Exp. Eye Res. (1979) 29,393-399

Ethanolamine Attack of the Bovine Rhodopsin Chromophore ROGER S. PAGER Department of Physiology, Uwiversity of Virgbaia Medid Charlottesville, Vu. 22901, U.S.A. L.

STUART

GOLDMAN

AND

Department of Chemistry, Uwiversity

of

School,

E. W. ABRAHAMSON

Guelph, Guelph, Odario, Canada

(Received18 March 1977 and in revisedform

15 March 1979, Zezc! York)

Ethanolamine attacks the rhodopsin chromophore, bleaching the visual pigment in the dark. Rhodopsin breakdown is first order under all conditions and the kinetic constants of breakdown are linear in ethanolamine concentration, suggesting that it is a specific attack on the retinal binding site, rather than a non-specific denaturation of the molecule. The rate of breakdown accelerated markedly at basic pH’s in the range of the pK’s of the protein’s

basic amino acids, implying that charge repulsion distorts the rhodopsin molecule, making ethanolamine Key words:

penetration ethanolamine;

easier. rhodopsin;

visual

pigment;

Schiff

‘a base;

retinal

binding

site.

1. Introduction Rhodopsin is the pigment of the retinal rods and receptor protein for scotopic vision. Light is absorbed by a retinal-based chromophore which is generally believed to be attached by a Schiff’s base linkage and buried deep in a hydrophobic pocket (Rimai, Kilponen and Gill, 19’73;Lewis, Pager and Abrahamson, 1973) basedon the resistance of rhodopsin to chemical attack by reagents such as hydroxylamine (Wald and Brown, 1954) and sodium borohydride (Bownds and Wald, 1965). In this study, we have investigated chemical attack by use of ethanolamine, which we viewed as a more

hydrophobic analogue of hydroxylamine, attack by a study of the kinetics.

and have investigated the mechanism of

2. Methods Preparation of rhodopsin

All operations until monitoring were carried out under dim red light. Water-washed retinal rods were prepared from 100 dark-adapted bovine retinas purchased from the George A. Hormel Co., Austin, Minnesota, by the method of Zorn and Futterman (1971) except for omission of a Tergitol wash. The rods were extracted with 75 ml of 1% Emulphogene BC 720 (obtained from General Aniline and Film Co., New York, N.Y.) for 1 hr at room temperature and the residue pelleted by centrifugation at 2’7 000 x g for 15 min in a Sorvall RC-2 centrifuge. The rhodopsin was concentrated to 30 ml on an Amicon Model 52 stirred cell with an XM-100A filter. This concentration method we fmd to be rapid and mild, and to give quantitative recovery of rhodopsin. The rhodopsin was purified by the method of Shichi, Lewis, Irreverre and Stone (1969) on a 2.5 cm diameter column packed with 15 g calcium phosphate and 20 g Celite. Purijication of ethanolamilze Reagent grade ethanolamine from Matheson Coleman by vacuum distillation at 5 mm pressure and 70°C. 00144835/79/109393+07

Bell, Norwood,

Q 1979 Academic

$01.00/0 393

Ohio, was purified

Press Inc. (London)

Limited

TWe wactiorb

mixture

The pH of the reaction mixture wits set by titration of the ethanolamine concentrate with HCI. Throughout the experiments reported, the ethanolamine itself was used to buffer the reaction. The Emulphogene concentration was ly& of the reaction mixture, 0.75 ml of rhodopsin solution added to 0.75 ml of ethanolamine solution in a 3 ml quartz cell with 1 cm path length. Monitoring

of the reaction

The reaction of the rhodopsin chromophore was monitored by disappearance of the 500nm band with a Cary 14 spectrophotometer. Reagents were mixed under red light (Kodak Wratten No. 1 filter) with rhodopsin added last. Spectrophotometric monitoring began 90 see after mixing the reagents.

Effect of

temperature

For the temperature-controlled experiment a Haake circulating bath was used. Both reagent and rhodopsin were kept in the temperature bath prior to mixing and during the reaction the temperature was maintained by use of a jacketed cell. The remainder of the experiments were run at room temperature, 26fl”C.

3. Results Time courseof

the reaction

The spectrophotometer (b), (c), (d)] are cIearly

readings plotted on a semilogarithmic first order at all pH’s and concentrations

scale [Pig.

l(a),

run,

-

l-00

(b

o-99

0.90

pH IO.25

5

0.96

0

2

4

0.85 6

8

IO

12

Time

14

0

2

4

6

8

(min)

FIG. 1. Time courz(e of disappearance of 600 nm band of bovine rhodopsin in the presence of various concentrations ofethanolamine at (a) pH 9.75, (b) pH 10.25, (c) pH IO.50 and (d) pH 10.75, all at 26&l% (0---0)0~33M;(~---.)0~67M;(~----~)1~00~.

ETHANOLAMINE

0.5

ATTACK

OF

RHODOPSIN

395

I.0

0 x L-

Concentration

FIG. 2. Kinetic constants of ethanolamine attack (b) pH 10.25, (c) pH 10.50 and (d) pH 10.75.

