- Email: [email protected]
Extractant effects on some properties of rhodopsin
EXTRACTANT BARBARA N. BAKE&’
EFFECTS ON SOME PROPERTIES OF R~~~~~S~~ WILLIAZV J. DO~OVAH’ and THEOIXNZP. WKLLG&~S~
‘.31nstitute of Mol~lar Biophysics, and %epartrnent of Psychology, Florida State University, Tallahassee, FL 32306, U.S.A. (Received 6 February 1976; in revised form 17 March 1977) Abstrac+--Tht photosensitivity, thermal stability and rate of metarhodopsin II production of rhodopsin solubilized in five commonly used extracting agents have been determined and compared with rod outer segment suspensions. An effect of .extracting agent on photosensitivity was found, indicating that the electronic interactions of the chromophore and protein are sensitive, io the miceLIar environment. Both the rate of thermal decay and metarhodopsin II production were found to be a function of the detergent. Onfv digitonin gaye rates which were comparable to those seen in rod outer segment suspensions.
Rhodopsin, the visual pigment of rod photoreceptors, is an intrinsic protein of the disc membrane and, like other proteins of this class, its solubilization requires the use of detergents. Over the years, a large number of detergents have been employed’ by various investigators in their studies of the bleaching behavior and molecular structure of rhodopsin. These have included both ionic and non-ionic detergents which contain hydrophobic aliphatic chains of varying length as we11as surfactants which contain bulky cyclic or aromatic structures as their hydrophobic moiety. Of the ionic detergents, ~tyltr~ethyl~onium bromide (CTAB) is perhaps the most commonly used (Bridges, 19.57; Heller, 1968) but the shorter chain, tetradecyl and dodecyl ammonium bromide (TT.AB, DTAB) have also been employed (Hong and Hubbell, 1972). The twitter-ionic detergent, Ammonyx LO (AL0 or LDAO) which is a mixture of tetradecyl and dodecyldimethylamine oxide, has been used by Ebrey (1971) and Busch, Applebury, Lamola and Rentzepis (1972) for probation and study of rhodopsin. Applebury, Zuckerman, Lamola and Jovin (1974) utilized the single com~nent, dod~yldimethyl~~e oxide, in preparing rh~o~in for r~ombinant studies. Among the non-ionic surfactabts, the most commonly used are T&on X-l@& (Crescitelli, 1967) and Emulphogene BC-720 (Shichi, Lewis, Irreverre and Stone I969), both of which are polyoxyethyleneglycol derivatives. The non-ionic detergent, digitonin, has been utilized for many years for extraction of rhodop sin from disc membranes (Wald and Brown, 1952). Along with the bile salts, sodium cholate and sodium taurodeoxycholate, digitonin is an example of a detergent which contains a bulky hydrophobic group. According to the present view of detergent action, soIubiI~ation of membranes involves the formation of mixed micelles with membrane lipids and of mixed micelles which contain both lipids and proteins ~eIenius and Simons, 1975). As the con~n~ation of
detefgent is increased above that required to induce the lamellar-rni~~e phase transition, separation of lipids from proteins occurs. Thus the degree to which lipids are dissociated is a function of the detergent concentration. In the case of ionic detergents, once lipids have been. displaced, further increase in detergent concentration leads to the binding of detergent to the protein thereby giving rise to an unfolded state of’the protein. The extent of unfolding is, of course, dependent on the detergent concentration (Tanford, 1958). The mild, non-ionic detergents by virtue of their lower CMC do not bind proteins in a cooperative manner, but the interaction is viewed as a complexing of detergent with the exposed hydrophobic portion of the protein thereby producing a soluble protein-detergent complex which may or may not contain iipid depending on the detergent concentration. Thus, the state of the membrane protein following. solubilization with detergent is a function of both the nature and concentration of the detergent. A wide variability in lipid content has been reported for rhodopsin extracted in the various detergents previously mention&. ~~~ylt~rne~yla~ monium bromide (Hong and Hubbell, 1972) and dod~yl~methyla~ne oxide (AppIebury et aL, 1974) have been shown to solubilize rhodopsin free of almost all phospholipids (0.5 mol phospholipid~mol of rhodopsin) following chromato~phy on a hydroxyapatite column. Osborne, Sardet and Helenius (1974) have reported the purification of rhodopsin devoid of lipid by use of the non-ionic detergents Triton X-100 and Emulphogene BC720. Complete removal of lipids necessitates that rod outer segment mem* branes be dissociated by at least 10 times their weight of these non-ionic detergents. At the other extreme, purification of rhodopsin solubilized in digitonin produces a system in which 90-11Omol pho~holipid/ mot rhodop~n have been reported (Shichi, 1974). In view of the established importance of lipids to regenerability of rhodopsin (Hong and Hubbell, 1972; Shichi, 1971) it seemed wo~hwhile to compare some other properties of rhodopsin extracted in commonly
1157
used detergents. In this paper. we report the measurement of photosensitivity, thermal stability and rate of metarhodopsin II production following light absorption in two ionic detergents. CTAB and LDAO; in two non-ionic aiiphatic detergents, Triton X-100, Emu~phogene BC-720 and in the non-ionic, bulky hydrophobic detergent, digitonin. For the sake of comparison, some measurements of thermal stability and rate of metarhodopsin II production have also been made in the more native rhodopsin environment, namely sonicated rod outer segment membranes. METHODS
A.VD 41ATERLkLS
Rod outer segments (ROS) were obtained from darkadapted bovine retinae (George Hormel Company, Austin, MN) by Hot&on on sucrose. Batches of 50 frozen retinae were homogenized in 45% sucrose, and were floated. Following the first flotation. ROS were pelleted by dilution of the sucrose with 67 mM phosphate buffer, pH 6.5 and centrifugation at 10,000~ for 15 min. The pelleted ROS were reiuspended in 40% sucrose, layered with buffer and suun at lO.OCi3 (1 for 30 min. The ROS were collected from the sucrose-b&er interface by means of a syringe fitted with a long needle. They were washed with 67 mM phosphate buffer, pH 6.5, spun, washed with distilled water, spun and resuspended in buffer. The suspension was then divided into six equal aliquots, spun and the resultant pellzts frozen at - IYC until used. Extraction of ROS was accomplished by adding deters.ent in the ratio of OScm’ detergent/retina. The suspension was gently homogenized and stirred in the dark for l-4 hr depending on &e extracting agent. AH extractants were prepared in 67 mM phosphate buffer, pH 6.5. Digitonin (Fisher) was used at a concentration of 2% (w/v). The CTAB concentration (Sigma) was 1% (w/v). Emulphogene BC-720 (gift of Geneial-Aniline Co.j was also utilized at lY/, concentration Iv/v). Triton Xl00 (rrift of Rohm & Haa$ was used at a cb&ntration of 2%&/v) for thermal stability measurements and 17; (v/v) for determination of the rate of meta II production. The higher conc~tration for thermal decay studies was utilized to prevent aggregation resulting during denaturation. Solutions of 1.5% LDAO and 3% LDAO (v/v) were prepared by dilution of a 30% stock solution provided by Onyx Chemical Company. The sonicated ROS preparation was made by suspension of the ROS pellet in 67% glycerol-buffer (S retina$6ml) and sonication for two 5&n periods with the microtip assembly of the Heat System-Ultrasonics, Inc. Sonifer, Model Wt39. The photosensitivity measurements were made at room temperature. The bleaching rate was detefined by monitoring the change in optical density at SOOnm, utilizing the Gilford Model 2000 spectrophotometer. Aliquots of rhodopsin in the various extractants were bleached using a 45 W Tungsten lamp passed through a 10 A interference filter of maximum wavelength 5145 4 (Thin Film Table 1. Photosensitivities
Products). Hydroxyinminc was added to a linal concentration of 0.10 RI prior to bleachinlz. The intensitv at I.& face of the cuv& was measured wyth the calibraied mlcrowatt head of the Tektronix J 16 photometer and it \\r\j kept low enough to prevent double hits on individual molecules. The thermal stability of rhodopsin was determined b> monitoring the loss of optical density on the Cary 14 spec trophotometer at 530 nm because we found that products of heat bleaching do not interfere at this wavelength. .\n aliquot of rhodopsin extracted in the particular surfactanl being investigated was injected into a special cell constructed of stainless strei. through u-hich was circulated water from a Haake ~ir~uIatin~ bath. The hoses from tht bath contained two Row channels; one by-passing the optical ccl1 and the other delivering water to it. Once the sample had been injected and the Cary 1-l activated. tie water was allowed to pass to the cell by moving a hose clamp from the cell channel to the by-pass channel. Temperature was equilibrated within 20sec so that very little of the reaction occurred during this short ~uiIibmtion period. For kinetic analysis, zero time was taken at 20 sec. The rate of production of metarhodopsin Ii was followed during flash photolysis of rhodopsin in the various extractants. The apparatus used was modified from the instrument described by Williams and Brie1 (1968). The modifications involve the use of a halogen-tungsten 45 V. monitoring lamp powered by a Hewlett-Packard Harrison 6332h d.c. poie; supply. The flash source was changed from the Honevweli 65 flash of I-msec duration (9941,dissipation) to the &-gap flash of the E.G. & G Mohel’j49-!I Microflash. This flash is of much shorter duration, namely 0.5 psec full-width at 1,3 man instantaneous output. The= changes allow the measurement of the extremely fast rares of meta II formation seen in the ionic and aiiphatic nonionic detergents. The rate of meta II production was monitored at 3SOnm. The sample was held in a semi-micro cuvette which was placed in a specially constructed compartment whose temperature was controlled to +O.?C. The sweep of a Tektronix oscilloscope (Model 565) n-as triggered simultaneously with the flash. The bandwidth of the oscilloscope amplifier was set so that the half-time of the reaction being followed was five times longer than rte response time of the electronics. The sweep representing transmission changes as a function of time was phor‘ographed using a Tektronix oscilloscope camera. The transmission changes were converted to optical densip changes based on the voltage displacement caused by a calibrated filter. The optical density changes were th:n plotted as a first-order reaction.
