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[49] Low-temperature spectrophotometry of intermediates of rhodopsin
[49]
LOW-TEMPERATURE
RHODOPSIN
[49] L o w - T e m p e r a t u r e Intermediates
SPECTROPHOTOMETRY
Spectrophotometry of Rhodopsin
333
of
By T6RU YOSHIZAWA and YOSHINOR1 S H I C H I D A It is well known that the absorption spectrum of rhodopsin in the visible range is in good agreement with the scotopic luminosity curve. 1 This fact gives us a basis for the proposition that the photochemical reaction of rhodopsin triggers the excitation of the visual cell. Since it takes a few milliseconds to generate the receptor potential of the visual cell after capture of a photon,2 the photoreaction of rhodopsin in less than a few milliseconds is important for elucidating the function of rhodopsin in visual transduction. Because such a fast photoreaction was so difficult to study at room temperature, most investigations had been carried out at low temperatures, 3'4 where the thermal reaction rate of intermediates of rhodopsin is remarkably reduced. The application of low-temperature spectrophotometry to the rhodopsin system has led to discovery of several •intermediates in the process of photobleaching of rhodopsin, as shown in Fig. 1. Thus it may safely be said that one of the most powerful tools for detection and identification of the intermediates of bleaching of rhodopsin is low-temperature spectrophotometry. In combination with this method, other spectroscopic measurements, such as CD, resonance Raman, and laser photolysis, also provide reliable information for analysis of the structure and kinetics of the intermediates of rhodopsin. Recently, lowtemperature spectrophotmetry has been applied to rhodopsin analogs prepared from cattle opsin and retinal analogs synthesized chemically. The studies on the photoreactions of rhodopsin analogs have given us invaluable information on structure of the intermediates of rhodopsin and its photoreceptive mechanism. 5-7 In the following, procedures for preparation of samples, techniques for low-temperature spectrophotometry, and absorption characteristics of the intermediates of rhodopsin will be described. i G. Wald and P. K. Brown, Science 127, 222 (1958). 2 T. E. Ogden and K. T. Brown, unpublished results, cited in K. T. Brown, Nature (London) 193, 958 (1962). 3 G. Wald, Science 162, 230 (1968). 4 T. Yoshizawa, in " H a n d b o o k of Sensory P h y s i o l o g y " (H. J. A. Dartnail, ed.), Vol. 7, Part 1, pp. 146-179. Springer-Verlag, Berlin and N e w York, 1972. 5 S. K a w a m u r a , S. Miyatani, H. Matsumoto, T. Yoshizawa, and R. S. H. Liu, Biochemistry 19, 1549 (1980). 6 S. K a w a m u r a , T. Yoshizawa, K. Horiuchi, M. Ito, A. K o d a m a , and K. Tsukida, Biochim. Biophys. Acta 548, 147 (1979). r y . Shichida, A. Kropf, and T. Yoshizawa, Biochemistry 20, 1962 (1981). Copyright © 1982by AcademicPress, Inc. METHODS IN ENZYMOLOGY, VOL. 81 All rights of reproduction in any form reserved. ISBN 0-12-181981-7
334
SPECTRAL RESPONSES OF VISUAL PIGMENTS
cattle rhodopsin (498 nm)* 3
squid rhodopsin (480 nm)"15
Zve < 15 psec 39 }
T1/e
hv
[49]
< 19 psec37 l hv
T
hypsorhodopsin (430 nm) 4 1"1~e
--- 50 psec39 I
> -251° C4
bathorhodopsin (543 nm) T1/e
-~ 40 nsec46 I
hypsorhodopsin (446 nm) 28
11
7"1/e
~- 50 psec37 I > - 2 3 8 ° C28
bathorhodopsin (534 nm)
> - 1 4 0 ° Cll
lumirhodopsin (497 nm) 23
7"1/e
26
300 nsec37 J > - 1 6 0 ° C 26
lumirhodopsin (515 nm) 26
/
T1/e
--~16sec 58 ]
> - 4 0 °C 11
metarhodopsin I (478 nm)
55
° C 26
LM-rhodopsin (486 nm)
/
!