(M)

of the bovine

rhodopain

chromophore

(a) pH 9.75

2400,

PH

3. pH dependence rhodopsin chromophore. FLC?.

of the molar

first order

rate constants

for ethanoiamine

attack

of the bovine

When the kin&c constants art: plottetl against the reagent cc~nccntration, it. is clear that t,he reaction rate is Iinear in reagent, concentration at all pH‘s (Fig. 2). lt can be seen that the rate of rhodopsin attack by ethanolamine rspitll\- acot~lttrates at, pH’s beyond 10 (Fig. 3). It should be noted t*hat the rapid chqy of rate (10~s wt accompany the titration of the ethanolamine. which possessesa pK of 9.5. At 1)1-I 9.25 where there is significant concentration of uncharged ethanolamine tht: reaction rate is essentially zero. Temperalure

dependence

Figure 4(a) showsat temperatures between 10 and 35°Cthe reaction (run at pH 10.5, 0.75 M-ethanolamine) is first order. Figure 4(b) showsthat since a plot of the log (kinetic constant) vs. 1/T is accurately linear over the temperature range studied. AH, the enthalpy of activation, is calculated to be 28.9 kcal/mol and dS *, the entropy of activation, calculated to be +31.7 entropy unit)s. 1

o-0

2

I 4

I 6 Time

8

1 3.4

IO

(mid

&

I 3-6

x IO3

Fro. 4. Temperature dependence of ethanolamine attack of the rhodopain chromophore. (Run at pH 10.5,0.75M ethanolamine.) (a) Time course of thereaction (O---O) 11°C; (a---e) 19°C; (n---o) 26°C; (H---m) 33°C; (b) activation plot.

4. Discussion There is a considerable body of evidence that the retinal chromophore of vertebrate rhodopsin is bound to the molecule by a Schiff’s base linkage. Denatured rhodopsin shows spectral behavior typical of a Schiff’s base (Morton and Pitt, 1955). Light-exposed rhodopsin undergoes oxime formation in the presence of hydroxylamine and is reduced by sodium borohydride (Bownds and Wald, 1965), reactions characteristic of Schiff bases.Rimai, Kilponen and Gill (1970) and Lewis, Fager and Abrahamson (1973) have shown that the retina has a Raman absorption band matching model Schiff basesof retinal in the protonated form. Rhodopsin in the native (i.e. not light exposed) form is also believed to possessa Schiff’s base linkage between retinal and lipoprotein. This is partiy based on the

ETHANOLAMINE

ATTACK

OF

RHODOPSIN

397

fact that when rhodopsin is denatured in various ways, the denatured form clearly is a retinal Schiff base (Hubbard, 1969; Poincelot, Pillar, Kimbel and Abrahamson, 1969). Native rhodopsin, however, lacks some of the typical chemical properties of a Sch.8 base, namely the spectrum is pH-independent over a range from pH 3 to pH 11, whereas a spectral shift of over 100 nm would be expected for a Schiff base (Morton and Pitt, 1955). Further, the rhodopsin molecule shows no reaction at pH 7 with hydroxylamine (Wald and Brown, 1954) or sodium borohydride (Bownds and Wald, 1965). This finding has been interpreted by most workers in the field as evidence that before light exposuxe, the chromophore is buried in a hydrophobic pocket deep in the molecule and is therefore inaccessible to polar reagents. However, the same evidence has prompted a few workers to suggest other forms of chemical linkage between retinal and the lipoprotein (Heller, 1968) although such suggestions are not compatible with the observed spectrum. kecent work in this laboratory (Pager, Sejnowski and Abrahamson, 1972; Fager et al., in preparation) has shown that if one uses sodium cyanoborohydride rather than sodium borohydride, a rapid and quantitative aqueous reduction of the rhodopsin chromophore occurs. Hall (1975) has shown borane dimethylamine can similarly penetrate and reduce the chromophore. This is made possible by the specificity of these reagents for protonated Schiff bases, their greater stability in acidic aqueous solutions and their greater hydrophobicity. Consistent with the earlier view, t,he binding site is lysine (Fager et al., 1972). The kinetic constants of reduction are linear in reductant concentration, so the rate-limiting step is first order in cyanoborohydride. This step, therefore, is more likely a reduction of the lipoprotein-chromophore linkage rather than denaturation preceeding the reduction of the exposed chromophore. Hydrolysis and clvomatographic identification of the chromophoric binding site shows binding only to an amino group of lysine. Tb.is is strong evidence supporting the idea of a retinal linkage through a Schiff base in a hydrophobic pocket of the molecule. In the experiments reported here we have shown that by using ethanolamine, a it is possible to demonstrate more hydrophobic molecule than hydroxylamine, chemical attack with kinetics and activation parameters coCst,ent with penetration and trsnsiminisation of the retinylidene chromophore. For a nonspecific denaturant such as methanol or urea, one would expect the rate constant of denaturation to be negligible at low concentrations and increase with a high power of the denaturant concentration, since denaturation in such a case is cooperative effect of multiple destabilizations. For a specific chemical attack, however, one would expect the rate limiting step to be first order in the attacking species and therefore for the rate constant of denaturation to be linear in denaturant concentration under all conditions. This is, in fact, the case for all conditions we have observed (see Fig. 2). The rate of reaction increases strongly in a temperature range where rhodopsin itself is quite stable (rhodopsin spectrum is unaffected by temperatures up to 50°C). The entropy and entbalpy of activation of 28.9 kcal and 31.7 e.u. respectively, are consistent with this view of a local attack by a small molecule rather than an overall denaturation since they are much smaller in magnitude than activation parameters of protein denaturation or even for the transformations between the rhodopsin photo-intermediates (Abrahamson and Ostroy, 1967). These &dings support the view that ethanolamine bleaches rhodopsin by penetration into the hydrophobic pocket in the molecule where retinal is bound in a Schiff bases linkage and that the

