Thermal stability of rhodopsin extracted with triton X-100 surfactant

tmon

Rrr.

Vol.

IO. pp

85-93

Pergamon

Press

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in Great

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THERMAL STABILITY OF RHODOPSIN EXTRACTED WITH TRITON X-100 SURFACTANTl RAYMOND

H. JOHNSONand THEODOREP. WILLIAMS

Department of Biological Sciences and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, U.S.A. (Received 20 June

1969)

INTRODUCTION

CRESCITELLI (1967) has explored rather thoroughly the usefulness of a series ofcommercially available surfactants, trade named Triton, as a means of extracting visual pigments. From his studies he concludes that photopigments extracted by Triton surfactants exhibit the same solution properties as do those extracted by digitonin. Furthermore, Triton is more easily soluble in water than digitonin and produces a more uniform solution than does digitonin. For these and other reasons he suggests that Triton may be the reagent of choice for visual pigment extraction. Triton compounds are alkyl aryl polyether alcohols, with an octyl- or nonylphenol hydrophobic end. Attached to the hydrophobic site is a variable length polyoxyethylene chain, whose ether oxygens are readily hydrated and thereby solubilized. Triton X-100, the surfactant used exclusively in the present experiments, is an iso-octylphenol combined linearly with 9-10 ethylene oxides. It is a colorless, viscous liquid freely soluble in water. The visual pigment, rhodopsin, is a chromoprotein formed by the combination of the protein, opsin and the moiety, 1I-cis retinal. There are, in general, two means by which the pigment may be bleached, a phenomenological observation noting the change of its color frqm red to light yellow. The first is absorption of a photon with the proper energy to isomerize the 11-cis configuration to an all-trans configuration. The latter is unable to conform to the geometry of opsin; the result is a sequence of thermal reactions terminating in the formation of all-trans retinal and opsin. Rhodopsin in digitonin solution may be regenerated by the dark incubation of the resulting opsin with 11-cis retinal, showing that photic bleaching does not denature the protein. A second mode of bleaching arises when the protein moiety is denatured by thermal or chemical means. It has been shown(HUBBARD, 19%) that the products of thermal bleaching are 11-cis retinal and denatured opsin. It is the intent of this article to present data and conclusions relative to several of the general properties of Triton X-100 solutions of cattle rhodopsin. Through kinetic measurements, for example, it is shown that such solutions are considerably less stable than rhodopsin solutions in digitonin. Also, we demonstrate that moderate amounts of hydroxylamine in Triton solutions of rhodopsin and photoreversed rhodopsin substantially increase thermal bleaching rates at slightly above ambient temperature. METHODS AND MATERIALS Cattle rhodopsin is prepared from retinas supplied by Hormel Company, Austin, Minnesota. The retinas are ground in 40 per cent sucrose solution in a mortar and the rod segments isolated by the usual flotation procedure. Following tanning in 4 per cent alum solution, the rod segments are washed twice with distilled water, divided into ‘This work supported by NINDS Grant NB-07140 and PHS Trainee ship Grant ITOI-MH-I 121801. 85

