Kinetics of cyanoborohydride reduction of bovine rhodopsin

Yisian Rrs Vol. 18. pp. 483 to 488 Q Pergamon Press Ltd 1978. Printed

in Great Britain

KINETICS

OF CYANOBOROHYDRIDE REDUCTION OF BOVINE RHODOPSIN

ROGER S. FAGER*, PATSY C. GENrncoREt and E. W. ABRAHAMSON~ *Department of Physiology, University of Virginia School of Medicine, Charlottesville, VA 22903, U.S.A.; and tDepartment of Chemistry, University of Guelph. Guelph, Ontario, Canada (Received 28 July 1977) ~yanoborohydride reduces bovine rhodopsin’s chromophore. The linear dependence of the kirietics on reductant concentration and the pH and temperature dependence of the reaction are evidence for penetration into the hydrophobic pocket of the native molecule. This supports earlier

Acted-S~ium

work indicating a SchifI’s base linkage between the retinal chromophore chain. Key Words-rhodopsin;

Bos domesticus; Schifl’s base.

INTRODUCTION

in a further precipitate very similar to that obtained by cooling. The two precipitates were pooled and resuspended in 85Oml ethyl acetate. To this was added 550ml p-dioxane. The entire solution was put in an ice-bath to cool and stirred for 1h during which time a precipitate appeared. This cooled solution was suction-filtered and the precipitated product was obtained. The wet product was dried in vacuum at 80°C for 2 days and stored in tightly stoppered flasks. At this point, sodium cyanoborohydride was a white crystalline solid.

The retinal-based chromophore of the visual pigment rhodopsin which resides in the rn~br~~ of the retinal rods isomer&s upon light absorption from il-cis form to tram and thereby initiates a series of chemical events which lead to visual excitation. Because of this, understanding the nature of the Iinkage between retinal and opsin and any changes in this linkage upon light exposure is a key question in understanding the visual process. In preliminary work (Fager, Sejnowski and Abrahamson, 1972) we have established that the reagent sodium cyanoborohydride (sodium hydrido cyanoborate) reduces the imino linkage of the retinylidine chromophore to rhodopsin in the dark. Basic hydrolysis showed only a single retinyl derivative-Nretinyl lysine. In the present study, we have investigated the reduction in terms of the reductant concentration, PI-I, temperature, and the nature of the deterSent used, to verify by a kinetic study that the reduction does, in fact, take place at the native binding site of the chromophore.

Assay of reagent

The assay for cyanoborohydride was based on that for sodium borohydride of Lyttle, Jensen and Struck (1952). This consisted of oxidizing cyanoborohydride with an excess amount of potassium iodate and back-titrating with sodium thiosulfate to determine the excess. The end-point is found by disappearance of color of a ICI/soluble starch indicator system. To make certain that the reagent was stable at the p&I’s used, the reagent was incubated at a concentration of Zmg/ml (titration had shown there to be 37% hydration), with 0.4M buffer, using sodium formate at pH 3, sodium acetate buffer at pH 4 and 5, and sodium phosphate at pH 6 and 7. Iodometric titrations were made after the solution stood for .5&20 and 65 hr to determine how much cyanoborohydride had decomposed.

EXPERIMENTAL PROCEDURE Purifkation ofthe

and a lysine in the peptide

NaBHsCN

Sodium cyanoborohydride was obtained from Alfa Inorganics of Beverly, MA. It was purified by a method based on that of Borch, Bernstein and Durst (1971). Purification was an absolute necessity, since the commercial material was highIy colored. Approximately 50 g of NaBHsCN were added to approximately 400 ml of tetrahydrofuran (THF), forming a dark brown solution. To this was added enough of a 1 M HCI methanol solution to bring the final mixture to a pH of 9.0, estimated by pH paper. This solution was mixed with 12SOml dioxane and a light brown precipitate was formed. The precipitate was suction-filtered, re-washed in approximately loo0 ml dioxane, and suction-filtered again. The wet solid was added to 12SOml ethyl acetate. After stirring for 23 h the solution was cooled in an ice-bath. The precipitate formed on cooling was suction filtered. 6CVml pdioxane were added to the filtrate, resulting