RESULTS
The results of photosensitivity measurements performed on rhodopsin extracted in the five surfactants are shown in Table 1. For the values in the first column, z is the extinction coefficient for a sing!e
of cattle rhodopsin in various extractants
Extractant
%&IJ (cm2 x IO”)
%l,,? (m’/mol)
Quantum efficiency 7
2% Digitonin 1% CTAB 1% Triton X-100 1% LDAO 1% Emulphogene B-720
10.5 k 0.06 10.4 f 0.06 10.3 It 0.14 10.0 & 0.09 10.0 + 0.19
2746 2720 2694 2615 2615
0.65 0.69 0.63 0.62 0.62
1159
Extractant effects on some properties of rhodopsin Thermal srabilir!:
The thermal stability of rhodopsin is a function of the solubilizing agent. This is shown in Fig. 1, in which is plotted the rate of loss of absorbance at 53Onm as a function of temperature for four of the five detergents investigated. Straight lines were fitted to the data points by a linear least squares method. Data could not be obtained in 1% Emulphogene BC-720 due to the development of turbidity as thermal bleaching proceeded. Rhodopsin solubilized in digitonin
lo-)
,\
I
2.90
3.00
I/T
1-K)
3.10 X IO’
,
I 3.20
Fig. 1. Arrhenius plot of the observed rate constant for disappearance of absorption at 530nm as a function of temperature in four of the five investigated detergents and sonicated ROS membranes. l, 1% CTAB; 0, 3% LDAO; A, 2’/, Triton X-100; A. sonicated ROS; n , 1.5% LDAO; q, 2”/, digitonin.
chromophore; that is, the area of the chromophore multiplied by the probability of absorption of a quantum falling in this area Column two gives the more familiar molar photosensitivity. In both column 1 and 2,~ is the quantum efficiency term of photosensitivity. The quantum efficiences (y) of column 3 were calculated utilizing published values of the molar extinction coefficient in the various solvents (Shichi et al., 1969; Shichi, 1970; Daemen, Borggreven and Bonting, 1970; Wald and Brown, 1953). It appears that the photosensitivity of rhodopsin is weakly dependent on the extracting agent utilized for solubilization. The differences seen are small but these values of quantum efficiency are in good agreement with those determined by Waddell, Yudd and Nakanishi (1976).
is more
stable than
rhodopsin
Metarhodopsin
II production
The rate of metarhodopsin II production is not a simple exponential process (cf. Abrahamson, 1973). The number of parallel first-order reactions reported has varied from two to four depending on the conditions of the experiment. The number of forms seen appears to be related to two factors, namely, the existence of two conformers of rhodopsin which gives rise to two rates of production in all systems so far investigated (Stewart, Baker and Williams, 1975) and the existence of heterogeneity of environment in detergents which at the concentrations utilized give incomplete dissociation of lipids. Therefore, in order to compare the rate of meta II production in the detergents under investigation, the value of l/tl:2 has been used
Table 2. Thermal decomposition of cattle rhodopsin-pH
6.5, 50°C
k
AHt
(set- *)
@al/mole)
ASt (e.u.)
AGi @al/mole)
1% CTAB 3.0% LDAOX-100 2% Triton
7.7 x 10-a 4.4 10-J 1.8 x lo-’
5SJO1 51,053 71.820
111.6 88.8 151.3
1.5% LDAO 2% Digitonin Sonicated ROS (67% glycerol-buffer)
1.5 x 10e3 3.3 x 1o-5
57.000 9-1.341
197.9 2 12.6
22,044 22,368 22,957 23,094 25.558
8.7 x 1o-6
54.808
181.1
26.3-15
Extractant
solubilized
in the other non-ionic detergent Triton X100, or the ionic surfactants CTAB and LDAO. This is not surprising, in view of the work of Tanford (1965) and others (Jirgensons and Capetillo, 1970; Meyer and Kauzmann, 1962) on the binding of ionic detergents to proteins and the resultant induction of conformational changes. Increasing the concentration of LDAO, the zwitterionic detergent, decreases the stability of rhodopsin. This probably occurs because more of the associated lipid is displaced and cooperative binding occurs as the detergent concentration is increased. As shown by Shichi (1974), the thermal stability of rhodopsin increases with the number of associated lipids. In agreement with this finding, rhodopsin in its native ROS membrane environment is more stable than any of the extracted rhodopsins. In Table 2, the activation parameters for the thcrma1 decomposition of cattle rhodopsin in the various extractants and in sonicated ROS are given. The enthalpy and entropy of activation vary considerably with the environment but the free energy of activation shows marked compensation (Rosenberg and Williams, 1971).
BUIBARA N. BAKER. W~LLLW
I I60
J. DONOV.U and
as a measure of rate. The possible errors inherent in this procedure will be discussed later. Figure 2 is an Arrhenius plot of the meta Ii rates as a function of temperature. Again. the straight lines are obtained by linear least squares fitting. The rate of production of meta I1 is seen to increase in the order ROS. digitonin IT, Triton X 100, 10, CTAB. l.YO LDAO. 1;;; Emulphogene BC-720. The activation parameters calculated from the Arrhenius plots are given in Table 3. Although the enthalpy and entropy of activation vary widely from extractant to extractant, the free energy of activation is again fairly constant.