86

RAYYOSD H. JOHNKM AND THEODORE P. WILLIA.MS

portions, and stored in a freezer until needed. As a general rule, rod segments representing the equivalent of four retinas have an optical density (OD) of about I.0 when extracted once with a 2.0 ml solution of 2 per cent Ttiton X-100. The extraction of rhodopsin is generally accomplished at room temperature in about two hours. Following extractions, the residues are spun down and the clear solution of pigment reserved for experimentation. Pigment solutions obtained in this fashion are stable for weeks when stored in the refrigerator; aged and freshly prepared solutions exhibit no discemable experimental variation in thermal stability. In the visible spectroscopic region the ratio of OD,, to OD,,, is usually about 0.25. In general, rhodopsin extractions in 2 per cent Triton are accomplished faster with greater yields, and the extracts have more satisfactory OD,i,;OD,,, ratios, than digitonin preparations carried out on the same lot of rod segments. The intensity of the absorption maximum at about 280 nm, attributed to aromatic amino acid residues of the protein moiety, cannot be determined, owing to the substantial absorption of Triton X-,100 itself in this region of the spectrum. Two per cent (w/v) solutions of Triton X-100 surfactant (Rohm and Haas, Philadelphia, Pa.) are made by suitable dilutions of a IO per cent stock solution. Phosphate buffers used in pH adjustments and NH,?H HCI reagent are also made up in 2 per cent Triton solutions. The latter reagent is prepared as a 2 M stock solutton of the hydrochloride salt and neutralized by NaOH immediately before use. Photoreversed rhodopsin is prepared by flashing a relatively concentrated solution (OD cc. 1.5-2.0, IO mm path length) of rhodopsin in 2 per cent Triton X-100 with the output of a Honeywell 65-C xenon flashgun. The sample is put into a quartz cuvette and its volume adjusted such that the irradiating flash will ‘see” an OD ofabout 0.2. The c’uvette is then cooled in an ice bath, placed directly upon the face of the flashgun, and immediately flashed. Under these conditions it may be assumed that each rhodopsin molecule absorbs at least one photon and, further. a finite probability exists that some molecules absorb two or more photons. Therefore, any rhodopsin remaining after such saturating flashes exists because it is photoregenerated from certain transient intermediates (WILLIA.\lS. 1964). Spectrophotometric measurements were made in quartz cuvettes on a Gilford Model 2000 spectrophotometer or on a Beckman Model DB-G spectrophotometer. The sample compartments of these instruments, when necessary. were held at constant temperature by means of an external thermostatted Haake Model F Circulator. Although we do not explicitly report the results, a number of kinetic measurements were made in I per cent and 3 per cent Triton solutions. No experimental differences in bleaching rates could be determined compared to 2 per cent Triton solutions, i.e. the stability of rhodopsin itself appears to be independent of Triton concentration in the l-3 per cent range. However, it was noted that in I per cent Triton, the solutions become cloudy in a shorter period of time following thermal bleaching than in the more concentrated Triton solutions. RESULTS

Thermal bleaching of rhodopsin in 2 per cent Triton X-100

Rhodopsin solutions in Triton become cloudy at elevated temperature; in the case of Triton X-100, at about 45°C. Such turbidity is reversible, in that cooling the sample to room temperature restores the clarity of the solution. Our data for the thermal stability of rhodopsin were obtained by injecting a stock sample of rhodopsin solution into a preheated test tube immersed in a constant temperature bath. After waiting 20 set to allow-the sample to come to thermal equilibrium, timed withdrawals were made and immediately cooled. Hydroxylamine (final concentration 0.1 M) solution was added and the optical density measured at 500 nm, both before and after light bleaching. This method thus allows measurements to be made at higher temperatures than 45°C even though the solution is turbid while the reaction is proceeding. Fig. 1 shows an integrated first-order rate plot of the data obtained from these experiments. Each set of points is essentially linear, confirming the first-order nature of the reaction at each temperature. The slope of each line, multiplied by 2.303, yields the first-order rate constant, k, at each temperature. These results are tabulated in Table 1. Included in the Table are the Arrhenius activation energy, E,, and the logto of the Arrhenius preexponential factor, A,. The temperature dependence of the rate constant isshown by the Arrhenius plots in Fig. 2. (Note: we have plotted all thermal bleaching results in Fig. 2 to facilitate the reader’s comparison of the several different systems. Reference is made to this Fig. throughout the text.) Curve A represents the results obtained for the thermal bleaching of rhodopsin in 2 per cent Triton. It is noted that the rate constants obey a linear relationship over about a

Thermal Stability of Rhodopsin Extracted with Triton X-100 Suriactant

87

FIG. I. Integrated, first-order rate plots of thermal bleaching of cattle rhodopsin in 2 per cent Triton X-100 (pH = 7.0) at five temperatures. OD, and OD, are the optical densities initially and at time, r. respectively. at 500 nm; all OD’s are corrected for residual absorption remaining after light bleaching. The line at 42.0” is dashed because it is the extrapolation of OD’s measured at times greater than those shown on the abscissa. However, the points from which this line is constructed are also linear.