T1/~-~ 200 msecS4l
>-15 °
C 11
metarhodopsin !1 (380 nrn) 55 T1~-~1 hr
>-65
!
/
l
> 0 °C55 /
¥
pararhodopsin (465 nm)
TI~ ~-10msec 6° [ > - 2 0 ° C26 acid metarhodopsin (482 nml ~ 61 +H+
11 iT
- H+
alkaline metarhodopsin (367 nm)* 61
> 5° C 55 all-trans-retinal (387 nm)* +
opsin FIG. 1. Photobleaching processes of cattle and squid rhodopsins. Photochemical reactions are denoted by wavy lines and thermal (dark) reactions by straight lines. Transition temperatures from one intermediate to another measured by low-temperature spectrophotometry are shown at the right sides of the straight lines and decay times of intermediates measured by flash photolysis at room temperatures are shown at the left sides of the straight lines. Absorption maxima are shown in parentheses. The absorption maxima indicated * are measured at room temperatures. S a m p l e for L o w - T e m p e r a t u r e
Spectrophotometry
Three kinds of rhodopsin preparations have been used, i.e., a whole r e t i n a , s-~° a s u s p e n s i o n o f r o d o u t e r s e g m e n t s TM ( R O S ) , a n d an e x t r a c t o f r h o d o p s i n . 11 W h o l e R e t i n a . A r e t i n a i s o l a t e d f r o m a d a r k - a d a p t e d f r o g e y e h as b e e n u s e d b e c a u s e o f e a s e in i s o l a t i o n o f an i n t a c t r e t i n a a n d t h e l a r g e a m o u n t
s F. 9 S. 10 S. 11 T.
Tokunaga, S. Kawamura, and T. Kawamura, F. Tokunaga, and T. Kawamura, F. Tokunaga, and T. Yoshizawa and G. Wald, Nature
Yoshizawa, Vision Res. 16, 633 (1976). Yoshizawa, Vision Res. 17, 991 (1977). Yoshizawa, Vision Res. 19, 879 (1979). (London) 197, 1279 (1963).
[49]
LOW-TEMPERATURE
RHODOPSIN
SPECTROPHOTOMETRY
335
of rhodopsin in the retina. After isolation of the retina in Ringer's solution, it is put between two sheets of filter paper with a hole (about 7 mm in diameter) at the center and then a small amount of glycerol is dropped on it. Afterward it is set in a sample cell of an optical cryostat described below. Suspension of Rod Outer Segments. In the case of the frog, two or three retinas isolated from dark-adapted frog eyes are gently shaken in Ringer's several times. ROS suspension thus obtained is centrifuged at 3000 g for 5 min in order to collect ROS as a precipitate. Afterward it is resuspended in a small amount of Ringer's containing 0.1 M hydroxylamine (pH 7.0) and then glycerol is added to give a final concentration of 75% or more. This mixture is used as a sample. Cattle ROS and squid microvilli both are prepared by a conventional sucrose flotation method 12-14 with or without lyophilization and extraction of lipid and retinal derivatives with petroleum ether. Glycerol is added to give a final concentration of 75% or more. Rhodopsin Extract. Vertebrate rhodopsin is extracted from ROS that have been purified by a conventional sucrose flotation method lz-14 followed by lyophilization and then extraction of lipid and retinal derivatives with petroleum ether. In the case of isolation of microvilli from squid retina, particular attention must be paid not to contaminate the inner segment fraction containing retinochrome. Here a procedure for preparation of squid rhodopsin is described as an example. Squid eyes are bisected and the rears are shaken in 0.1 M phosphate buffer (pH 6.8) or 0.1 M HEPES buffer (pH 7.0) to separate the outer limbs (microvilli) of the photoreceptor cells. The microvilli suspension thus obtained is filtrated through double sheets of gauze to remove the retinal debris, mixed with an equal volume of 80% sucrose solution (w/v in distilled water), and then centrifuged at 14,000 g for 1 hr. The precipitate is suspended in 38% sucrose solution (w/v in 0.1 M phosphate buffer, pH 6.8), followed by the centrifugation. The supernatant is diluted with more than 3 vol of phosphate buffer and centrifuged in order to get a precipitate composed of microvilli. The 38% sucrose flotation is repeated. The precipitate thus obtained is frozen and thawed several times, and is then layered on a sucrose linear gradient (25-50% in distilled water) followed by centrifugation at 100,000 g for more than 4 hr. The microvilli fraction containing rhodopsin floats at about the 29% sucrose density region, whereas the retinochrome fraction, if contaminated, floats at about the 36% sucrose density region with a small amount of micro12 E. K i m u r a , Jpn. J. Physiol. 3, 250 (1952). 13 D. S. P a p e r m a s t e r and W. J. Dreyer, Biochemistry 13, 2438 (1974). 14 R. H u b b a r d , P. K. Brown, and D. B o w n d s , this series, Vol. 18, Part C, pp. 615-653.