Preparation of rhodopsin

All steps, until monitoring of the reduction. were carried out under dim red light. 200 dark-adapted bovine eyes from the Geo. Hormef Co., Austin, MN. were suspended by homogen~ation in 0.047M sodium phosphate/42% sucrose. Sufficient sucrose buffer was stirred in to bring the total volume to 280 ml. This was divided among eight 50ml centrifuge tubes; 4ml of buffer without sucrose were layered on the top of each tube and the tubes centrifuged for 15 min at 27,ooOg in a Sorvall Superspeed Refrigerated Centrifuge. This brought the rods to the interface boundary from which they were aspirated up by pipette and bulb. The rods were then washed three times by suspending in buffer without sucrose, and pelleted by centrifuging at 27,000~ for 15 min. They were then washed twice in chilled distilled water by homogenizing the pellet into 80ml of distilled water and centrifuging at 27,OOOgfor 30min. 483

484

ROGERS. FAGER. PATSY C. GENTILCOREand E. W. ABWAHAMSOS

The rod preparation was extracted with lOOmI of Emulphogene buffer (Shichi. Lewis. lrreverre and Stone. 1969) (19, Emulphogene BC 720, 0.01 M sodium phosphate. pH 7.0) by stirring for 1hr at room temoerature in the dark and pelleting- the unextracted residue at 27,000y for 15min. The spectral ratio (OD2so/OD,,,I of the crude extract was 3.5. The rhodopsin was concentrated on an Amicon Model 52 stirred cell with an XM-1OOAfilter to a volume of 50ml. This extract was purified in two equal amounts on a calcium phosphate column based on the preparation of Shichi et al. (1969). 15 g calcium phosphate and 20 g Celite were mixed in I”/, Emulphogene. 0.005 M sodium phosphate pH 7. The adsorbant, in dense slurry, was poured into a column 2.5cm in diameter and allowed to-pack under a pressure of 25psi nitrogen. The rhodopsin was aoolied to the .* column and was driven through at the same pressure. Samples of 25ml were collected from the column and a spectrum taken of each. The purest rhodopsin was collected and concentrated. Fractions with an 0D,,,/OD2,, ratio better than 1.85 were pooled and the spectral ratio of these pooled fractions (which amounted to about 75”, of the starting material) was 1.75. The ratio OD,,,/OD,,, was 0.22. Monitoring of

the reduction

The reduction was observed at three pH’s-3.30, 4.30 and 5.00. For each pH, the concentration of NaBH,CN was varied such that the half-life of reduction ranged from approximately 2 to 5 min. The reaction was buffered with 0.08 M sculium formate at pH 3.3 and 0.08 M sodium acetate at pH 4.3. At pH 5.0. where much higher levels 01 reducing agent were used, 0.25 M sodium acetate was used as the buffer. The buffer was mixed with the reducing agent titrated to the desired pH and taken up to volume. Reaction mixtures of 2.5 ml were used in a 3 ml quartz cuvette. The rhodopsin was added last, the mixture shaken and immediately put into the Cary 14, monitoring for the disappearance of the 500 nm band. The reactions were run for approximately 10 min. At this time the reaction mixture was light-exposed, and an A, was obtained, which is the slight absorption of NaBH,CN at 5OOnm. This absorbance is used to calculate the corrected absorbance to plot on a logarithmic scale. Control

of temperaiure

Reactions were run in a 5 cm water-jacketed cylindrical cell; the temperature was controlled by a Haake Circulating Temperature Bath and both rh+opsin and reagent solutions were immersed in the bath prior to mixing. Effecf of detergent The above experiments were performed by use of rhodopsin in Emulphogene BC 720. A check was made to see the effect of different detergents on the reaction. In addition to Emulphogene (GAF Industries. New York) CTAB (cetyltrimethylammonium bromide, from Eastman Chemicals, Rochester, New York), digitonin (Nutritional Biochemicals. Cleveland, Ohio), and Triton X-100 (Rohm & Haas, Philadelphia) were also investigated. A 1% final solution of each detergent was used buffered with 0.08 M sodium acetate. pH 4.3.