1O’C range. For comparison, we show by curve F the results obtained by HUBBARD (1958) for the thermal bleaching of rhodopsin in 2 per cent aqueous digitonin. She reports an E, value of 100 kcal mole-t; we calculate from her data that the logto of A, is 62.5. Extrapolation of curves A and F indicates that at about 55°C digitonin solutions are approximately lOO-fold more stable towards thermal bleaching than Triton solutions. If we further assume that the rate constants for both the Triton and digitonin systems continue to obey their respective linear relationships as the temperature is increased, another parameter, the isokinetic temperature, may be calculated from the preceding data. The isokinetic temperature is defined as the temperature at which the rate constants are equal. For these two systems, the isokinetic temperature is 101°C. Above this temperature, the reaction with the greater activation energy (rhodopsin in digitonin) proceeds faster; below this temperature, the reaction with the lesser activation energy (rhodopsin in Triton) has the greater rate. Thermal bleaching of rhodopsin and photoreversed rhodopsin in Triton X-100 solutions with added hydroxylamine

We have studied the thermal bleaching of rhodopsin in Triton solutions in the presence of hydroxylamine, a reagent commonly used in the spectrophotometric analysis of visual TABLE I. FIRST+RDER RATE CONSTANTS FOR THE THERMAL BLMCHISG OF RHOWPSIN IN 2% TRITON X-100, pH = 7.0

Temperature, “C 42.0 49,7 50.6 51.7 52.5

k x lO’,sec-’ 0.102 I.45 2.24 2.80 464

E,, kcal mole- 1

log,,A,. see-’

75.5

48.2

88

RAYMOSDH. JOHSUSOS ANDTHEODORE P.

WILLIAM

FIG. 2. Arrhenius plots of the rate constants from Tables I and 2. The slope’of each line multiplied by 2.303R yields the Arrhenius activation energy, E,. (A) In 2 per cent Triton. pH 7.0; (B) 0.5 M NH,OH. pH 7.3: (C) 0.5 M NH,OH. pH 5.5: (D) 0.5 M NH,OH. pH 8.2: (E) photoreversed rhodopsin. 0.5 M NH20H. pH 7.0: (F) data from. HUBBARD(1958). in 2 per cent aqueous digitonin. pH 6.1

pigments. It is generally believed this reagent, at least in small quantities, does not enhance the bleaching rate of cattle rhodopsin in digitonin solution. The experiments reported here were run on solutions of rhodopsin in Triton in which the hydroxylamine concentration was constant at 0.5 M. Thus, we report pseudo first-order rate constants for the thermal bleaching reaction. Separate determinations were made in buffered solutions at acidic, nearneutral and basic pH’s. The results of the experiments are compiled in Table 2 and indicate the sizeable decrease in the activation ener,T and the pre-exponential factor when rhodopsin is thermally bleached in the presence of hydroxylamine, compared to its absence. Arrhenius plots of the reactions are shown in Fig. 2 by curves B through D. The effect of pH is to decrease slightly the activation energy for thermal bleaching at near-neutrality (pH 7.2). It has been recognized for some time that rhodopsin in digitonin solution, when subjected to an intense quanta1 flux, is not completely bleached. In fact, the bleaching limit is about 50 per cent, at ordinary temperatures (WILLIAMS, 1965). The phenomenon occurs because the &initialphotoproducts resulting from the 114s to all-trans isomerization of the chromophore can themselves absorb a second quantum of light and revert to rhodopsin (and isorhodopsin), provided the time course of the flash is short compared to their thermal lifetimes. The same result is observed for rhodopsin solutions in Triton when irradiated by saturating flashes. We obtain a 57 per cent bleach at 2’C and a 82 per cent bleach at room temperature. As shown by curve E in Fig. 2, the thermal stability of photoreversed rhodopsin parallels that of rhodopsin under similar solution conditions. This result is not unexpected, in that BAKER and WILLUMS (1968) found experimentally identical first-order rate constants for cattle rhodopsin and photoregenerated rhodopsin in digitonin.

