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Thermal stability of squid rhodopsin in photoreceptor membrane and their triton extracts
Vision Rex. Vol. 17. pp. 169 to 173. Pergamon Press1977.Printed in GreatBritain.
THERMAL STABILITY OF SQUID RHODOPSIN IN PHOTORECEPTOR MEMBRANE AND THEIR TRITON EXTRACTS V. L.
SHNYROV, A. L. BERMAN, U. A. LAZAREV
and R. N.
ETING~F
I.
M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the U.S.S.R., Leningrad, U.S.S.R. and Institute of Biological Physics, Academy of Sciences of the U.S.S.R. Pushino (Moscow region), U.S.S.R. (Received 25 February 1976)
Abstract-Kinetic parameters of the thermal decay of squid rhodopsin in isolated photoreceptor membranes and in a 1% Triton X-100 solution have been determined. It has been shown that the treatment with Triton X-100 considerably decreases thermal stability of rhodopsin. Addition of hydroxylamine (0.5 M) to Triton X-100 solutions leads to further decrease of rhodopsin thermostability. When Triton extracts are exposed to light, bleaching of visual pigments occurs at room temperature.
temperature of -20°C for 4-7 months. The rhabdomeres isolation procedure was conducted in dim red light at a temperature of +4”C. Just before rhabdomeres isolation the squid eyes were placed in 2.7% NaCl solution and thawed. The eyeball was dissected with a scissors, the crystalline lens removed, and the eyeballs were turned inside out and slightly stirred in the 2.7% NaCl solution. The suspension obtained was filtered through two layers of gauze, and rhabdomeres were separated from the filtrate according to the scheme described earlier (Berman and Chirkivskaya, 1974) with some modifications. Supernatant obtained after centrifugation of homogenate (1 min, 100 g) was repeatedly centrifuged (10 min at 20,OOOg); the residue suspended in 1.3 M sucrose was layered under the steplike density gradient of sucrose solutions (0.7-0.9-1.1 M). The rhabdqmere fraction was selected after centrifugation (1 hr, 100,000 g) between the 0.9-1.1 M layers of sucrose solution. The content of rhodopsin was calculated according to determination of retinal by the thiobarbituric acid procedure (Zorn and Futterman. 1971); the protein content was measured by a burette method modified by us (Berman and Chirkovskaya, 1974). The molecular weight of rhodopsin was assumed to be equal to 70,000 (Abrahamson and Fager, 1973). The purity of the rhabdomere fraction was characterized by the rhodopsin protein to total protein ratio, the ratio constituted 4&50x. While studying temperature stability of rhosopsin directly in the photoreceptor membranes, the 0.1 ml suspension of rhabdomeres (0.05 M potassium phosphate buffer, pH 6.9, 4°C) was placed in an empty prewarmed test tube. The sample was incubated in an ultrathermostat for a certain period of time. Then the test tube was put on ice and 1.7 ml of precooled buffer solution together with 0.2 ml of a 10% Triton X-100 solution were added (final concentration of detergent was 1%). The samples were kept at 0°C for 10 min, centrifugated for 10 min at 10,000g, and rhodopsin content was determined spectrophotometritally in the supernatant thus obtained, by registration of absolute absorption spectrum in the region from 350 to 700 nm (Specord-UV-spectrophotometer, Karl Zeiss, Jena). The quantity of thermally bleached rhodopsin was continuously determined as the difference in optic densities at 490nm between the sample under study and the same METHODS AND MATERIALS preparation previously exposed to white light (So00 lx) for The squid (Ommasthrephes sloanei pacificus) eyes were 10 min at 25°C. used in the study. In most cases, animals’ were darkIt was found that after such an exposure to light rhodopadapted for some hours before enucleating the eye. Then sin was totaly bleached and only non-photosensitive imthe eyes were frozen, protected from light and kept at the purities were responsible for absorption at 490nm. 169
decay of visual pigments is the important feature which allows us to compare rhodopsin properties of various animals. Meanwhile the data existing are extremely scarce. Only cattle rhodopsin has been investigated in detail. It has been shown that thermal stability of visual pigment in a photoreceptor membrane differs from that in detergent solutions (Hubbard, 1958; Johnson and Williams, 1970). Recent work of Williams, Baker and McDowell (1974) has shown that temperature stability of the derivatives of cattle visual pigment is determined by properties of protein and membrane lipid components as well. As to visual pigments of other animals, only a few indications are available. These data were obtained using digitonin extracts of rhodopsin (Lythgoe and Quilliam, 1938; Hubbard and St. George, 1957-1958; Williams and Mirby, 1962; Crescitelli, 1974) and therefore cannot characterize the thermostability of the visual pigment in the membrane. The squid rhodopsin is known to display some peculiarities in photo-induced transformations (Hubbard and St. George, 1957; Kropf, Brown and Hubbard, 1959; Yoshizawa and Wald, 1964) as well as differences in the phospholipid patterns of photoreceptor membranes compared with vertebrates (Mason, Fayes and Abrahamson, 1973; Berman and Chirkovskaya, 1974). The present paper deals with the determination of the kinetic parameters of thermal decay of rhodopsin in its natural state (i.e. in the membrane) and when solubilized by Triton X-100. The choice of the detergent was specified by the earlier experiments in which significant difference in the temperature stability of cattle and squid rhodopsins solubilized by this detergent was shown (Berman and Chirkovskaya, 1974). The kinetics of the thermal