RESULTS Reagent stability

Table 1 shows that the breakdown negligible

in the reaction

periods.

of reagent is

pH: 5f hr 20 hr 65 hr

Table I. Stability of cyanoborohydrldc __-._ ._. _ Percentage of initial reagent present as shown by iodometric titration 3.42 4.30 4.85 5.56 6.60 74”, 54 13

92”, 90 70

loo”,, I W., 100 IIN) 03 87 --_____-.---

I(K)“,,

/oo 96 .-._

pH and concentration dependence Figure I(a, b. c) shows the time course of the reducchromophore at various reductant concentrations and at pH’s 3.30, 4.30 and 5.00. Throughout the entire course of the reaction the

tive attack on the rhodopsin

logarithmic plots show that the reaction is quite accurately first order. Secondary plots in Fig. 2( a, b, c) show that the kinetic constants are linear in redudant concentration for all three cases. For pH 4.3 and 5.0 they extrapolate quite close to the origin; for pH 3.3 the intercept corresponds to an appreciable reaction velocity. The slope (the molar rate constant with OD,,, used as a unitless quantity) is 17.0min-’ mole- ’ for pH 3.3, 1.70min-‘mol-’ for pH 4.3, and 0.4min-’ mol-’ for pH 5. Figure 2d shows that the plot of log molar rate constant vs pH is a straight line, and that therefore the reduction rate is proportional to hydrogen ion concentration under all conditions examined. Temperature dependence

Figure 3a shows the reaction velocity at 11.5, 20.0 and 25.o”C. Figure 3b shows an activation plot from this data. AH* is 22.5 kcal/mole and AS* is 14.7 entropy units. Dependence of rate on detergent

The molar rate constant at pH 4.3 was 0.023 mole - ’ min - ‘ for digitonin and 0.27 mole-’ min-’ for Triton X-100, as compared to 1.70min-’ mole- ’ for Emulphogene at the same conditions. With rhodopsin in CTAB there was a massive precipitation. The same precipitation occurred with only CTAB and cyanoborohydride and no rhodopsin: apparently an insoluble uricharged complex formed between the hydrophobic anion and the hydrophobic cation. DISCUSSION

The first thorough-going series of chemical studies supporting the idea of a Schiff base chromophoric binding site was the work of Morton and his coworkers (Morton and Pitt, 1955) who clearly established that the spectral behavior of acid-denatured rhodopsin matched that of synthesized Schiff bases of retinal with various amines. Similar experiments by Hubbard (1%9) using guanidine denaturation have supported the idea of Schiff base binding. Bownds and Wald (1965) demonstrated that while sodium borohydride had no effect on native rhodopsin, it rapidly reduced the chromophoric linkage after light exposure. Bownds (1967) and Akhtar, Bless and Dewhurst (1968) identified the binding site after reduction

under these conditions

as an amino

group

Reduction of bovine rhodopsin

0

I

I

I

I

I

I

I

2

3

4

5

6

Ttme,

mln

Time,

min

2

I I l

I

I

I

I

2

3

4

5

Time,

I 6

min

Fig. 1. Time course of reduction of bovine rhodopsin by cyanoborohydride as a function of reductant concentration. a. pH 3.3: x----x----x, 1.80 x lo-‘M; n ----m----m, 1.44 x lO-2 M; 0 ----r-~---. 0,

1.08 x lo-’ M; O---+---O. 0.72 x IO-* M; o----o----o, 0.36 x IO-’ M. b. pH 4.3: x---- x----x. c1----Cl----Cl, 1.80 x 10-l M; W---&---m, 1.44 x IO-‘M; 1.08 x 10-l M; .----+---., 0.72 x 10-l M; O----O----O, 0.36 x 10-I M. c. pH 5: B----&---m, 7.0 x 10-l M; O----O----D. 5.2 x 10-I M; e----.----O,

3.5 x 10-l M; o---o---o,

of a lysine residue of the protein chain, On the basis of the binding site in the light-exposed form, it was assumed that lysine also served as the binding-site in the native form. Workers in this laboratory (Poin~lot, Millar, Kimbe1 and Abrahamson, 1969, 1970) found that when lyophihzed native bovine rod cells or lyophilized native rhodopsin freed from detergent were extracted with acidified methanol, the rhodopsin retinal chromophore was solubilized as a SchitT base, which was identified after sodium borohydride reduction by a variety of &mica1 and physical methods to be N-retinyl ph~p~tidyl ethano~ine, i.e. the reduced

1.8 x 10-l M.