Thermal

Stability

of Rhodopsin

Extracted

with Triton

X-100 Surfactant

89

TABLE 2. PSEUDO FIRST-ORDER RATE CONSTANTS AND ARRHESIL'S PARAMETERS FOR THERMAL BLEACHISGOFRHODOPSIS ASD PHOTOREVERSED RHODOPSIN 13 TRITON X-loO,+T FOUR pH’s IS THE PRESESCE,OF@~ M HYDROIYLAMISE

PH

Temperature.

5.5

31.0 34.7 37.1 39.1 41.2

I.64 3.82 7.53 IO.7 16.4

J-l.9

28.4

32.8 35.5 38. I 40.7

40.3

25. I

42.I

2.84 3.54 6.65 14, I 16.4

31.0 33.2 36.2 39.1 41.0

2.62 4.74 9.03 15.3 26.8

44.0

28.3

34.3 37.3 42. I

I.31 5.10 12.1

38.9

24. I

7.3

8.2

Photoreversed 7.0

C

!i ‘ 10J.sec-1

E,, kdt mole-1

log,,AO,

set-’

Kinetic order of the hydro.xylamine-induced thermal bleaching of rhodopsin It is of interest to evaluate the exponent, m, in the equation,

rate = k [NH,OH]”

[Rhodopsin]”

in which k is the specific rate constant and m and n are the powers to which the concentrations of hydroxylamine and rhodopsin, respectively, must be raised to satisfy the equation. Taking the logarithm of both sides and rearranging, the above equation is recast as log rate = m log [NH,OH]

+ constant

Consequently, a plot of the log rate of thermal bleaching, at constant temperature and rhodopsin concentration, against the log concentration of hydroxylamine (Fig. 3a), yields a direct measure of m from the sldpe of the line. In Fig. 3a, the slope is O-92approximately unity. Therefore, the thermal bleaching of rhodopsin is first-order in hydroxylamine concentration. Because the bleaching reaction is first-order in hydroxylamine concentration, a plot of rate against hydroxylamine concentration is also linear at constant temperature. Moreover, such a plot, shown in Fig. 3b, may be extrapolated to zero hydroxylamine concentration. In the Fig., the extrapolated line passes almost through the origin. It is apparent that at 37.2°C the hydroxylamine concentration is in fact the predominant factor in governing the rate of the bleaching reaction. Isomeric state of retinal oxime following hydroxylamine-induced

thermal bleaching

It is pertinent to ascertain if the retinal oxime formed as a result of thermal bleaching in the presence of 0.5 M hydroxylamine is predominantly in a trans or cb configuration. It is to be anticipated that heat bleaching produces the cis oxime. To determine this, rhodopsin in 2 per cent Triton (pH 7.0, [NH,OH] = 0.5 M) was bleached in stepwise

90

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P.

WILLIAMS

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FIG. 3. {a) Plot of the tog rate of thermal bleaching of cattle rhodopsin in 2 per cent Triton X-100 (3?.2”C, pH 7-O) against log concentration of hydroxylamine. The slope of the line is 0,92approximately unity. (b)The same data plotted (method of least squares) to show the linear relationship between rate constant and hydroxylamine concentration.

fashion by immersing a cuvette containing the sample in a water bath at 35’C. Successive spectra made initially and after each thermal bleach showed a loss of optical density at 500 nm and an increase in optical density in the u.v., maximal at 365 nm. The spectra generated an isosbestic point at 407 nm, indicating the presence of only two chemical species during the course of bleaching. After the final thermal bleach, the ratio of AODjes to AOD,, was O-67. To conclude the experiment, the sample was irradiated exhaustively with U.V. light at 3600~ wavelength, whereupon the optical density at 365 nm increased. Furthermore, the spectrum recorded after irradiation no longer passed through the isosbestic point, but instead was shifted bathochromicaily with reference to it. By contrast, light bleaching should produce the trans oxime. Therefore, a similar experiment was undertaken, except that the hydroxylamine concentration was reduced to 0.1 M (causing negligible bleaching at room temperature) and the rhodopsin solution was bleached by orange light. Under these conditions, the isosbestic point was at 409 nm, the U.V.optical .density increase was maximal at 370 nm and the ratio of AOQ370 to AOD,,, was 0.87. Now, exhaustive U.V.irradiation caused the optical density at 370 nm to decrease and the spectrum of the irradiated solution was shifted hypsochromicaliy with reference to the isosbestic point. Ali the above data are consistent with the concept that thermal bleaching of T&on solutions of rhodopsin results in the formation of a cis retinal oxime: (I) whose. molar kxtinction is less than that of an all-rrans oxime; (2) whose h,,, occurs at a shorter wavelength than that of an all-rruns oxime; (3) whose optical density increases following U.V. irradiation, owing to the formatidn of a mixiure of cis-truns isomers. Thus, these experiments are consistent with the faq that the 1 1-cis oxime is produced by thermal bleaching. Visual pigment regeneration in Triton solutions