170
V. L.
Time,
min
SHNYR~V
._
Fig. 1. Kinetics of thermal bleaching of squid rhodopsin in photoreceptor membranes at four temperatures. OD, and OD, are the optical densities initially and at time t. respectively, at 490 nm. pH 6.9. When studying the thermal decay of illuminated rhodopsin, the fraction of rhabdomeres was illuminated at 0°C with the white light (50001x) for 10 min. For studying the thermal stability of rhodopsin in Triton extracts, the latter were obtained in the following way: l-l.Sml of 1% Triton X-100 solution was added to the sediment of rhabdomere fraction (10 mg protein); the suspension was incubated for 30 min at 4°C and centrifuged during 10 min at 10,000 g. Then, 0.05 ml samples of supernatant, presenting the rhodopsin extract in 1% Triton X-100 solution were put into a prewarmed thermostable cuvettes with 2 ml of 1% Triton X-1000 solutions. The mixture was stirred and an absolute absorption spectrum of rhodopsin was recorded at a particular time interval. Temperature measurements in the cuvette were taken continuously using calibrated thermistor. In all the experiments performed, the time for temperature equilibrium in the test tube or in the cuvette after introduction of samples did not exceed 20sec. RESULTS Figures 1 and 2 show the kinetics of thermal decay of rhodopsin in photoreceptor membranes and extracted by Triton X-100 solution at various temperatures. The decrease of absorption at 490nm observed was caused by rhodopsin decay, as during this process a product with a absorption maximum at 385 nm appears due to the complete decomposition of visual pigment and release of retinal. Special experiments showed also that the screening pigment of the squid retina, ommin (absorption maximum 5lOnm) presented in the form of impurities in rhabdomeres preparations (Hagins, 1973), proved to be more thermally stable than rhodopsin and did not decay in these conditions. The data presented in Figs. 1 and 2 clearly illustrate that the reaction of rhodopsin thermal decay in both cases follows first-order kinetics. Some calculations similar to those used by Hubbard (1958) have made it possible to characterize the changes in free energy, entropy, activation energy (E,) as well as the temperature at which a half of the total amount of rhodopsin decayed during 10 min (Table 1). The considerable decrease in the stability of extracted rhodopsin as compared to the native one
t’f ~1.
revealed in the reduction of activation energ) ;tnd considerable decrease of the temperature at ~l~clr denaturation of half of the visual pigment occurs 111 10 min. So, in this respect modifications of the squid rhodopsin under the influence of Triton X-I(K) ucrc approximately similar to those of the cattle rhodopsin (Johnson and Williams, 1970). However. in our cxperiments the squid rhodopsin treated by Triton X-100 showed the entropy change reduced. by contrast to the cattle rhodopsin (Hubbard. 1958). In the presence of hydroxylamine the thermal stability of the squid rhodopsin reduced (Fig. 3, Table I ), similar to that of the cattle rhodopsin (Johnson and Williams, 1070). A linear dependence has been revealed (Fig. 4b) for the rate of the decay of the rhodopsin on various concentrations of hydroxylamine at a constant temperature (Fig. 4a). Extrapolation of this line to the zero concentration point of hydroxylamine led us to conclude that the Triton extract is not stable :tt tcmperatures as low as room temperature, which is in line with the data of Berman and Chirkovskaya ( 1974). In studying the kinetics of the thermal decay of rhodopsin preparations exposed to light (in Triton X-100 solution) the curves characterizing a decay reaction had a clearly expressed break (Fig. 5). which indicated the existence of two different products. Kinetic parameters of the two stages of the process differed greatly from each other (Table I). DISCUSSION Parameters characterizing the thermal decay of rhodopsin enable us to compare the properties of visual pigments of various animals. Fragmentary data available at the present moment are summed up in Table 2. The most obvious and convincing parameter characterizing rhodopsin resistance to heating is the temperature threshold of its denaturation. In this respect, a considerable difference (21°C) in thermostability of cattle and squid rhodopsins may be of interest. These data were obtained when rhodopsin was investigated in its natural environment, i.e. in the photoreceptor membranes. However, this difference is retained.