Schiff base of retinal and the phosphatidyl ethanolamine. When native rhodopsin was heat-denatured in the dark in the presence of borohydride, again the product observed was N-retinyl phosphatidyl ethanolamine. When the same experiments were performed after light exposure. a lysine binding site was demonstrated, consistent with the earlier work. When dark-adapted lyophiliied rods were light exposed, the reaction proceeded as far as metarhodopsin I and stopped, since water is required for the metarhodopsin I-+metarhodopsin II reaction, which fell in a time range after light exposure which made it a good candidate for the reaction linking light and the chemi-

486

ROGER S. FAGER. PATSY C. GFNTILCORE and E. W. ABRAHAMSON

pH

3.3

I

I

I

I

05

I

IO

Concentration,

I 15

MXl02

I

I

I

2

3

\ 4

I

I

5

6

65(b)

64-

MXIO

Concentration,

al 0 E . $ Y

63-

:: c I*\

62-

2

61

Concentration,

M

60

I

I

I

3.4

3.5

3.6

-

I

T(‘K)

x IO

3

Fig. 3. Temperature dependence of reduction of bovine rhodopsin by cyanborohydride. a. Time course: c)----c) ---- 0. 10°C; .___+__*, 20°C; q____lJ____o, 26°C. b. Activation plot. cal response

m

1

molar

kinetic

constant

Fig. 2. Kinetic constants reduction of bovine rhodopsin at various pH’s and reductant concentrations. a. pH 3.3, b. pH 4.3, c. pH 5.0, d. Molar rate constants as a function of pH.

leading to vision. It formed a very appealing model for the excitation process. Although the initial observations are reproducible, later work in various laboratories has made a lipid binding site in native rhodopsin untenable. Hall and Bacharach (1970) Anderson (1970). Borggreven, Rotmans, Bonting and Daeman (1971) and Hubbell (personal communication) presented evidence that native rhodopsin could be stripped of phospholipid to the degree that there was considerably less than 1 mole of ethanolamine phospholipid per mole of rhodopsin under conditions which maintain rhodopsin’s native

487

Reduction of bovine rhodopsin spectrum. Work in this Iaboratory indicated that the binding site of squid rhodopsin was a lysyl residue both before and aRer light (Fager, Kimbel and Abrahamson, 1971). Zorn (1971) and Girsch and Rabinovitch (1971) published reports of aqueous sodium borohydride reduction at 37°C in the presence of a high urea concentration. Both lysine and phosphatidyl ethanolamine binding were found. We attempted to find a reductant which could penetrate to the binding site better than sodium borohydride, This meant a more hydrophobic borohydride derivative, since the evidence indicated the retinal binding site lay within a hydrophobic pocket of the molecule. Cyanoborohydride seemed ideal since, in addition to being more hydrophobic than borohydride, it was much more acid stable and highly specific for protonated Schiff bases (Borch et al., 1971). The data in Table 1 demonstrate that cyanoborohydride is stable for days under conditions where borohydride would break down to hydrogen in seconds. Cyanoborohydride does undergo a similar breakdown, but the ratio of reducing power to breakdown is more favorable by a factor of IO5 (Borch et al., 1971). This yields an additional advantage: since gas production is insignificant. continuous monitoring of the kind we have carried out is possible. In preliminary work we found cyanoborohydride would reduce bovine rhodopsin under mild conditions (PH 5. 3°C) and that the only retinyl derivative found after basic hydrolysis was N-retinyl lysine (Fager et nl.. 1972). At the same reductant concentration and at pH 7, where cyanoborohydride has little reducing power, the 500nm band of rhodopsin was stable. Likewise. rhodopsin itself is quite stable at pH 5. This implied that cyanoborohydride was directly reducing the rhodopsin chromophore rather than first denaturing rhodopsin, thereby exposing the ~hromophore for reduction. Hall (1975) has shown that rhodopsin can also be reduced by borane dimethylamine, a hydrophobic boron hydride of similar chemistry. However, the conclusion could not be made with complete rigor, since it was possible that cyanoborohydride acted as a non-specific denaturant only at acid pH’s. The question of whether there is a direct attack on the binding site or a nonspecific denaturation followed by reduction is a key one, because it is essential to know this to conclude that the reduced product is the native binding site. This question can be settled with considerable certainty by studying the detailed kinetics of reduction. If cyanoborohydride were a non-specific denaturant, then one would expect a very low rate constant of reduction at low concentrations and for the rate to rapidly accelerate upward with a high power of the concentration since the denaturation would be by the cooperative interaction of destabilization at many points of the molecule. Hubbard (1969) observed just such an effect with guanidine hydrochloride denaturation. On the other hand, if the reaction was a direct attack on the native binding site, one would expect the kinetic constant of rhodopsin reduction to be first order in cyanoborohydride concentration. From Fig. 2 it is clear this, in fact, is the case;