336
SPECTRAL RESPONSES OF VISUAL PIGMENTS
[49]
villi fraction. Addition of neutralized hydroxylamine (pH 7.0) to the microvilli fraction is recommended as a test of whether or not they contain retinochrome. Since rhodopsin is stable in the presence of hydroxylamine (0.1 M) but retinochrome is not, 1~one can check the contamination of retinochrome by measuring the descrease of absorbance at 480 nm. Digitonin solution (2%) is usually used for extraction of rhodopsin. For low-temperature spectrophotometry, the extract is mixed with glycerol in a final concentration of6611 or 75%, s by which the absorption spectrum of rhodopsin is not distorted. The mixture can be frozen to a clear glass without cracks above about - 100°, but below this temperature some cracks are usually formed in the mixture. Since the absorbance of rhodopsin at hmax should be more than 0.5 in a 2-mm light path for low-temperature spectrophotometry, the rhodopsin extract is concentrated by means of ultracentrifuge at 105,000 g for more than 12 hr or a millipore filter such as Amicon, if necessary. When a sample cannot be concentrated to such a high absorbance, it is recommended that the glycerol concentration in the sample be lowered to about 50%. The reason is that a 50% glycerol mixture forms a large amount of microcrystals on warming from liquid nitrogen temperatures (about - 195°, 77 K) to about - 500,TM so that the absorbance of rhodopsin in the mixture increases about 10 times because of multiple reflection of the measuring light by the microcrystals. Since the rnicrocrystals is stable at any temperature below - 5 0 °, one can measure the absorption spectrum at these temperatures using an opal glass. On application of this technique, photochemical reactions of iodopsin were studied at liquid nitrogen 17 and liquid helium temperatures TM (about - 269°, 4 K). The reversible photochemical process between iodopsin and bathoiodopsin at 4 K is shown in Fig. 2. Optical Cryostats For measurement of the absorption spectrum at low temperatures an optical cryostat must be used. 11 As an example, a cryostat we have used at liquid nitrogen temperature or above is shown in Fig. 3. The sample cell consists of three parts: a front quartz window, a middle silicone rubber ring, (1-5 mm thick) and a back opal glass. A visual pigment sample is filled into the sample cell, which is fixed in a sample cell holder (made of unoxygenated copper) by a screw-on ring. Then the cell holder is screwed to a copper tube at the bottom of the cold finger made of Pyrex glass. The 15 T. 16 T. 17 T. 18 y .
Hara and R. Hara, Proc. 1SCERG Syrup. Jpn. Ophthalmol. Vol. 10, Suppl. 22 (1966). Yoshizawa and G. Wald, Nature (London) 212, 483 (1966). Yoshizawa and G. Wald, Nature (London) 214, 566 (1967). Tsukamoto, S. Horiuchi, and T. Yoshizawa, Vision Res. 15, 819 (1975).