0.60
-
0
I
lke.
min
2
3
Fig. 2. Kinetics of thermal bleaching of squid rhodopsin in l”/, Triton X-100 solution at five temperatures. See legend to Fig. I.
171
Thermal stability of squid rhodopsin Table 1. Thermal bleaching of squid rhodopsin in various conditions
Decay rate constant (k x 103, set-‘)
Temperature (“C)
Experiment conditions
AFf E: (kcal/mole) (kcal/mole)
ASf (Cal/mole degree)
Temperature for halfdenaturation in 10 min (“C)
Photoreceptor membranes
50.0 51.6 53.0 54.2
1.5 2.9 4.3 6.7
77
23
164
49
1% Triton X-100 solution
35.0 37.3 40.3 42.2 45.0
2.7 6.7 13.8 27.6 52.2
63
21
131
32
26.0 28.2 30.3 31.8 34.0
0.8 1.3 2.3 3.7 8.2
55
21
107
27.5
24.0 27.0 30.0
2.1 4.4 13.0
55
21
112
21
24.0 27.0 30.0
0.4 0.7 1.1
29
22
21
1% Triton X-100 solution +
0.5 M hydroxylamine 1% Triton X-100 solution, preparation exposed to light, Fig. 5, Stage I
Stage
II
* See Fig. 6.
though less pronounced (14”C), in digitonin extracts of visual pigments that confirms the idea of differences in rhodopsin protein component of both kinds of animals (Hubbard, 1958). The biological significance of these differences is not clear yet. It is possible that this property of rhodopsin results from structural peculiarities of photoreceptor membranes and molecules of visual pigments in invertebrates and vertebrates. Independently of the
0
2
4
6
6 Time,
IO
12
14
16
min
Fig. 3. Kinetics of thermal bleaching of squid rhodopsin in 1% Triton X-100 solution in the presence of hydroxylamine (0.5 M) at five temperatures. See legend to Fig. 1.
reason for these differences, it is evident that thermal stability of visual pigment may be a useful parameter for comparing rhodopsin properties of animals found in various environment. In our opinion the thermostability of visual pigments is to be studied on photoreceptor membranes rather than in detergent extracts, since when dealing with detergents it is difficult to say whether the properties observed are inherent to the pigment itself or need to be referred to the extraction procedure (Crescitelli, 1974). Treatment with detergents, in particular Triton X-100 (see Table 2), leads to “labilization” of the visual pigment molecules that is manifested in the reduction of the thermostability in comparison with the membrane form of the pigment. Triton X-100 considerably decreased the activation energy of thermal decay of squid rhodopsin in comparison with visual pigment in the native membrane, which reminds its action on cattle rhodopsin (Table 2). Digitonin appeared to be a milder agent as compared with Triton X-100. Addition of hydroxylamine to Triton X-100 extracts of both rhodopsins studied led to further reduction of their thermal stabilities. So, the sensitivity of cattle and squid rhodopsins to the agents disturbing their native structure was approximately identical. As far as the other kinetic parameters are concerned, the most inexplicable one is the change in entropy. We observed decrease of entropy change as the result of various treatments of rhodopsin; it may be
172
V. L. SHNYROV Table
Rhodopsin experiment
2. Kinetic
origin and conditions
(kcat;ole)
SQUID Photoreceptor membranes I”,: digitonin solution IS, Triton X-100 solution I”‘, Triton X-100 solution +0.5 M NH,OH 1” Triton X-100 solution (bikached preparation)* CATTLE Photoreceptor membranes I”,, digitonin solution 2”,, Triton X-100 solution
process
of thermal
AFS (kcal/mole)
decay
AS: (Cal/mole degree)
of various
animals
Temperature for halfdenaturation in IO min
( CI
Reference
71 12 63 55
23 23 21 21
I64 IS? 131 107
49.0 43.x 32.0 27 s
Present Hubbard Present Present
55 29
21 22
II2 21
21.0
Present
100 100 7s
26 24
214 225
72.0 60.0
24
63
54.6
2’!:, Triton X-100 solution f0.5 M NH,OH FROG l”‘<,,digitonin solution * Two-stage
parameters
u/ d.