Preliminary experiments have established that dark incubation of mixtures of 11-cLs retinal and opsin solubilized by Triton does not regenerate rhodopsin. Identical experiments using digitonin solutions of the two components do exhibit rhodopsin regeneration. One may speculate that the dissimilar behavior of the two solvent systems arises from gross differences in the opsin-surfac~nt miceilular structure. Further expe~men~tion is in progress with the goal of clarifying and extending these results.

Thermal Stability of Rhodopsin Extracted with Triton X-100 Surfactant

91

DISCUSSION

A representation of the overall reaction by which oxime formation proceeds is shown in the accompanying equation, in which the terminal C of retinal is attached by a Schiff base covalent bond to a N on the protein opsin or, as POINCELOT et al. (1969) have recently proposed, to the N of phosphatidylethanolamine.

“27Cl9 \

c

=N

lR

"27Cl9

\ C=N’

--+-

/ H

H

OH

+ R - NH2

/

t R = opsin or phosphatidylethanolamine The Schiff base C, owing to its electrophilic character;presents a locus for attack by the nonbonding electrons on N in NH,OH. Any factor increasing the electrophilicity of C or the nucleophilicity of the attacking N would, in this scheme, increase the rate of the reaction, other reaction parameters remaining constant. It may be seen from the data in Table 2 that E, is dependent upon the pH of the solution, increasing at pH 5.5 and 8.2 and achieving a lower numerical value at essentially neutral pH. The rationale of such a pH dependence of &may be explained as follows: (1) increasing the acidity leads to greater electrophilicity of the Schiff base C, because of protonation of the imine N. In fact, in one of the limiting resonance species shown below, the C possesses a full positive charge;

(2) an increase in acidity decreases the nu~leophiIicity of the hydroxylamine too, is protonated:

N, because it,

NH,OH + H+ f NH,OH+ The net effect of these two opposing reaction parameters optimizes oxime formation at near-neutral condition, a situation reflected in the lowering of the magnitude of Ea. The reasons for changes in A,, the pre-exponential factor, for the different chemical systems described in this paper, are less clear. In terms of transition rate theory, A, can be used to calculate the entropy change that accompanies the transition of a molecule from the ground state to the activated complex. It is our belief that the use of transition state theory parameters is not realistic in view of the present state of knowledge of protein denaturations in general or the bleaching mechanism for rhodopsin in particular. We have therefore employed only the simple pre-exponential factor, i$,, throughout this paper.