[49]
LOW-TEMPERATURE RHODOPS1N SPECTROPHOTOMETRY
337
Iodop#ln540nma 4 K batholoc;opBIn
1.0" 0.8" 0.6"
4 2'
7.1
0.4
o.z a
o
o 1.0
i
i
Bathoiodopsln )64Ohm, 4 K Iodopsln
0.8 0.6 0.4 0.2
b 0
400
500
600
700
Wavelength (nrn)
FIG. 2. Course of reversible photoconversion between iodopsin and bathoiodopsin at 4 K. Iodopsin/2% digltonin/50% glycerol mixture was cooled to 77 K and gradually warmed to 215 K for formation of microcrystals in the mixture, followed by cooling to 4 K for measuring the spectrum [curve 1 in (a)]. The absorbance of iodopsin in the mixture (Amax: 0.06) was intensified about 12 times (Amax: 0.7). (a) Iodopsin/2% digitonin/50% glycerol mixture (curve 1) was successively irradiated with green light at 540 nm for a total of 4, 8, 16, 32, 64, 128, and 256 sec (curves 2-8). In these irradiations, iodopsin changed to bathoiodopsin. The final curve (curve 8) represents a photosteady state mixture composed of iodopsin and bathoiodopsin. (b) Curve 8 in (a) was redrawn as curve 1, which was successively irradiated with red light at wavelengths longer than 640 nm for a total of 0.2, 0.5, 1, 2, 4, and 8 sec (curves 2-7). Bathoiodopsin converted to mainly iodopsin with a small amount of isoiodopsin. [From Y. Tsukamoto, S. Horiuchi, and T. Yoshizawa, Vision Res. 15, 819 (1975).]
cold finger with the sample cell holder is put into the glass jacket and then filled with liquid nitrogen. Immediately after that, the space between the sample cell holder and the glass jacket is evacuated by a rotary pump. The temperature of the sample is monitored by a copper-Constantan thermocouple attached to the sample cell holder. When the experiments are carded out at a desired temperature above 190°, a small volume of liquid nitrogen is dropped into the cold finger. The temperature can be kept at a constant temperature within _-_2° if one manages carefully. When dry ice/acetone is used instead of liquid nitrogen, the sample can be kept at about - 7 3 ° or above. Figure 4 shows a double vacuum glass cryostat for spectrophotmetry at liquid helium temperatures. The container of liquid helium is surrounded by that of liquid nitrogen through the vacuum space in order to -
338
[49]
SPECTRAL RESPONSES O F V I S U A L PIGMENTS
Thermocouple (copper vs. constantan) Flanges ~ E .....
3
:_--_ --.
Liquid nitrogen-..
, Vacuum
X 7-_" ~'-: _%
Glassjacket
,t..J,
---S
~ Cold finger
-=
.-_-
Cobar \
Coppe
__-~': ~.~. Screw-on ring .~. % =
Copper
y_-
- tube
\I ,I~ I
. Screw-on ring
I
Light
Cell holder
Quartz window J
Thermocouple
k ~ I
. . . .
5 cm
=
Opal glass Silicone rubber Sample Quartz plate
t
Samplecell
FIG. 3. Diagram of an optical cryostat for measuring the absorption spectrum at liquid nitrogen temperature or above. Details are given in the text. [From T. Yoshizawa and S. Horiuchi., Bunko Kenkyu 20, 206 (1971).]
insulate the heat radiation from the outside. The sample cell and its holder are the same as those of Fig. 3. Monitoring light from a spectrophotometer falls on the sample cell through the outer quartz window and the vacuum space. A volume of the container of liquid helium is about 1.2 liter. The sample can be kept at about 7 K for more than 12 hr if the cryostat is evacuated to about 10 -s mm Hg by a diffusion pump. Calculations of Absorption Spectra of I n t e r m e d i a t e s When a visual pigment is irradiated at a low temperature, a photosteady state mixture composed of the original pigment, its isopigment and intermediates is formed because of overlapping of their absorption spectra at a wavelength of irradiation. Accordingly the absorption spectra of
[49]
LOW-TEMPERATURERHODOPSIN SPECTROPHOTOMETRY
he,iuil
Cupro-nickelpipe
Liquid
339
~1
~
Balloon
~
Liquidnitrogen,~
~
Vacuum
~, Liquidnitrogen
llJ J:}l r
/
0o,0r. mp,e
Ce,, ho,der
FIG. 4. Diagram of an optical cryostat for measuring the absorption spectrum at liquid helium temperature or above. Details are given in the text. [From F. Tokunaga, N. Sasaki, and T. Yoshizawa, Photochem. Photobiol. 32, 447 (1980).]