paper
Hubbard (1958) Hubbard (1958) Johnson and Williams (1970) Johnson and Williams (1970)
41
45
paper (1958) paper paper
Hubbard,
(1958)
(see Fig. 5). J30.0' 060-
0
2
4
6
6 Time.
IO
12
14
16
min
0
5
15
IO Time,
min
Fig. 5. Kinetics of thermal bleaching of squid rhodopsin exposed to light in a 1% Triton X-100 solution at three temperatures. See legend to Fig. I
I 3.
I
I 3.2
I 3.3
3
I
lIToK I: IO3
IN&OH],
moles/l
Fig. 4. (a) Kinetics of thermal bleaching of squid rhodopsin in 1% Triton X-100 solution in the presence of various concentrations of hydroxylamine at 30°C. See legend to 1. Hydroxylamine concentration (M): Aa.06; Fig. B--0.12; C-0.25; D--0.3; E-OS. (b) The relationship between rate constant and hydroxylamine concentration. The data from Fig. 4a.
Fig. 6. Arrhenius plots of the rate constants 1. The slope of each line multiplied by 2.303 stant yields the Airhenius activation energy photoreceptor membranes, pH 6.9; B-in 1% 6.9; C-in 1% Triton + 0.5 M NH,OH, pH 1% Triton. exposed to light; stage I; E-the 11. pH 6.9.
from Table x gas conE,. A---in Triton, pH 6.9; D-in same, stage
Thermal stability of squid rhodopsin supposed that in these conditions the structure of visual pigment molecules becomes less orderly and its denaturation is accomplished with a smaller degree of contingency. However, taking into account the complexity of interpretation of thermodynamic transformation in visual pigment, the validity of such a suggestion is not yet clear. Two-stage process of the thermal decay of illuminated rhodopsin (Fig. 5) may point to the existence in this preparation of two products with different thermostabilities. This problem needs further investigation. REFERENCES
Abrahamson E. W. and Fager R. S. (1973) The chemistry of vertebrate and invertebrate visual photoreceptors. Curr. Topics Bioengng 5, 125-200.
Berman A. L. and Chirkovskaya E. V. (1974) The content of rhodopsin and phospholipids in photoreceptor membranes of the squid Ommasthrephes sloanei pacificus. J. Euol. Biochem. Physiol. 10, 566572 (in Russian). Crescitelli F. (1974) The gecko visual pigments: the thermosensitive property. Msion Res. 14, 243-259. Hagins F. M. (1973) Purification and partial characterization of the protein component of squid rhodopsin. J. biol. Chem. 248, 3298-3304.
173
Hubbard R. (1958) The thermal stability of rhodopsin and opsin. .I. gen. Physiol. 42, 259-280. Hubbard R. and St. George R. C. (1957) The rhodopsin system of the squid. J. gen. PhysioI. 41, 501-528. Johnson R. H. and Williams T. P. (1970) Thermal stability of rhodopsin extracted with Triton X-100 surfactant. Vision Res. 10. 85-93. Kropf A., Brown P. K. and Hubbard R. (1959) Action of light on visual pigments: lumi and meta-rhodopsins of squid and octopus. Nature, Lond. 183, 446448. Lythgoe R. L. and Quilliam J. P. (1938) The thermal decomposition of visual purple. J. Physiol., Lond. 93. 24-31.
Mason W. T., Fager R. S. and Abrahamson E. W. (1973) Characterization of the lipid composition of squid rhabdome outer segments. Biochim. Biophys. Acta 306, 67-73. Williams T. P., Baker B. N. and McDowell G. H. (1974) The influence of lipids on dynamic properties of rhodopsin. Expl Eye Res. 18, 69-77. Williams T. P. and Mirby S. E. (1968) The thermal decomposition of some visual pigments. Vision Res. 8, 359-367. Yoshizawa T. and Wald G. (1964) Transformations of squid rhodopsin at low temperatures. Nature, Lond. 201, 34c-345.
