4o9-412