intermediates can be estimated by subtracting the spectra of the original pigment and its isopigment from the photosteady state mixture. Absorption Spectrum of lsopigment. Isopigment can be prepared by adding 9-cis-retinal to an opsin TM and then incubating in the dark, or by irradiating rhodopsin at low temperatures. 2°'21 In the following a typical experiment by the latter method will be described. 2~ Frog ROS containing rhodopsin was irradiated with light at wavelengths longer than 540 nm at a liquid nitrogen temperature (about 77 K) to produce a photosteady state mixture composed mainly of isorhodopsin 19R. Hubbard and G. Wald, J. Gen. Physiol. 36, 269 (1952). s0 y . Kito, M. Ishigami, and T. Yoshizawa, Biochim. Biophys. Acta 48, 287 (1961). zl T. Yoshizawa and G. Wald, Nature (London) 201, 340 (1964). z~ S. Kawamura, S. Wakabayashi, A. Maeda, and T. Yoshizawa, Vision Res. 18, 457 (1978).
340
[49]
SPECTRAL RESPONSES OF VISUAL PIGMENTS
with small amounts of rhodopsin and bathorhodopsin. Then the photosteady mixture w a s irradiated with light at wavelengths longer than 610 nm to convert the bathorhodopsin to rhodopsin and isorhodopsin. After warming the mixture to room temperature, the spectrum was compared with that of the original rhodopsin. The intersection point between them is an isosbestic point (508 nm) between rhodopsin and isorhodopsin because the preparation contains only rhodopsin and isorhodopsin. In order to estimate the amount of isorhodopsin in the preparation, it was successively irradiated at room temperature with orange light at wavelengths longer than 540 nm until completely bleached and each time the spectrum was recorded (Fig. 5a). Under this irradiation, rhodopsin bleached much more rapidly than isorhodopsin. Then the difference in absorbance at wavelength of the isosbestic point (508 nm) between each spectrum (curves 1-7) and the final spectrum completely bleached (curve 8) was plotted on the semilogarithmic scale against the time of irradiation as shown in Fig. 5b. A straight line in the later stage of the irradiation is due to the bleaching of isorhodopsin. Extrapolating the straight line to zero time yields a measure of the proportion of isorhodopsin present in i
/
0.4
/+x
I
\
'
Rh. ~ 5~ , , = , o ~,Ret.oxime
I00
i
i
i
I
I
I
20
,0
i
i
i
i
i
50
O3
"6 I0 t)
~t o.I
'6 4~
..~ ~o
~bo''~
Wavelength (nm)
b
I
I
80
,00
,b0
Time ( s e c )
FIo. 5. (a) Successive irradiation of a mixture of rhodopsin and isorhodopsin in frog ROS at room temperature. The mixture (curve 1) of rhodopsin and isorhodopsin was prepared by irradiation of dark-adapted frog ROS suspension in 75% glycerol/0.1 M phosphate buffer (pH 7.0) with orange light (>540 nm) for 30 min at 77 K. After warming to room temperature, the mixture was successively irradiated with orange light ( > 540 nm) for 5, 5, 10, 20, 40 and 80 sec (curves 2-7) until finally the residual pigments were completely decomposed (curve 8). (b) Kinetics of bleaching of the mixture of rhodopsin and isorhodopsin. Difference absorbances at 508 nm (an isosbestic point between rhodopsin and isorhodopsin) between curves 1-7 and curve 8 in (a) are calculated and then normalized by the difference absorbance between curves 1 and 8. Filled circles represent the experimental values. A solid line shows a slow component of the kinetics that has been calculated by the least-squares method. The value obtained by extrapolating the solid line to zero gives the molar percentage of isorhodopsin. [From S. Kawamura, S. Wakabayashi, A. Maeda, and T. Yoshizawa, Vision Res. 18, 457 (1978).]