Zorn M. and Futterman S. (1971) Properties of rhodopsin dependent in associated phospholipid. J. biol. Chem. 246, 881-886.
THERMAL STABILITY OF SQUID RHODOPSIN IN PHOTORECEPTOR MEMBRANE AND THEIR TRITON EXTRACTS V. L.
SHNYROV, A. L. BERMAN, U. A. LAZAREV
and R. N.
ETING~F
I.
M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the U.S.S.R., Leningrad, U.S.S.R. and Institute of Biological Physics, Academy of Sciences of the U.S.S.R. Pushino (Moscow region), U.S.S.R. (Received 25 February 1976)
Abstract-Kinetic parameters of the thermal decay of squid rhodopsin in isolated photoreceptor membranes and in a 1% Triton X-100 solution have been determined. It has been shown that the treatment with Triton X-100 considerably decreases thermal stability of rhodopsin. Addition of hydroxylamine (0.5 M) to Triton X-100 solutions leads to further decrease of rhodopsin thermostability. When Triton extracts are exposed to light, bleaching of visual pigments occurs at room temperature.
temperature of -20°C for 4-7 months. The rhabdomeres isolation procedure was conducted in dim red light at a temperature of +4”C. Just before rhabdomeres isolation the squid eyes were placed in 2.7% NaCl solution and thawed. The eyeball was dissected with a scissors, the crystalline lens removed, and the eyeballs were turned inside out and slightly stirred in the 2.7% NaCl solution. The suspension obtained was filtered through two layers of gauze, and rhabdomeres were separated from the filtrate according to the scheme described earlier (Berman and Chirkivskaya, 1974) with some modifications. Supernatant obtained after centrifugation of homogenate (1 min, 100 g) was repeatedly centrifuged (10 min at 20,OOOg); the residue suspended in 1.3 M sucrose was layered under the steplike density gradient of sucrose solutions (0.7-0.9-1.1 M). The rhabdqmere fraction was selected after centrifugation (1 hr, 100,000 g) between the 0.9-1.1 M layers of sucrose solution. The content of rhodopsin was calculated according to determination of retinal by the thiobarbituric acid procedure (Zorn and Futterman. 1971); the protein content was measured by a burette method modified by us (Berman and Chirkovskaya, 1974). The molecular weight of rhodopsin was assumed to be equal to 70,000 (Abrahamson and Fager, 1973). The purity of the rhabdomere fraction was characterized by the rhodopsin protein to total protein ratio, the ratio constituted 4&50x. While studying temperature stability of rhosopsin directly in the photoreceptor membranes, the 0.1 ml suspension of rhabdomeres (0.05 M potassium phosphate buffer, pH 6.9, 4°C) was placed in an empty prewarmed test tube. The sample was incubated in an ultrathermostat for a certain period of time. Then the test tube was put on ice and 1.7 ml of precooled buffer solution together with 0.2 ml of a 10% Triton X-100 solution were added (final concentration of detergent was 1%). The samples were kept at 0°C for 10 min, centrifugated for 10 min at 10,000g, and rhodopsin content was determined spectrophotometritally in the supernatant thus obtained, by registration of absolute absorption spectrum in the region from 350 to 700 nm (Specord-UV-spectrophotometer, Karl Zeiss, Jena). The quantity of thermally bleached rhodopsin was continuously determined as the difference in optic densities at 490nm between the sample under study and the same METHODS AND MATERIALS preparation previously exposed to white light (So00 lx) for The squid (Ommasthrephes sloanei pacificus) eyes were 10 min at 25°C. used in the study. In most cases, animals’ were darkIt was found that after such an exposure to light rhodopadapted for some hours before enucleating the eye. Then sin was totaly bleached and only non-photosensitive imthe eyes were frozen, protected from light and kept at the purities were responsible for absorption at 490nm. 169
decay of visual pigments is the important feature which allows us to compare rhodopsin properties of various animals. Meanwhile the data existing are extremely scarce. Only cattle rhodopsin has been investigated in detail. It has been shown that thermal stability of visual pigment in a photoreceptor membrane differs from that in detergent solutions (Hubbard, 1958; Johnson and Williams, 1970). Recent work of Williams, Baker and McDowell (1974) has shown that temperature stability of the derivatives of cattle visual pigment is determined by properties of protein and membrane lipid components as well. As to visual pigments of other animals, only a few indications are available. These data were obtained using digitonin extracts of rhodopsin (Lythgoe and Quilliam, 1938; Hubbard and St. George, 1957-1958; Williams and Mirby, 1962; Crescitelli, 1974) and therefore cannot characterize the thermostability of the visual pigment in the membrane. The squid rhodopsin is known to display some peculiarities in photo-induced transformations (Hubbard and St. George, 1957; Kropf, Brown and Hubbard, 1959; Yoshizawa and Wald, 1964) as well as differences in the phospholipid patterns of photoreceptor membranes compared with vertebrates (Mason, Fayes and Abrahamson, 1973; Berman and Chirkovskaya, 1974). The present paper deals with the determination of the kinetic parameters of thermal decay of rhodopsin in its natural state (i.e. in the membrane) and when solubilized by Triton X-100. The choice of the detergent was specified by the earlier experiments in which significant difference in the temperature stability of cattle and squid rhodopsins solubilized by this detergent was shown (Berman and Chirkovskaya, 1974). The kinetics of the thermal