THEODORE
P. WILLI,A_~~S
lo:
IO
IO’ 7
P
2
-1-J ;
DISCLSSIOS
IO2 As shown in Table 1, small differences in photosensitivity were seen as an effect of the solvating agent. This suggests that detertent interactions with the protein have caused subtle datortions of the chromophore-protein electronic interactions even though these are not extensive enough to change the Emax(not shown). Further evidence of such action by detergents has been given recently by Waddell er al. (1976). They report that the ratio of molar ellipicity to optical density in both the r and p band of the CD spectrum of rhodopsin is sensitive to the extracting agent utilized. In addition. the rotational strength ratio, that is. RJR,, is also a function of the detergent used to solubilize rhodopsin. Furthermore. they demonstrated that the quantum efficiency and bleaching product were also related to extracting agent. Their results, combined with ours, indicate that the micellar environment can alter the photochemical properties of rhodopsin. This could result from changes in the twist of the retinal or changes in the electronic interactions of the protein and the chromophore brought about by varying protein conformation. No attempt was made in this study to measure separately the components of photosensitivity, namely extinction coefficient and quantum efficiency. For this reason we cannot comment directly on the report of Rabinotitch (1974) of a quan turn efficiency of rhodopsin bleaching in CTAB of over 2.0. The generally accepted quantum efficiency of bleaching both in extracts and in situ is distinctly below one; probably 0.65 (Dartnall, 1972). If the quantum efficiency of bleaching in CTAB is as high as 2.0, the close correspondence in our experiments of bleaching rate in CTAB
compared
with
the other
extracting
agents
Table 3. Rate of metarhodopsin
Extractant 1’5;Emulphogene BC-720 1.5% LDAO 1% Cl-AB 1% Triton X-100 27; Digitonin Sonicated ROSbuffer Sonicated ROS, (67% glycerol, buffer)
IO'
---L-b-
‘“50
3.40
3.50 AQp
3.60
Fig. 2. Arrhenius plot of rate of menrhodopsin II production as a function of temperature in five detergents and sonicated ROS membranes. l, 1% CTAB; O. 1% Emulphogene BC-720; A, 1% Triton X-100; A, 1%; LDAO: n , sonicated ROS buffer; 0, sonicated ROS glycerolbuffer; V. 2% digitonin.
could only come about by a drastic lowering of the extinction coefficient of rhodopsin in that extractant. Although a lower extinction coefficient for rhodopsin solubilized in CTAB has been reported by Heller (196S), we have been unable to support such a finding when rod outer segments are simply extracted with CTAB (Williams and Baker, 1970). Therefore, the extremely high value of quantum efficiency determined by Rabinovitch is not supported by our results over the last few years. The ordering of thermal stabilities of rhodopsin in the various extractants is not unexpected (Tanford. 1968) with the possible exception that 1.504 LDAO. a zwitter-ionic detergent, lies below 2% Triton X-100.
II production-pH
6.5. 2O’C
AM (Cal/mole)
Mt (calideg mole)
4Gt (caljmole)
10” 10’ lo3 IO3 IO’
16,996 19,239 31,417 48,490 56.612
15.3 35.6 65.8 121.3 143.8
11.631 11.732 12.138 12,949 14.479
7.29 x 10’
40,424
SY.3
i-l.552
3.15 x IO’
Ad.171
99.3
15.079
k (set-‘) 1.17 x 9.75 x 4.77 x 1.17 x 8.66 x
Extractant effects on some properties of rhodopsin
a presumed mild non-ionic detergent. This may in part reflect the almost non-ionic character of LDAO at the pH employed. The pK of the amine oxide is approximately 5 and the thermal stability was studied at pH 6.5. Therefore, under our conditions, this detergent should probably be considered non-ionic. Another factor which may explain the transposition of 2% Triton X-100 and 1.5% LDAO is the longer alkyl chain length found in Triton X-100. The extent of conformational change upon binding of detergents to proteins has been shown to be dependent on hydrocarbon chain length (Reynolds, Herbert, Polet and Steinhardt. 1967). They showed that the binding of octyl and decyl sulfates and of octyl, decyl and dodecyl sulfonates occurred without drastic conformational change but when alkyl sulfates and sulfonates with longer hydrocarbon chains were bound, a marked conformational change was induced as judged by optical rotation and intrinsic viscosity. Pontus and Delmelle (1975) have recently labeled rhodopsin in ROS with the spin probe, 3 maleimido 2.2,5,5, tetramethyl-1-pyrrolidifloxyl (ML) and determined the effect of using various detergents on the ESR spectrum of the labeled rhodopsin. These investigators had previously demonstrated (Delmelle and Pontus, 1974) that the ESR spectra result mainly from ML molecules bound to rhodopsin sulfhydryl groups. They interpret the detergent effects seen on the ESR spectrum as perturbations of the label environment and consequent changes in its state of immobilization. Therefore, these changes indicate effects on the conformation of rhodopsin. Their results show a minimal change with digitonin and an increasing change in the order Triton X-100 5 Emulphogene BC-720 % CTAB B SDS. This ranking fits well with our results for the thermal stability of rhodopsin. The thermal stability of rhodopsin appears to depend on two factors. First, the degree to which lipid, normally associated with the protein, can be dissociated by the detergent, and second, the ability of the detergent, having once displaced lipid, to substitute for lipid without inducing conformational changes in the protein. The effect of extractants on meta II rates is more difficult to understand. It seemed possible that using t,/r values as a description of overall rate might reflect effect of detergent on the conformer equilibrium. So rate analysis have been done on double exponential plots utilizing the “peeling off” method. While the calculated rate constants for the slow and fast forms d&r from each other by a factor of five. and the single rate constant determined by using the t1,2 method lies between these two rates, the activation parameters are very similar. There is no. change in the ranking of detergents by their effect on rate of meta II production. We have, therefore, chosen to report the rate as a single value to aid in comparison with existing data in the literature (Applebury et al., 1974; Rapp, 1971; Sengbusch and Stieve, ,197l). Because ROS meta II rates have been previously determined in both aqueous glycerol and buffer sonicates we have utilized both media in our investigation. The agreement with previously reported values is good but it should be pointed out that unlike the other investigators we find that meta II production in both sonicates is double exponential even though
1161
a single k is reported due to the use of the t,:, method. It does appear that detergents which are effective in displacing lipids do produce environments which lead to faster meta II rates and less thermally stable rhodopsin. However, there appears to be no real correspondence between thermal stability and rate of meta II production. The cationic CTAB which is viewed by many protein chemists as the most denaturing of the detergents used does not yield meta II rates as fast as those seen in either the zwitter-ionic, LDAO or the non-ionic, Emulphogene BC-720. Triton X-100, considered by many workers to be a mild, non-denaturing detergent causes a 16-fold increase in rate when compared to that seen in the more native sonicated ROS buffer preparation. The rate of meta II production in digitonin is very close to that seen in the native ROS. This is probably due to the inability of digitonin to displace lipid so that the resulting micelle is mixed lipid-detergent and little change of native environment occurs. This is supported by our recent study of meta II rates of phospholipid-free rhodopsin in digitonin (Stewart, Baker. Plante and Williams, 1976) which showed that meta II is produced about 100 times more slowly in digitonin preparations which contain no detectable phospholipid. A possible answer to the detergent effects on the rate of meta II production may he in the nature of the meta I to meta II transition. The existence of a tautomeric equilibrium between meta I and meta II in digitonin has been demonstrated (Matthews, Hubbard, Brown and Wald, 1963) and its properties have been investigated. The thermodynamic parameters reported by Matthews et al., namely AIY = 13.1 kcal/mole and AS = 46.5 e.u., suggest that there is considerable randomization of the protein configuration in going from meta I to meta II. More recently, in this laboratory (Baker and W’illiams, 1971), we have shown that the transition from meta I to meta II can be dissected into two separate steps; namely a conversion of meta I to meta I’: and a further reaction of meta I to meta II. The thermodynamic parameters measured for these two reactions suggest that meta I to meta I’ with its enthalpy of 11.3 kcal/mole and entropy of 42.3 e.u., is the step which involves significant protein conformational change. The transition from meta I’ to meta II with its small enthalpy (1.8 k&/mole) and entropy (7.3 e.u.) may well be the depronation of the Schiffs base suggested by the spectral blue shift of the meta I-meta II conversion. Thus, the difficulty of understanding the extractant effects on meta II rates may arise from the fact that this is a two-step conversion involving three molecular species. On the basis of the present data, it is not possible to evaluate whether these detergents influence only one or both of these steps. Studies of the meta I-meta II equilibrium in both rod outer segments and detergent-rhodopsin systems will be necessary in order to gain further insights into the effect of detergents on meta II rates. Experiments of this nature are presently being undertaken. The data presented in this paper suggest that the use of these detergents either as tools for preparing lipid-protein recombinants or as solubilizing agents for physical studies must be undertaken with caution. The differing susceptibilities of the rhodopsin proper-
116’
B~B,u(.I N. BAKER. WILLLAI J. DONOVANand
ties investigated here make the evaluation of reeombinant s&&es extremely difTicult. Recombination with particular lipids might well restore regenerabiiity but fail to restore some other measureable property of rhodopsin such as meta II rate. The ~vestiga~ons of the size and shape of rhodopsin such as those of Sardet, Tardieu and Luzzati (1976) must be evaluated with the view that few of the properties of the protein, aside from the spectrum, remain unaltered when extracted from the disc membrane even when the socalled mild, non-denaturing detergents are employed.