[49]
LOW-TEMPERATURE
RHODOPSIN
SPECTROPHOTOMETRY
341
[5-
1.0-
•
I
3
o
2
5 0.5-
0.0-
460
~6o
660
700
Wovelength (rim)
FIG. 6. A n experiment for calculation of absorption spectrum of squid lumirhodopsin. Absorption spectrum of rhodopsin/2% digitonin/66% glycerol mixture ( p H 10.5) was measured at - 85 ° ( c u r v e l). After cooling to - 188 °, the preparation was irridiated with blue light (437 nm) for 40 min, and then warmed to - 85 ° for measurement of spectrum (curve 2). After incubation at 2 ° for 30 min, the spectrum was measured at - 85 ° ( c u r v e 3). Finally, the preparation was irradiated with light at wavelengths longer than 510 nm at 2 °, and then recooled to -85°to measure the spectrum (curve 4). [From Y. Shichida, F. Tokunaga, and T. Y o s h i z a w a , Biochim. Biophys. Acta 504, 413 (1978).]
the preparation. Thus the absorption spectrum of isorhodopsin can be obtained by subtracting the absorption spectrum of the rhodopsin in the preparation from that of the mixture. Absorption Spectra of Lumi- and Meta-intermediates. W h e n a visual pigment is irradiated at about - 80 °, it changes to a photosteady state mixture composed of visual pigment, isopigment and an intermediate, the latter of which has been called lumi-intermediate.23 However, the lumi-intermediate is not always a single isomer, but may be a mixture of isomers that have all-trans-, 13-cis- and 7-cis-retinals as their chromophores according to analysis by high-performance liquid chromatography of cattle and squid lumirhodopsins 24,25 (Fig. 10c). In order to get the absorption spectrum of lumirhodopsin (all-trans), rhodopsin must be irradiated at liquid nitrogen temperatures, followed by warming to about - 8 0 °, because the irradiation of rhodopsin at liquid nitrogen temperatures does not yield other isomers than all-trans-bathorhodopsin (Fig. 10b), which converts to all-trans-lumirhodopsin on the warming. Figure 6 shows a typical experiment from which the absorption spectrum of squid lumirhodopsin was calculated, z6 All the spectra were mea23 R H u b b a r d , P. K. B r o w n , a n d A. K r o p f , Nature (London) 183, 442 (1959). 24 A. M a e d a , T. O g u r u s u , Y. S h i c h i d a , F. Tokunaga, and T. Yoshizawa, FEBS Lett. 92, 77 (1978). z5 A. M a e d a , Y. Shichida, and T. Yoshizawa, Biochemistry 18, 1499 (1979). 26 y . S h i c h i d a , F. Tokunaga, and T. Yoshizawa, Biochim. Biophys. Acta 504, 413 (1978).