170
V. L.
Time,
min
SHNYR~V
._
Fig. 1. Kinetics of thermal bleaching of squid rhodopsin in photoreceptor membranes at four temperatures. OD, and OD, are the optical densities initially and at time t. respectively, at 490 nm. pH 6.9. When studying the thermal decay of illuminated rhodopsin, the fraction of rhabdomeres was illuminated at 0°C with the white light (50001x) for 10 min. For studying the thermal stability of rhodopsin in Triton extracts, the latter were obtained in the following way: l-l.Sml of 1% Triton X-100 solution was added to the sediment of rhabdomere fraction (10 mg protein); the suspension was incubated for 30 min at 4°C and centrifuged during 10 min at 10,000 g. Then, 0.05 ml samples of supernatant, presenting the rhodopsin extract in 1% Triton X-100 solution were put into a prewarmed thermostable cuvettes with 2 ml of 1% Triton X-1000 solutions. The mixture was stirred and an absolute absorption spectrum of rhodopsin was recorded at a particular time interval. Temperature measurements in the cuvette were taken continuously using calibrated thermistor. In all the experiments performed, the time for temperature equilibrium in the test tube or in the cuvette after introduction of samples did not exceed 20sec. RESULTS Figures 1 and 2 show the kinetics of thermal decay of rhodopsin in photoreceptor membranes and extracted by Triton X-100 solution at various temperatures. The decrease of absorption at 490nm observed was caused by rhodopsin decay, as during this process a product with a absorption maximum at 385 nm appears due to the complete decomposition of visual pigment and release of retinal. Special experiments showed also that the screening pigment of the squid retina, ommin (absorption maximum 5lOnm) presented in the form of impurities in rhabdomeres preparations (Hagins, 1973), proved to be more thermally stable than rhodopsin and did not decay in these conditions. The data presented in Figs. 1 and 2 clearly illustrate that the reaction of rhodopsin thermal decay in both cases follows first-order kinetics. Some calculations similar to those used by Hubbard (1958) have made it possible to characterize the changes in free energy, entropy, activation energy (E,) as well as the temperature at which a half of the total amount of rhodopsin decayed during 10 min (Table 1). The considerable decrease in the stability of extracted rhodopsin as compared to the native one
t’f ~1.
revealed in the reduction of activation energ) ;tnd considerable decrease of the temperature at ~l~clr denaturation of half of the visual pigment occurs 111 10 min. So, in this respect modifications of the squid rhodopsin under the influence of Triton X-I(K) ucrc approximately similar to those of the cattle rhodopsin (Johnson and Williams, 1970). However. in our cxperiments the squid rhodopsin treated by Triton X-100 showed the entropy change reduced. by contrast to the cattle rhodopsin (Hubbard. 1958). In the presence of hydroxylamine the thermal stability of the squid rhodopsin reduced (Fig. 3, Table I ), similar to that of the cattle rhodopsin (Johnson and Williams, 1070). A linear dependence has been revealed (Fig. 4b) for the rate of the decay of the rhodopsin on various concentrations of hydroxylamine at a constant temperature (Fig. 4a). Extrapolation of this line to the zero concentration point of hydroxylamine led us to conclude that the Triton extract is not stable :tt tcmperatures as low as room temperature, which is in line with the data of Berman and Chirkovskaya ( 1974). In studying the kinetics of the thermal decay of rhodopsin preparations exposed to light (in Triton X-100 solution) the curves characterizing a decay reaction had a clearly expressed break (Fig. 5). which indicated the existence of two different products. Kinetic parameters of the two stages of the process differed greatly from each other (Table I). DISCUSSION Parameters characterizing the thermal decay of rhodopsin enable us to compare the properties of visual pigments of various animals. Fragmentary data available at the present moment are summed up in Table 2. The most obvious and convincing parameter characterizing rhodopsin resistance to heating is the temperature threshold of its denaturation. In this respect, a considerable difference (21°C) in thermostability of cattle and squid rhodopsins may be of interest. These data were obtained when rhodopsin was investigated in its natural environment, i.e. in the photoreceptor membranes. However, this difference is retained.