.-lcknowle~ge~enf-Thjs work was supported by NSF grant BMSW24655 and in part by an ERDA grant to the Institute of Molecular Biophysics AT-(-IO-l)-2690. The authors wish to thank J. P. P. Webbers for design and construction of the thermal ceil used in this study. REFERMCES
Abrahamson E. W. (1973) The kinetics of early intermediate processes in the photolysis of visual pigments. Biochemistry and Physiology of Visual Pigments, pp. 47-56. Springer, Berlin. Applebury M., Zuckerman D., Lamola A. and Jovin T. (f 974) Purification and recombination with phospholipids assayed by the metarhodopsin I -+ metarhodopsin 11 transition. Bi~he~istry 13. 3448-3453. Baker B. N. and Williams T. P. (1971) Photolysis of metarhodopsin I: rate and extent of conversion to rhodopsin. Vision Res. 11, 449-458. Bridges C. D. B. (1957) Cationic extracting agents for rhodopsin and their mode of action. &&em. J. 66, 375-3s;. Busch G. E., Applebury M. L., Lamola A. A. and Rentzepis P. M. (1972). Formation and decay of prelumirhodopsin at room temperature. Proc. nam. Acad. Sci. U.S.A. 69, 2802-2806.
Ctescitelli F. (1967) Extraction of visual pigments with certain alkyd phenoxy-polyethoxy-ethanol surface-active compounds. Vision Res. 7, 683-693. Daemen F. I. M., Borggreven J. M. P. M. and Bonting S. L. (1970) Molar absorbance coefhcient of rhodops~n. Narure, Load. 227, 1258-1239. Dartnali H. J. A. (1968) The photosensitivities of visual pigments in the presence of hydroxylamine. Vision Res. 8, 339-358.
Dartnall H. J. A. (1972) Photosensitivity. In handbook uf Sensory Physiology, Vof VII/t (Edited by Dartnail H. J. A.f, IYD. h-145. Sorinaer. Beriin. Delmelle M. and PO& LG. (1974) Magnetic reSOWIce study of spin-labeled rhodopsin. Eiochim. biophys. Acta .I
363, 47-56.
Ebrey T. G. (1971) The use of Ammonyx LO in the purification of rhodopsin and rod outer segments. Vision Res. 11, 1007-1009. Helen& A. and Simons K. (1975) Solubiliiation of membranes by detergents. 3iochi~ biophys. Acta 415, 29-79. Heller 3. (1968) Structure of visual pigments I. Purification, molecular weight and composirion of bovine visual pigment. Biochemistry 7, 2906-2913. Hong K. and Hubbell W. (1972) Preparation and properties of phospholipid biayers containing rhodopsin. Proc. nam. Acad. Sci. U.S.A.
69, 2617-2621.
Jergenson B. and Capetillo S. (1970) Effect of sodium dodecyl sulfate on circular dichroism of some nonhelical proteins. Biochim. biophys. Acra 214, l-5.
THEOWRE
P.
WILLIAMS
Matthews R. G.. Hubbard R., Brown P. K. and Wald G. (1963) Tautomeric forms of metarhodopsin. J. gen. Ph_rsiol. 38, 265-315.
Meyer M. L. and Kauzmann W. (1962) The effects of detergents and urea on the rotatory dispersion of ovalbumin. Archs Biochem.
Biophys. 99, 348-349.
Osborne H. 3.. Sardet C. and Helenius A. (19’74) Bovine rhodopsin; characterization of the complex formed with Triton X-100. Eur. J. Biochem. 44, 383-3%. POROUSIM. and Delmelle M. (1975) Effect of detergents on the conformation of spin-labeled rhodopsin. .&pl Eye Res. 20, 599-603. Rabinotitch B. (1974) The quantum efficiency of photo bleaching of the rh~opsin/~ty~~methyIammonium bromide micefle. Studio biophysics 43, l-12. Rapp J. (197 I) Ph.D. thesis, Case Western Reserve University, Cleveland, Ohio. Reynolds 1. A., Herbert S., Polet H. and Steinhardt J. (1967) The binding of diverse detergent anions to bovine serum albumin. B~oc~e~~s~ry6, 935-947. Rosenberg 8. and Williams T. P. (1971) The thermal decomposition of visual pigments as a compensation law process. Vision Res. 11, 613-615. Sardet C., Tardieu A. and Luuati V. (1976) Shape and size of bovine rhodopsin: a small angle X-ray scattering study of a rhodopsin-detergent complex. J. molec. Biol. 105, 383-407. Sengbusch G. Von, and Stieve H. (1971) Flash photo&is of rhodopsin. I. M~surements on bovine rod outer sep ments. 2. Narurf: 266, 4881159. Shicki H. (1970) Spectrum and purity of bovine rhodopsin. Biochemistry 9, 1973-1977. Shichi H. (1971) Biochemistry of visual pigments. i1. Phosphohpid requirement and opsin conformation for regeneration of bovine rhodopsin. J. biol. Chem. 246. 6178-;6182.
Shichi H. (1974) Conformational aspects of rhodopsin associated with disc membranes. E?cpl Eye Res. 17. 533-543. Shichi H., Lewis M. S., Irreverre F. and Stone A. L. (1969) Biochemistry of visual pigments I. Purification and properties of bovine rhodopsin. J. biol. Chem 244. 529-536.
Stewart J. G., Baker 8. N. and Williams T. P. (1975) Kinetic evidence for a conformational transition in rhodop sin. il’ature, Land. 2%. 89. Stewart J. G., Baker B. N., Plante E. 0. and Williams T. P. (1976) Effect ‘of phospholipid removai on the kinetics of the metarhodopsin I to metarhodopsin II reaction. A&s Biochem. Biopkys. 172. 246.
Tanford C. (1968) Protein denaturation. In Aduutrces in Protein Chemistry, Vol. 23, pp. 122-282. Academic Press. New York. Waddeli W. H., Yudd A. P. and Sakanishi K. (19761 Micellar effects on the photochemistry of rhodopsin. J. Am. them. Sot. 98, 238-239. Watd G. and Brown P. K. (1952) The role of sulfydrl groups in the bieaching and synthesis of rhodopsin. J. gen. Physiol. 35, 797-821. Waid G. and Brown P. K. (1953) The molar extinction of rhodopsin. J. gen. Physiof. 37, 189-200. Williams T. P. and Baker B. N. (19iO) An hypothesis on the extinction and color of rhodopsin. Vision Res. 10. 901-903. Williams f. P. and Brie1 S. J. (1968) Kiietic me~urerne~t~ on rhodopsin solutions during inrense flashes. Vision Res. 8, 777-786.