RAYHOSD H. JOHM~H AND

92

THEODORE P. WILLI.*=

Increase in thermal rates of bleaching occasioned by hydroxylamine in Triton solutions may perhaps be explained by the following argument: Neither the presence nor absence of hydroxylamine causes a change in the position of A,,, or the half-band width of the main band of rhodopsin. This lack of effect implies that the active site of the protein-chromophore interaction is not altered in any essential aspect by hydroxylatine. However, from the enhancement of bleaching rates observed in Triton solutions containing this reagent, one may conclude that the molecule as a whole is more sensitive to thermal denaturation. In essence, hydroxylamine causes the protein in its micellular environment to become “partially denatured”, at a point so far removed from the active site that the spectroscopic parameters of the molecule are unchanged. The thermal stability is, however, decreased. In summary, the experiments reported here lead to the following conclusions: (1) cattle rhodopsin extracts in Triton display many of the same features as digitonin extracts, including the phenomenon of photoreversibility. However, rhodopsin cannot be regenerated by the dark incubation of its components, opsin and 1 1-cis retinal, in Triton solution. (2) the rate of thermal bleaching of Triton solutions of rhodopsin is about 100 times faster at moderate temperatures than similar solutions in digitonin. (3) the presence of hydroxylamine in Triton solutions makes the visual pigment distinctly more sensitive to thermal bleaching, the rate being first-order with respect to hydroxylamine. (4) the oxime resulting from thermal bleaching with hydroxylamine is predominately in a cis configurationprobably 11-cis. REFERENCES BAKER.B. N. and WILLIAMS. T. P. (1968). Thermal decomposition of rhodopsin. photoregenerated rhodopsin and P-170. Visiwr Res. 8. 1467-1469. CRESCITELLI. F. (1967). Extraction of visual pigments with certain.alkyl phenoxy polyethoxy ethanol surfaceactive compounds. Vision Res. 7. 685-693. HUBBARD.R. (1958). The thermal stability of rhodopsin and opsin. J. gm. Ph~siol. 42. 259-X30. POISCELOT.R. P.. M~LLAR,P. G., KNBEL. R. L. and ABRAHAUSOS. E. W. (1969). Lipid to protein chromophore transfer in the photolysis of visual pigments. .Vuture. Loud. 221. 25&X7. WILLIASIS.T. P. (1964-I).Photoreversal of rhodopsin bleaching. J. gem Ph)siol. 17. 679-688. WILLIAXIS. T. P. (1965). Rhodopsin bleaching: Relative effectiveness of high and low intensit? flashes. Vrxion Res. 5. 633-638.

Abstract-Thermal stabilities of T&on-extracted solutions of rhodopsin and photoreversed rhodopsin are determined and evaluated with reference to rhodopsin solutions in digitonin. It is found that the Arrhenius activation energy and pre-exponential factor are less in the Triton system with respect to the thermal bleaching reaction. Hydroxylamine (0.5 M) in Triton solutions causes rhodopsin to be distinctly more labile to thermal bleaching; the oxime product is predominantly in an 1I-cis configuration. Dark incubation of Triton solutions of I I-cis retinal and opsin does not yield regenerated rhodopsin. R&me-Sur des solutions extraites au Triton de rhodopsine et de rhodopsine d&colorie B la lumiere, on determine la stabiliti thermique et on la compare h celle de solutions de rhodopsine dans la digitonine. On trouve que I’energie d’activation d’Arrhinius et le facteur pr&exponentiel sont moindres dans le syst&me au Triton en ce qui conceme la reaction thermique de dSroloration. L’hydroxylamine (0.5 M) en solutions de Triton rend la rhodopsine nettement plus labile ti la dtioloration thermique: I’oliime produit est en predominance de configuration I I-cis. L’incubation dans I’obscuritC de solutions dans le Triton de ritinal I I-& et d’opsine ne rig&ire pas de rhodopsine.

Thermal Stability of Rhodopsin Extracted with friton

X-t@) Surfactant

95

Z~menf~u~g-Die The~albest~ndi~eit der mit Triton extrahierten Sehpurpur- und ~ichtumkehrsehpu~uri~sungen wird bestimmt und mit jener der DigitoninI~sungen des Sehpurpurs verglichen. Es wird gefunden. dess die Arrheniussche Aktivietungsenetgie und der praexponentielle KoelIizient im Tritonsystem kleiner sind, soweit es sich urn die Thermalausbleichungsreaktion handelt. Hydroxylamin (O-S M) in TritonlBsungen machr den Sehpurpur empfindlicher fbr die Thermalausbleichung das Oximprodukt besteht hauptskhlich .aus der I I-ci&?usammensetzung. Wenn Tritonliisungen des I I-c&Retinal mit Opsin im Dunkeln inkubiert werden, gibt es keine Sehpurpumeubildung.

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