0.60
-
0
I
lke.
min
2
3
Fig. 2. Kinetics of thermal bleaching of squid rhodopsin in l”/, Triton X-100 solution at five temperatures. See legend to Fig. I.
171
Thermal stability of squid rhodopsin Table 1. Thermal bleaching of squid rhodopsin in various conditions
Decay rate constant (k x 103, set-‘)
Temperature (“C)
Experiment conditions
AFf E: (kcal/mole) (kcal/mole)
ASf (Cal/mole degree)
Temperature for halfdenaturation in 10 min (“C)
Photoreceptor membranes
50.0 51.6 53.0 54.2
1.5 2.9 4.3 6.7
77
23
164
49
1% Triton X-100 solution
35.0 37.3 40.3 42.2 45.0
2.7 6.7 13.8 27.6 52.2
63
21
131
32
26.0 28.2 30.3 31.8 34.0
0.8 1.3 2.3 3.7 8.2
55
21
107
27.5
24.0 27.0 30.0
2.1 4.4 13.0
55
21
112
21
24.0 27.0 30.0
0.4 0.7 1.1
29
22
21
1% Triton X-100 solution +
0.5 M hydroxylamine 1% Triton X-100 solution, preparation exposed to light, Fig. 5, Stage I
Stage
II
* See Fig. 6.
though less pronounced (14”C), in digitonin extracts of visual pigments that confirms the idea of differences in rhodopsin protein component of both kinds of animals (Hubbard, 1958). The biological significance of these differences is not clear yet. It is possible that this property of rhodopsin results from structural peculiarities of photoreceptor membranes and molecules of visual pigments in invertebrates and vertebrates. Independently of the
0
2
4
6
6 Time,
IO
12
14
16
min
Fig. 3. Kinetics of thermal bleaching of squid rhodopsin in 1% Triton X-100 solution in the presence of hydroxylamine (0.5 M) at five temperatures. See legend to Fig. 1.
reason for these differences, it is evident that thermal stability of visual pigment may be a useful parameter for comparing rhodopsin properties of animals found in various environment. In our opinion the thermostability of visual pigments is to be studied on photoreceptor membranes rather than in detergent extracts, since when dealing with detergents it is difficult to say whether the properties observed are inherent to the pigment itself or need to be referred to the extraction procedure (Crescitelli, 1974). Treatment with detergents, in particular Triton X-100 (see Table 2), leads to “labilization” of the visual pigment molecules that is manifested in the reduction of the thermostability in comparison with the membrane form of the pigment. Triton X-100 considerably decreased the activation energy of thermal decay of squid rhodopsin in comparison with visual pigment in the native membrane, which reminds its action on cattle rhodopsin (Table 2). Digitonin appeared to be a milder agent as compared with Triton X-100. Addition of hydroxylamine to Triton X-100 extracts of both rhodopsins studied led to further reduction of their thermal stabilities. So, the sensitivity of cattle and squid rhodopsins to the agents disturbing their native structure was approximately identical. As far as the other kinetic parameters are concerned, the most inexplicable one is the change in entropy. We observed decrease of entropy change as the result of various treatments of rhodopsin; it may be
172
V. L. SHNYROV Table
Rhodopsin experiment
2. Kinetic
origin and conditions
(kcat;ole)
SQUID Photoreceptor membranes I”,: digitonin solution IS, Triton X-100 solution I”‘, Triton X-100 solution +0.5 M NH,OH 1” Triton X-100 solution (bikached preparation)* CATTLE Photoreceptor membranes I”,, digitonin solution 2”,, Triton X-100 solution
process
of thermal
AFS (kcal/mole)
decay
AS: (Cal/mole degree)
of various
animals
Temperature for halfdenaturation in IO min
( CI
Reference
71 12 63 55
23 23 21 21
I64 IS? 131 107
49.0 43.x 32.0 27 s
Present Hubbard Present Present
55 29
21 22
II2 21
21.0
Present
100 100 7s
26 24
214 225
72.0 60.0
24
63
54.6
2’!:, Triton X-100 solution f0.5 M NH,OH FROG l”‘<,,digitonin solution * Two-stage
parameters
u/ d.