EFFECTS ON SOME PROPERTIES OF R~~~~~S~~ WILLIAZV J. DO~OVAH’ and THEOIXNZP. WKLLG&~S~
‘.31nstitute of Mol~lar Biophysics, and %epartrnent of Psychology, Florida State University, Tallahassee, FL 32306, U.S.A. (Received 6 February 1976; in revised form 17 March 1977) Abstrac+--Tht photosensitivity, thermal stability and rate of metarhodopsin II production of rhodopsin solubilized in five commonly used extracting agents have been determined and compared with rod outer segment suspensions. An effect of .extracting agent on photosensitivity was found, indicating that the electronic interactions of the chromophore and protein are sensitive, io the miceLIar environment. Both the rate of thermal decay and metarhodopsin II production were found to be a function of the detergent. Onfv digitonin gaye rates which were comparable to those seen in rod outer segment suspensions.
Rhodopsin, the visual pigment of rod photoreceptors, is an intrinsic protein of the disc membrane and, like other proteins of this class, its solubilization requires the use of detergents. Over the years, a large number of detergents have been employed’ by various investigators in their studies of the bleaching behavior and molecular structure of rhodopsin. These have included both ionic and non-ionic detergents which contain hydrophobic aliphatic chains of varying length as we11as surfactants which contain bulky cyclic or aromatic structures as their hydrophobic moiety. Of the ionic detergents, ~tyltr~ethyl~onium bromide (CTAB) is perhaps the most commonly used (Bridges, 19.57; Heller, 1968) but the shorter chain, tetradecyl and dodecyl ammonium bromide (TT.AB, DTAB) have also been employed (Hong and Hubbell, 1972). The twitter-ionic detergent, Ammonyx LO (AL0 or LDAO) which is a mixture of tetradecyl and dodecyldimethylamine oxide, has been used by Ebrey (1971) and Busch, Applebury, Lamola and Rentzepis (1972) for probation and study of rhodopsin. Applebury, Zuckerman, Lamola and Jovin (1974) utilized the single com~nent, dod~yldimethyl~~e oxide, in preparing rh~o~in for r~ombinant studies. Among the non-ionic surfactabts, the most commonly used are T&on X-l@& (Crescitelli, 1967) and Emulphogene BC-720 (Shichi, Lewis, Irreverre and Stone I969), both of which are polyoxyethyleneglycol derivatives. The non-ionic detergent, digitonin, has been utilized for many years for extraction of rhodop sin from disc membranes (Wald and Brown, 1952). Along with the bile salts, sodium cholate and sodium taurodeoxycholate, digitonin is an example of a detergent which contains a bulky hydrophobic group. According to the present view of detergent action, soIubiI~ation of membranes involves the formation of mixed micelles with membrane lipids and of mixed micelles which contain both lipids and proteins ~eIenius and Simons, 1975). As the con~n~ation of
detefgent is increased above that required to induce the lamellar-rni~~e phase transition, separation of lipids from proteins occurs. Thus the degree to which lipids are dissociated is a function of the detergent concentration. In the case of ionic detergents, once lipids have been. displaced, further increase in detergent concentration leads to the binding of detergent to the protein thereby giving rise to an unfolded state of’the protein. The extent of unfolding is, of course, dependent on the detergent concentration (Tanford, 1958). The mild, non-ionic detergents by virtue of their lower CMC do not bind proteins in a cooperative manner, but the interaction is viewed as a complexing of detergent with the exposed hydrophobic portion of the protein thereby producing a soluble protein-detergent complex which may or may not contain iipid depending on the detergent concentration. Thus, the state of the membrane protein following. solubilization with detergent is a function of both the nature and concentration of the detergent. A wide variability in lipid content has been reported for rhodopsin extracted in the various detergents previously mention&. ~~~ylt~rne~yla~ monium bromide (Hong and Hubbell, 1972) and dod~yl~methyla~ne oxide (AppIebury et aL, 1974) have been shown to solubilize rhodopsin free of almost all phospholipids (0.5 mol phospholipid~mol of rhodopsin) following chromato~phy on a hydroxyapatite column. Osborne, Sardet and Helenius (1974) have reported the purification of rhodopsin devoid of lipid by use of the non-ionic detergents Triton X-100 and Emulphogene BC720. Complete removal of lipids necessitates that rod outer segment mem* branes be dissociated by at least 10 times their weight of these non-ionic detergents. At the other extreme, purification of rhodopsin solubilized in digitonin produces a system in which 90-11Omol pho~holipid/ mot rhodop~n have been reported (Shichi, 1974). In view of the established importance of lipids to regenerability of rhodopsin (Hong and Hubbell, 1972; Shichi, 1971) it seemed wo~hwhile to compare some other properties of rhodopsin extracted in commonly
1157
used detergents. In this paper. we report the measurement of photosensitivity, thermal stability and rate of metarhodopsin II production following light absorption in two ionic detergents. CTAB and LDAO; in two non-ionic aiiphatic detergents, Triton X-100, Emu~phogene BC-720 and in the non-ionic, bulky hydrophobic detergent, digitonin. For the sake of comparison, some measurements of thermal stability and rate of metarhodopsin II production have also been made in the more native rhodopsin environment, namely sonicated rod outer segment membranes. METHODS
A.VD 41ATERLkLS
Rod outer segments (ROS) were obtained from darkadapted bovine retinae (George Hormel Company, Austin, MN) by Hot&on on sucrose. Batches of 50 frozen retinae were homogenized in 45% sucrose, and were floated. Following the first flotation. ROS were pelleted by dilution of the sucrose with 67 mM phosphate buffer, pH 6.5 and centrifugation at 10,000~ for 15 min. The pelleted ROS were reiuspended in 40% sucrose, layered with buffer and suun at lO.OCi3 (1 for 30 min. The ROS were collected from the sucrose-b&er interface by means of a syringe fitted with a long needle. They were washed with 67 mM phosphate buffer, pH 6.5, spun, washed with distilled water, spun and resuspended in buffer. The suspension was then divided into six equal aliquots, spun and the resultant pellzts frozen at - IYC until used. Extraction of ROS was accomplished by adding deters.ent in the ratio of OScm’ detergent/retina. The suspension was gently homogenized and stirred in the dark for l-4 hr depending on &e extracting agent. AH extractants were prepared in 67 mM phosphate buffer, pH 6.5. Digitonin (Fisher) was used at a concentration of 2% (w/v). The CTAB concentration (Sigma) was 1% (w/v). Emulphogene BC-720 (gift of Geneial-Aniline Co.j was also utilized at lY/, concentration Iv/v). Triton Xl00 (rrift of Rohm & Haa$ was used at a cb&ntration of 2%&/v) for thermal stability measurements and 17; (v/v) for determination of the rate of meta II production. The higher conc~tration for thermal decay studies was utilized to prevent aggregation resulting during denaturation. Solutions of 1.5% LDAO and 3% LDAO (v/v) were prepared by dilution of a 30% stock solution provided by Onyx Chemical Company. The sonicated ROS preparation was made by suspension of the ROS pellet in 67% glycerol-buffer (S retina$6ml) and sonication for two 5&n periods with the microtip assembly of the Heat System-Ultrasonics, Inc. Sonifer, Model Wt39. The photosensitivity measurements were made at room temperature. The bleaching rate was detefined by monitoring the change in optical density at SOOnm, utilizing the Gilford Model 2000 spectrophotometer. Aliquots of rhodopsin in the various extractants were bleached using a 45 W Tungsten lamp passed through a 10 A interference filter of maximum wavelength 5145 4 (Thin Film Table 1. Photosensitivities
Products). Hydroxyinminc was added to a linal concentration of 0.10 RI prior to bleachinlz. The intensitv at I.& face of the cuv& was measured wyth the calibraied mlcrowatt head of the Tektronix J 16 photometer and it \\r\j kept low enough to prevent double hits on individual molecules. The thermal stability of rhodopsin was determined b> monitoring the loss of optical density on the Cary 14 spec trophotometer at 530 nm because we found that products of heat bleaching do not interfere at this wavelength. .\n aliquot of rhodopsin extracted in the particular surfactanl being investigated was injected into a special cell constructed of stainless strei. through u-hich was circulated water from a Haake ~ir~uIatin~ bath. The hoses from tht bath contained two Row channels; one by-passing the optical ccl1 and the other delivering water to it. Once the sample had been injected and the Cary 1-l activated. tie water was allowed to pass to the cell by moving a hose clamp from the cell channel to the by-pass channel. Temperature was equilibrated within 20sec so that very little of the reaction occurred during this short ~uiIibmtion period. For kinetic analysis, zero time was taken at 20 sec. The rate of production of metarhodopsin Ii was followed during flash photolysis of rhodopsin in the various extractants. The apparatus used was modified from the instrument described by Williams and Brie1 (1968). The modifications involve the use of a halogen-tungsten 45 V. monitoring lamp powered by a Hewlett-Packard Harrison 6332h d.c. poie; supply. The flash source was changed from the Honevweli 65 flash of I-msec duration (9941,dissipation) to the &-gap flash of the E.G. & G Mohel’j49-!I Microflash. This flash is of much shorter duration, namely 0.5 psec full-width at 1,3 man instantaneous output. The= changes allow the measurement of the extremely fast rares of meta II formation seen in the ionic and aiiphatic nonionic detergents. The rate of meta II production was monitored at 3SOnm. The sample was held in a semi-micro cuvette which was placed in a specially constructed compartment whose temperature was controlled to +O.?C. The sweep of a Tektronix oscilloscope (Model 565) n-as triggered simultaneously with the flash. The bandwidth of the oscilloscope amplifier was set so that the half-time of the reaction being followed was five times longer than rte response time of the electronics. The sweep representing transmission changes as a function of time was phor‘ographed using a Tektronix oscilloscope camera. The transmission changes were converted to optical densip changes based on the voltage displacement caused by a calibrated filter. The optical density changes were th:n plotted as a first-order reaction.