R. S. FAGER,

398

S. L. COLTJI\IAN

AND

K:. R. ABRAHAMSOS

failure of hydroxylamine to attack the chromophore is due to its failure to peneht P. At basic pH’s there is some attack of rhodopsin chromophore by hydroxylamine. hut is much less than that of ethanolamine. for instance at pH 10.5 the bleaching of rhodopsin with hydroxylamine is approximately 30-fold slower than with the corresponding concentration of ethanolamine. Johnson and Williams (1970) studied hydroxylamine attack on rhodopsin in Triton X-100 which, like emulphogene, is a polyethylene oxide detergent. As we do, they see a first order dependence on the nueleophile and a general increase in reaction velocity at basic pH’s, although the pH ranges in the two studies do not overlap. The kinetic parameters are much smaller than those we see with ethanolamine, consistent with our own tests with hydroxylamine. Shichi and Somers (1975) studied the transfer of retinal from phosphatidyl ethanolamine to opsin. They found that no such transfer takes place with ethanolamine Schiff’s base. This is consistent with the irreversible reaction we see. We would expect there to be a steric factors as well as factors of hydrophobicity in the penetration of the hydrophic pocket. That is, if the amine molecule is too large, it might not penetrate readily. This is consistent with Bridges’ findings (1957) that rhodopsin is stable in the presence of long chain amines but that with short chain amines the chromophore is bleached. The pH dependence of the reaction (see Pig. 3) shows that while the uncharged form of ethanolamine is undoubtedly the attacking form: the rapid rise in rate with rise in pH is considerably shifted to the basic side of its pK of 9.5. Therefore, the titration of the lysine and arginine residues of the protein backbone is probably involved. It seems likely that increase of net negative charge on the molecule as the basic amino acids are titrated introduces strains on the molecule which open the structure and assist penetration. (There are, however, no significant spectral shifts of the rhodopsin chromophore in this pH range.) Bleaching of rhodopsin by penetration into the binding site, then, is affected by at least three factors, the polarity of the attackin, n reagent, the size of the attacking reagent and the conformation of the rhodopsin molecule at the pH used. ACKNOWLEDGMENTS

This work was supported by Grants EY-00471 and EY-00209 from the National Eye Institute, National Institutes of Health to EWA and EY-01505 to RSF. REFERENCES Abrahamson, E. W. and Ostroy, S. E. (1967). The photochemical and macromolecuiar aspects of vision. Prog. Biophys. Molec. Biol. 17, 179-218. Bownds, D. and Wald, G. (1965). Reaction of the rhodopsin chromophore with sodium borohydride. Nature (London) 205,254. Bridges, C. D. B. (1957). Cationic extracting agents for rhodopsin and their mode of action. Biochem. J. 66,375-83. Fager, R. S., Sejnowski, P. and Abrahamson, E. W. (1972). Aqueous cyanohydridoborate reduction of the rhodopsin chromophore. Biochem. Biophys. Res. Commun. 47, 1244. Hall, M. 0. (1975). Reduction of retinal-opsin in intact frog outer segments. ARVO Abstract, 32-4. Heller, J. (1968). Structure of the visual pigments II. Binding of retinal and conformational changes on light exposure in bovine visual pigment,,,. Biochemistry 7, 2914. Hubbard, R. (1969). Absorption spectrum of rhodopsin: 500 nm absorption band. Nature (London)

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ATTACK

OF

RHODOPSIN

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H., Lewis, M. S., Irreverre, F. and Stone, A. L. (1969). Biochem. of visual pigments Purification and properties of bovine rhodopsin. J. Biol. Chem. 244,529-000. Shichi, H. and Somers, R. L. (1975). Rhodopsin regeneration from retinylidine phospholipid. PhotochewL. Photobiol. 22, 187-91. Wald, G. and Brown, P. K. (1954). The molar extinction of rhodopsin. J. Ben. Physiol. 37,189-000. Zorn, M. and Futterman, S. (1971). Properties of rhodopsin dependent on associated phospholipid. J. Biol. Chem. 246, 881-000.

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