the kinetic constants of pH 3.3, 4.3 and 5.0 are accurately linear in reductant and therefore one can conclude with considerable certainty that the reduction at these conditions is at the native binding center. The data at pH 3.3, where denaturation is clearly taking place, shows an interesting combination of nonspecific denaturation and direct reduction. At pH 3.3. the kinetic constant can be described as the sum of a constant term and a term proportional to cyanoborohydride. The latter term is evidence for a similar direct attack; the former is a non-specific denaturation due only to the pH, as evidenced by the fact that rhodopsin breaks down at this rate with no reductant present. Therefore, at pH 4.3 and 5.0 one can view with confidence the reduction at the native binding site and, likewise at pH 3, provided the reductant concentration is high enough that non-specific denaturation is negligible in comparison to direct reduction. Therefore this. coupled with our earlier findings and those of other laboratories, strongly argue that lysine is the chromophore binding-site of rhodopsin. The temperature dependence of the reaction is also consistent with a small molecule penetration and inconsistent with denaturation. Typical activation parameters for denaturation are AS* of 6&70, eu AH* of 100 kcal/mole. Here. the AH* of 22.5 k~l/mole and AS* of 14 eu point to no major change in the macromolecular structure of rhodopsin, but rather to a simple specific attack at one point in the molecule. The rate of reduction increases sharply with decrease in pH. At all pH’s the rate of reduction is proportional to the hydrogen ion concentrations. This proportionality has been seen for small molecule reductions by this reagent. The differences of behavior for the different detergents might be expected from other measurements. Rhodopsin solubilized in digitonin retains its ability to regenerate the native spectrum after bleaching in the presence of added II-cis retinal, and retains the circular dichroism behavior of rhodopsin in the rod membranes (Shichi, 1971). Therefore, it is believed that the conformation of rhodopsin in digitonin is a good approximation of that in the membrane. For Triton X-100 or Emulphogene BC-720 neither of these properties are retained although, as in digitonin, the native visible spectrum is identical to that in the membrane. Therefore, it is not surprising that for Triton X-100 and Emulphogene BC-720, where the molecule is somehow distorted from the native form, that reductive penetration is much faster than in digitonin. Aeknowledyements-This work was supported by research grants from the Nationai Eye Institute of the National Institutes of Health. EY-00209 to E.W.A. and EY-01505 to R.S.F. REFERENCES

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Borch R. F.. Bernstein cyanohydridoborate

M. D. and Durst H. D. (1971) The anion as a selective reducing agent, J. .Am clwn. Sm. 93, ‘897 2904. Borggreven J. M. P. M.. Rotmans J. P.. Bontlng S. L. and Daemen F. J. M. (1971) The role of phospolipids in cattle rhodopsin studied with phospholypase c. .4r~,/1,s Biochm. Biophvs. 145. ?9l$ 299. Bownds D. (1967) Site of attachment of retinal in rhodopsin. Nuru,r 216, 1178 11x1. Bownds D. and Wald G. (I 9651 Reaction of the rhodopsin Chromophore with sodium borohydride. Nature 205, 154.-257. Fager R. S., Kimbel R. L. and Abrahamson E. W. (1971) Molecular properties of squid rhodopsin. .4hstr. Biophys. SW. 45a. Fager R. S.. Sejnowski P. and Abrahamson E. W. (1972) Aqueous cqanohydridoborate reduction of the rhodopsin chromophore. Bioc~hcm. hiophp. Rm. Commun. 47, 1243 1247. Girsch S. J. and Rabinovitch B. (1971) Bleaching of rhodopsin in the dark. Biochrm. hiophvs. Rrs. Commun. 44, 550 556. Hall M. 0. (1975) Reduction of the retinal-oosin linkare in intact frog rod outer segments. .3sroc. ~CS. O@hl. lis., Abstr. 31-4. Hall M. 0. and Bacharach A. D. E. (1970) Linkage of

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