RESULTS
The results of photosensitivity measurements performed on rhodopsin extracted in the five surfactants are shown in Table 1. For the values in the first column, z is the extinction coefficient for a sing!e
of cattle rhodopsin in various extractants
Extractant
%&IJ (cm2 x IO”)
%l,,? (m’/mol)
Quantum efficiency 7
2% Digitonin 1% CTAB 1% Triton X-100 1% LDAO 1% Emulphogene B-720
10.5 k 0.06 10.4 f 0.06 10.3 It 0.14 10.0 & 0.09 10.0 + 0.19
2746 2720 2694 2615 2615
0.65 0.69 0.63 0.62 0.62
1159
Extractant effects on some properties of rhodopsin Thermal srabilir!:
The thermal stability of rhodopsin is a function of the solubilizing agent. This is shown in Fig. 1, in which is plotted the rate of loss of absorbance at 53Onm as a function of temperature for four of the five detergents investigated. Straight lines were fitted to the data points by a linear least squares method. Data could not be obtained in 1% Emulphogene BC-720 due to the development of turbidity as thermal bleaching proceeded. Rhodopsin solubilized in digitonin
lo-)
,\
I
2.90
3.00
I/T
1-K)
3.10 X IO’
,
I 3.20
Fig. 1. Arrhenius plot of the observed rate constant for disappearance of absorption at 530nm as a function of temperature in four of the five investigated detergents and sonicated ROS membranes. l, 1% CTAB; 0, 3% LDAO; A, 2’/, Triton X-100; A. sonicated ROS; n , 1.5% LDAO; q, 2”/, digitonin.
chromophore; that is, the area of the chromophore multiplied by the probability of absorption of a quantum falling in this area Column two gives the more familiar molar photosensitivity. In both column 1 and 2,~ is the quantum efficiency term of photosensitivity. The quantum efficiences (y) of column 3 were calculated utilizing published values of the molar extinction coefficient in the various solvents (Shichi et al., 1969; Shichi, 1970; Daemen, Borggreven and Bonting, 1970; Wald and Brown, 1953). It appears that the photosensitivity of rhodopsin is weakly dependent on the extracting agent utilized for solubilization. The differences seen are small but these values of quantum efficiency are in good agreement with those determined by Waddell, Yudd and Nakanishi (1976).
is more
stable than
rhodopsin
Metarhodopsin
II production
The rate of metarhodopsin II production is not a simple exponential process (cf. Abrahamson, 1973). The number of parallel first-order reactions reported has varied from two to four depending on the conditions of the experiment. The number of forms seen appears to be related to two factors, namely, the existence of two conformers of rhodopsin which gives rise to two rates of production in all systems so far investigated (Stewart, Baker and Williams, 1975) and the existence of heterogeneity of environment in detergents which at the concentrations utilized give incomplete dissociation of lipids. Therefore, in order to compare the rate of meta II production in the detergents under investigation, the value of l/tl:2 has been used
Table 2. Thermal decomposition of cattle rhodopsin-pH
6.5, 50°C
k
AHt
(set- *)
@al/mole)
ASt (e.u.)
AGi @al/mole)
1% CTAB 3.0% LDAOX-100 2% Triton
7.7 x 10-a 4.4 10-J 1.8 x lo-’
5SJO1 51,053 71.820
111.6 88.8 151.3
1.5% LDAO 2% Digitonin Sonicated ROS (67% glycerol-buffer)
1.5 x 10e3 3.3 x 1o-5
57.000 9-1.341
197.9 2 12.6
22,044 22,368 22,957 23,094 25.558
8.7 x 1o-6
54.808
181.1
26.3-15
Extractant
solubilized
in the other non-ionic detergent Triton X100, or the ionic surfactants CTAB and LDAO. This is not surprising, in view of the work of Tanford (1965) and others (Jirgensons and Capetillo, 1970; Meyer and Kauzmann, 1962) on the binding of ionic detergents to proteins and the resultant induction of conformational changes. Increasing the concentration of LDAO, the zwitterionic detergent, decreases the stability of rhodopsin. This probably occurs because more of the associated lipid is displaced and cooperative binding occurs as the detergent concentration is increased. As shown by Shichi (1974), the thermal stability of rhodopsin increases with the number of associated lipids. In agreement with this finding, rhodopsin in its native ROS membrane environment is more stable than any of the extracted rhodopsins. In Table 2, the activation parameters for the thcrma1 decomposition of cattle rhodopsin in the various extractants and in sonicated ROS are given. The enthalpy and entropy of activation vary considerably with the environment but the free energy of activation shows marked compensation (Rosenberg and Williams, 1971).
BUIBARA N. BAKER. W~LLLW
I I60
J. DONOV.U and
as a measure of rate. The possible errors inherent in this procedure will be discussed later. Figure 2 is an Arrhenius plot of the meta Ii rates as a function of temperature. Again. the straight lines are obtained by linear least squares fitting. The rate of production of meta I1 is seen to increase in the order ROS. digitonin IT, Triton X 100, 10, CTAB. l.YO LDAO. 1;;; Emulphogene BC-720. The activation parameters calculated from the Arrhenius plots are given in Table 3. Although the enthalpy and entropy of activation vary widely from extractant to extractant, the free energy of activation is again fairly constant.