paper
Hubbard (1958) Hubbard (1958) Johnson and Williams (1970) Johnson and Williams (1970)
41
45
paper (1958) paper paper
Hubbard,
(1958)
(see Fig. 5). J30.0' 060-
0
2
4
6
6 Time.
IO
12
14
16
min
0
5
15
IO Time,
min
Fig. 5. Kinetics of thermal bleaching of squid rhodopsin exposed to light in a 1% Triton X-100 solution at three temperatures. See legend to Fig. I
I 3.
I
I 3.2
I 3.3
3
I
lIToK I: IO3
IN&OH],
moles/l
Fig. 4. (a) Kinetics of thermal bleaching of squid rhodopsin in 1% Triton X-100 solution in the presence of various concentrations of hydroxylamine at 30°C. See legend to 1. Hydroxylamine concentration (M): Aa.06; Fig. B--0.12; C-0.25; D--0.3; E-OS. (b) The relationship between rate constant and hydroxylamine concentration. The data from Fig. 4a.
Fig. 6. Arrhenius plots of the rate constants 1. The slope of each line multiplied by 2.303 stant yields the Airhenius activation energy photoreceptor membranes, pH 6.9; B-in 1% 6.9; C-in 1% Triton + 0.5 M NH,OH, pH 1% Triton. exposed to light; stage I; E-the 11. pH 6.9.
from Table x gas conE,. A---in Triton, pH 6.9; D-in same, stage
Thermal stability of squid rhodopsin supposed that in these conditions the structure of visual pigment molecules becomes less orderly and its denaturation is accomplished with a smaller degree of contingency. However, taking into account the complexity of interpretation of thermodynamic transformation in visual pigment, the validity of such a suggestion is not yet clear. Two-stage process of the thermal decay of illuminated rhodopsin (Fig. 5) may point to the existence in this preparation of two products with different thermostabilities. This problem needs further investigation. REFERENCES
Abrahamson E. W. and Fager R. S. (1973) The chemistry of vertebrate and invertebrate visual photoreceptors. Curr. Topics Bioengng 5, 125-200.
Berman A. L. and Chirkovskaya E. V. (1974) The content of rhodopsin and phospholipids in photoreceptor membranes of the squid Ommasthrephes sloanei pacificus. J. Euol. Biochem. Physiol. 10, 566572 (in Russian). Crescitelli F. (1974) The gecko visual pigments: the thermosensitive property. Msion Res. 14, 243-259. Hagins F. M. (1973) Purification and partial characterization of the protein component of squid rhodopsin. J. biol. Chem. 248, 3298-3304.
173
Hubbard R. (1958) The thermal stability of rhodopsin and opsin. .I. gen. Physiol. 42, 259-280. Hubbard R. and St. George R. C. (1957) The rhodopsin system of the squid. J. gen. PhysioI. 41, 501-528. Johnson R. H. and Williams T. P. (1970) Thermal stability of rhodopsin extracted with Triton X-100 surfactant. Vision Res. 10. 85-93. Kropf A., Brown P. K. and Hubbard R. (1959) Action of light on visual pigments: lumi and meta-rhodopsins of squid and octopus. Nature, Lond. 183, 446448. Lythgoe R. L. and Quilliam J. P. (1938) The thermal decomposition of visual purple. J. Physiol., Lond. 93. 24-31.
Mason W. T., Fager R. S. and Abrahamson E. W. (1973) Characterization of the lipid composition of squid rhabdome outer segments. Biochim. Biophys. Acta 306, 67-73. Williams T. P., Baker B. N. and McDowell G. H. (1974) The influence of lipids on dynamic properties of rhodopsin. Expl Eye Res. 18, 69-77. Williams T. P. and Mirby S. E. (1968) The thermal decomposition of some visual pigments. Vision Res. 8, 359-367. Yoshizawa T. and Wald G. (1964) Transformations of squid rhodopsin at low temperatures. Nature, Lond. 201, 34c-345.
Zorn M. and Futterman S. (1971) Properties of rhodopsin dependent in associated phospholipid. J. biol. Chem. 246, 881-886.