THEODORE
P. WILLI,A_~~S
lo:
IO
IO’ 7
P
2
-1-J ;
DISCLSSIOS
IO2 As shown in Table 1, small differences in photosensitivity were seen as an effect of the solvating agent. This suggests that detertent interactions with the protein have caused subtle datortions of the chromophore-protein electronic interactions even though these are not extensive enough to change the Emax(not shown). Further evidence of such action by detergents has been given recently by Waddell er al. (1976). They report that the ratio of molar ellipicity to optical density in both the r and p band of the CD spectrum of rhodopsin is sensitive to the extracting agent utilized. In addition. the rotational strength ratio, that is. RJR,, is also a function of the detergent used to solubilize rhodopsin. Furthermore. they demonstrated that the quantum efficiency and bleaching product were also related to extracting agent. Their results, combined with ours, indicate that the micellar environment can alter the photochemical properties of rhodopsin. This could result from changes in the twist of the retinal or changes in the electronic interactions of the protein and the chromophore brought about by varying protein conformation. No attempt was made in this study to measure separately the components of photosensitivity, namely extinction coefficient and quantum efficiency. For this reason we cannot comment directly on the report of Rabinotitch (1974) of a quan turn efficiency of rhodopsin bleaching in CTAB of over 2.0. The generally accepted quantum efficiency of bleaching both in extracts and in situ is distinctly below one; probably 0.65 (Dartnall, 1972). If the quantum efficiency of bleaching in CTAB is as high as 2.0, the close correspondence in our experiments of bleaching rate in CTAB
compared
with
the other
extracting
agents
Table 3. Rate of metarhodopsin
Extractant 1’5;Emulphogene BC-720 1.5% LDAO 1% Cl-AB 1% Triton X-100 27; Digitonin Sonicated ROSbuffer Sonicated ROS, (67% glycerol, buffer)
IO'
---L-b-
‘“50
3.40
3.50 AQp
3.60
Fig. 2. Arrhenius plot of rate of menrhodopsin II production as a function of temperature in five detergents and sonicated ROS membranes. l, 1% CTAB; O. 1% Emulphogene BC-720; A, 1% Triton X-100; A, 1%; LDAO: n , sonicated ROS buffer; 0, sonicated ROS glycerolbuffer; V. 2% digitonin.
could only come about by a drastic lowering of the extinction coefficient of rhodopsin in that extractant. Although a lower extinction coefficient for rhodopsin solubilized in CTAB has been reported by Heller (196S), we have been unable to support such a finding when rod outer segments are simply extracted with CTAB (Williams and Baker, 1970). Therefore, the extremely high value of quantum efficiency determined by Rabinovitch is not supported by our results over the last few years. The ordering of thermal stabilities of rhodopsin in the various extractants is not unexpected (Tanford. 1968) with the possible exception that 1.504 LDAO. a zwitter-ionic detergent, lies below 2% Triton X-100.
II production-pH
6.5. 2O’C
AM (Cal/mole)
Mt (calideg mole)
4Gt (caljmole)
10” 10’ lo3 IO3 IO’
16,996 19,239 31,417 48,490 56.612
15.3 35.6 65.8 121.3 143.8
11.631 11.732 12.138 12,949 14.479
7.29 x 10’
40,424
SY.3
i-l.552
3.15 x IO’
Ad.171
99.3
15.079
k (set-‘) 1.17 x 9.75 x 4.77 x 1.17 x 8.66 x
Extractant effects on some properties of rhodopsin
a presumed mild non-ionic detergent. This may in part reflect the almost non-ionic character of LDAO at the pH employed. The pK of the amine oxide is approximately 5 and the thermal stability was studied at pH 6.5. Therefore, under our conditions, this detergent should probably be considered non-ionic. Another factor which may explain the transposition of 2% Triton X-100 and 1.5% LDAO is the longer alkyl chain length found in Triton X-100. The extent of conformational change upon binding of detergents to proteins has been shown to be dependent on hydrocarbon chain length (Reynolds, Herbert, Polet and Steinhardt. 1967). They showed that the binding of octyl and decyl sulfates and of octyl, decyl and dodecyl sulfonates occurred without drastic conformational change but when alkyl sulfates and sulfonates with longer hydrocarbon chains were bound, a marked conformational change was induced as judged by optical rotation and intrinsic viscosity. Pontus and Delmelle (1975) have recently labeled rhodopsin in ROS with the spin probe, 3 maleimido 2.2,5,5, tetramethyl-1-pyrrolidifloxyl (ML) and determined the effect of using various detergents on the ESR spectrum of the labeled rhodopsin. These investigators had previously demonstrated (Delmelle and Pontus, 1974) that the ESR spectra result mainly from ML molecules bound to rhodopsin sulfhydryl groups. They interpret the detergent effects seen on the ESR spectrum as perturbations of the label environment and consequent changes in its state of immobilization. Therefore, these changes indicate effects on the conformation of rhodopsin. Their results show a minimal change with digitonin and an increasing change in the order Triton X-100 5 Emulphogene BC-720 % CTAB B SDS. This ranking fits well with our results for the thermal stability of rhodopsin. The thermal stability of rhodopsin appears to depend on two factors. First, the degree to which lipid, normally associated with the protein, can be dissociated by the detergent, and second, the ability of the detergent, having once displaced lipid, to substitute for lipid without inducing conformational changes in the protein. The effect of extractants on meta II rates is more difficult to understand. It seemed possible that using t,/r values as a description of overall rate might reflect effect of detergent on the conformer equilibrium. So rate analysis have been done on double exponential plots utilizing the “peeling off” method. While the calculated rate constants for the slow and fast forms d&r from each other by a factor of five. and the single rate constant determined by using the t1,2 method lies between these two rates, the activation parameters are very similar. There is no. change in the ranking of detergents by their effect on rate of meta II production. We have, therefore, chosen to report the rate as a single value to aid in comparison with existing data in the literature (Applebury et al., 1974; Rapp, 1971; Sengbusch and Stieve, ,197l). Because ROS meta II rates have been previously determined in both aqueous glycerol and buffer sonicates we have utilized both media in our investigation. The agreement with previously reported values is good but it should be pointed out that unlike the other investigators we find that meta II production in both sonicates is double exponential even though
1161
a single k is reported due to the use of the t,:, method. It does appear that detergents which are effective in displacing lipids do produce environments which lead to faster meta II rates and less thermally stable rhodopsin. However, there appears to be no real correspondence between thermal stability and rate of meta II production. The cationic CTAB which is viewed by many protein chemists as the most denaturing of the detergents used does not yield meta II rates as fast as those seen in either the zwitter-ionic, LDAO or the non-ionic, Emulphogene BC-720. Triton X-100, considered by many workers to be a mild, non-denaturing detergent causes a 16-fold increase in rate when compared to that seen in the more native sonicated ROS buffer preparation. The rate of meta II production in digitonin is very close to that seen in the native ROS. This is probably due to the inability of digitonin to displace lipid so that the resulting micelle is mixed lipid-detergent and little change of native environment occurs. This is supported by our recent study of meta II rates of phospholipid-free rhodopsin in digitonin (Stewart, Baker. Plante and Williams, 1976) which showed that meta II is produced about 100 times more slowly in digitonin preparations which contain no detectable phospholipid. A possible answer to the detergent effects on the rate of meta II production may he in the nature of the meta I to meta II transition. The existence of a tautomeric equilibrium between meta I and meta II in digitonin has been demonstrated (Matthews, Hubbard, Brown and Wald, 1963) and its properties have been investigated. The thermodynamic parameters reported by Matthews et al., namely AIY = 13.1 kcal/mole and AS = 46.5 e.u., suggest that there is considerable randomization of the protein configuration in going from meta I to meta II. More recently, in this laboratory (Baker and W’illiams, 1971), we have shown that the transition from meta I to meta II can be dissected into two separate steps; namely a conversion of meta I to meta I’: and a further reaction of meta I to meta II. The thermodynamic parameters measured for these two reactions suggest that meta I to meta I’ with its enthalpy of 11.3 kcal/mole and entropy of 42.3 e.u., is the step which involves significant protein conformational change. The transition from meta I’ to meta II with its small enthalpy (1.8 k&/mole) and entropy (7.3 e.u.) may well be the depronation of the Schiffs base suggested by the spectral blue shift of the meta I-meta II conversion. Thus, the difficulty of understanding the extractant effects on meta II rates may arise from the fact that this is a two-step conversion involving three molecular species. On the basis of the present data, it is not possible to evaluate whether these detergents influence only one or both of these steps. Studies of the meta I-meta II equilibrium in both rod outer segments and detergent-rhodopsin systems will be necessary in order to gain further insights into the effect of detergents on meta II rates. Experiments of this nature are presently being undertaken. The data presented in this paper suggest that the use of these detergents either as tools for preparing lipid-protein recombinants or as solubilizing agents for physical studies must be undertaken with caution. The differing susceptibilities of the rhodopsin proper-
116’
B~B,u(.I N. BAKER. WILLLAI J. DONOVANand
ties investigated here make the evaluation of reeombinant s&&es extremely difTicult. Recombination with particular lipids might well restore regenerabiiity but fail to restore some other measureable property of rhodopsin such as meta II rate. The ~vestiga~ons of the size and shape of rhodopsin such as those of Sardet, Tardieu and Luzzati (1976) must be evaluated with the view that few of the properties of the protein, aside from the spectrum, remain unaltered when extracted from the disc membrane even when the socalled mild, non-denaturing detergents are employed.
.-lcknowle~ge~enf-Thjs work was supported by NSF grant BMSW24655 and in part by an ERDA grant to the Institute of Molecular Biophysics AT-(-IO-l)-2690. The authors wish to thank J. P. P. Webbers for design and construction of the thermal ceil used in this study. REFERMCES
Abrahamson E. W. (1973) The kinetics of early intermediate processes in the photolysis of visual pigments. Biochemistry and Physiology of Visual Pigments, pp. 47-56. Springer, Berlin. Applebury M., Zuckerman D., Lamola A. and Jovin T. (f 974) Purification and recombination with phospholipids assayed by the metarhodopsin I -+ metarhodopsin 11 transition. Bi~he~istry 13. 3448-3453. Baker B. N. and Williams T. P. (1971) Photolysis of metarhodopsin I: rate and extent of conversion to rhodopsin. Vision Res. 11, 449-458. Bridges C. D. B. (1957) Cationic extracting agents for rhodopsin and their mode of action. &&em. J. 66, 375-3s;. Busch G. E., Applebury M. L., Lamola A. A. and Rentzepis P. M. (1972). Formation and decay of prelumirhodopsin at room temperature. Proc. nam. Acad. Sci. U.S.A. 69, 2802-2806.
Ctescitelli F. (1967) Extraction of visual pigments with certain alkyd phenoxy-polyethoxy-ethanol surface-active compounds. Vision Res. 7, 683-693. Daemen F. I. M., Borggreven J. M. P. M. and Bonting S. L. (1970) Molar absorbance coefhcient of rhodops~n. Narure, Load. 227, 1258-1239. Dartnali H. J. A. (1968) The photosensitivities of visual pigments in the presence of hydroxylamine. Vision Res. 8, 339-358.
Dartnall H. J. A. (1972) Photosensitivity. In handbook uf Sensory Physiology, Vof VII/t (Edited by Dartnail H. J. A.f, IYD. h-145. Sorinaer. Beriin. Delmelle M. and PO& LG. (1974) Magnetic reSOWIce study of spin-labeled rhodopsin. Eiochim. biophys. Acta .I
363, 47-56.
Ebrey T. G. (1971) The use of Ammonyx LO in the purification of rhodopsin and rod outer segments. Vision Res. 11, 1007-1009. Helen& A. and Simons K. (1975) Solubiliiation of membranes by detergents. 3iochi~ biophys. Acta 415, 29-79. Heller 3. (1968) Structure of visual pigments I. Purification, molecular weight and composirion of bovine visual pigment. Biochemistry 7, 2906-2913. Hong K. and Hubbell W. (1972) Preparation and properties of phospholipid biayers containing rhodopsin. Proc. nam. Acad. Sci. U.S.A.
69, 2617-2621.
Jergenson B. and Capetillo S. (1970) Effect of sodium dodecyl sulfate on circular dichroism of some nonhelical proteins. Biochim. biophys. Acra 214, l-5.
THEOWRE
P.
WILLIAMS
Matthews R. G.. Hubbard R., Brown P. K. and Wald G. (1963) Tautomeric forms of metarhodopsin. J. gen. Ph_rsiol. 38, 265-315.
Meyer M. L. and Kauzmann W. (1962) The effects of detergents and urea on the rotatory dispersion of ovalbumin. Archs Biochem.
Biophys. 99, 348-349.
Osborne H. 3.. Sardet C. and Helenius A. (19’74) Bovine rhodopsin; characterization of the complex formed with Triton X-100. Eur. J. Biochem. 44, 383-3%. POROUSIM. and Delmelle M. (1975) Effect of detergents on the conformation of spin-labeled rhodopsin. .&pl Eye Res. 20, 599-603. Rabinotitch B. (1974) The quantum efficiency of photo bleaching of the rh~opsin/~ty~~methyIammonium bromide micefle. Studio biophysics 43, l-12. Rapp J. (197 I) Ph.D. thesis, Case Western Reserve University, Cleveland, Ohio. Reynolds 1. A., Herbert S., Polet H. and Steinhardt J. (1967) The binding of diverse detergent anions to bovine serum albumin. B~oc~e~~s~ry6, 935-947. Rosenberg 8. and Williams T. P. (1971) The thermal decomposition of visual pigments as a compensation law process. Vision Res. 11, 613-615. Sardet C., Tardieu A. and Luuati V. (1976) Shape and size of bovine rhodopsin: a small angle X-ray scattering study of a rhodopsin-detergent complex. J. molec. Biol. 105, 383-407. Sengbusch G. Von, and Stieve H. (1971) Flash photo&is of rhodopsin. I. M~surements on bovine rod outer sep ments. 2. Narurf: 266, 4881159. Shicki H. (1970) Spectrum and purity of bovine rhodopsin. Biochemistry 9, 1973-1977. Shichi H. (1971) Biochemistry of visual pigments. i1. Phosphohpid requirement and opsin conformation for regeneration of bovine rhodopsin. J. biol. Chem. 246. 6178-;6182.
Shichi H. (1974) Conformational aspects of rhodopsin associated with disc membranes. E?cpl Eye Res. 17. 533-543. Shichi H., Lewis M. S., Irreverre F. and Stone A. L. (1969) Biochemistry of visual pigments I. Purification and properties of bovine rhodopsin. J. biol. Chem 244. 529-536.
Stewart J. G., Baker 8. N. and Williams T. P. (1975) Kinetic evidence for a conformational transition in rhodop sin. il’ature, Land. 2%. 89. Stewart J. G., Baker B. N., Plante E. 0. and Williams T. P. (1976) Effect ‘of phospholipid removai on the kinetics of the metarhodopsin I to metarhodopsin II reaction. A&s Biochem. Biopkys. 172. 246.
Tanford C. (1968) Protein denaturation. In Aduutrces in Protein Chemistry, Vol. 23, pp. 122-282. Academic Press. New York. Waddeli W. H., Yudd A. P. and Sakanishi K. (19761 Micellar effects on the photochemistry of rhodopsin. J. Am. them. Sot. 98, 238-239. Watd G. and Brown P. K. (1952) The role of sulfydrl groups in the bieaching and synthesis of rhodopsin. J. gen. Physiol. 35, 797-821. Waid G. and Brown P. K. (1953) The molar extinction of rhodopsin. J. gen. Physiof. 37, 189-200. Williams T. P. and Baker B. N. (19iO) An hypothesis on the extinction and color of rhodopsin. Vision Res. 10. 901-903. Williams f. P. and Brie1 S. J. (1968) Kiietic me~urerne~t~ on rhodopsin solutions during inrense flashes. Vision Res. 8, 777-786.