342
SPECTRAL RESPONSES OF VISUAL PIGMENTS
[49]
sured at - 85° to counteract the effects of temperature on absorption spectra. To begin with, the spectrum of rhodopsin was recorded at - 8 5 ° (curve 1). Then the preparation was cooled to a liquid nitrogen temperature and irradiated with 437 nm light until the photosteady state mixture was formed. Warming to - 8 5 ° converted bathorhodopsin in the mixture to lumirhodopsin (curve 2). After incubation at 2° for 30 min for bleaching of lumirhodopsin to alkaline metarhodopsin, the spectrum was measured at - 85° (curve 3). Then it was irradiated at 2° with yellow light at wavelengths longer than 510 nm to bleach the residual rhodopsin and isorhodopsin. The final spectrum was measured at - 8 5 ° (curve 4). The difference spectrum between lumirhodopsin and alkaline metarhodopsin was calculated by subtracting curve 3 from curve 2. The amount of lumirhodopsin can be estimated by the formula (A1 - An)/(A1 - A4), where A is the absorbance at the isosbestic point (490 nm) between rhodopsin and isorhodopsin and the suffixes are the curve numbers. Thus the absorption spectrum of lumirhodopsin (curve 5 in Fig. 8) can be obtained by adding curve 4 to the difference spectrum corrected to 100% conversion. Absorption spectrum of metarhodopsin I can be obtained by the same procedures except that all the spectra are measured at about - 40° where metarhodopsin I is stable. Absorption Spectrum o f Batho-intermediate. Batho-intermediate is produced by irradiating visual pigment or isopigment at liquid nitrogen temperature or below. Since the glycerol mixture containing visual pigment forms a clear glass with some cracks on cooling to a liquid nitrogen temperature, the spectrum of batho-intermediate cannot be calculated by the same procedure as that of lumi-intermediate, because the second cooling from room temperature to the liquid nitrogen temperature freezes to a glass having a different number of cracks than the first one. Thus the spectrum of batho-intermediate must be calculated by either of the methods described below. The first is the application of the rapid cooling technique described in Article [85], this volume, by which a clear glass without any cracks can be prepared at liquid nitrogen temperature or below. Experimental method and calculation for the absorption spectrum of batho-intermediate are almost the same as those for lumi-intermediate except that the spectral measurement is carried out at liqui d nitrogen temperatures, z7 The second precedure can be applied when the absorption spectra of visual pigment and its isopigment at liquid nitrogen temperatures are already known, z6"28The photosteady state mixture composed of visual pig27 S. Horiuchi, F. Tokunaga, and T. Yoshizawa, Biochim. Biophys. Acta 591, 445 (1980). 2a y . Shichida, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 29, 343 (1979).
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY
343
ment, isopigment, and batho-intermediate is produced by irradiating the visual pigment with light at liquid nitrogen temperatures, and then warmed to room temperatures for bleaching of the batho-intermediate. The amounts of residual visual pigment and isopigment in the sample can be estimated by the successive irradiation of the sample with light at wavelengths longer than 540 nm, followed by plotting the decrease of absorbance at an isosbestic point between the original pigment and its isopigment on the semilogarithmic scale against the time of irradiation as described previously (Fig. 5). The amount of batho-intermediate is estimated from the difference in absorbance at the isosbestic point between the original sample and the sample after warming. Thus the absorption spectrum of batho-intermediate can be calculated by subtracting the absorption spectrum of the sum of the residual pigment and its isopigment from that of the photosteady state mixture. Absorption Spectrum of Hypsorhodopsin. Hypso-intermediate has been detected only in the rhodopsin system. 4'29 Irradiation of rhodopsin with yellow light at liquid helium temperatures (about 4 K) yields hypsorhodopsin. The experimental procedure for calculating the absorption spectrum is almost the same as that for bathorhodopsin except for the measuring temperature. However, one must pay attention to the contamination of bathorhodopsin in a photosteady state mixture, which is produced by irradiating rhodopsin at liquid helium temperatures. By irradiating the photosteady state mixture with longer wavelengths of light than 610 nm, the bathorhodopsin can be removed from the mixture, because the bathorhodopsin converts to rhodopsin and isorhodopsin. Thus, a mixture composed of only rhodopsin, isorhodopsin, and hypsorhodopsin can be prepared. Using this mixture, one can calculate the spectrum of hypsorhodopsin. Spectral Changes in Conversion of One Intermediate to Another. In order to solve the question of how many intermediates exist in the photobleaching process for rhodopsin, the spectral change in the thermal conversion of one intermediate to another must be measured, because if an isosbestic point exists in the conversion, it may safely be said that no intermediate exists between them. For measuring the spectral change, all the absorption spectra must be recorded at the same temperature to eliminate the effect of temperature on spectrum. In fact, the cooling from room temperature to liquid nitrogen temperature causes a shift of the hma x toward the longer wavelengths (cattle rhodopsin: about 8 nm) and increases its extinction coefficient about 1.1 times. 4 Figure 7 shows a spectral change in thermal conversion of squid lumi29 T. Yoshizawa and S. Horiuchi, in "Biochemistry and Physiology of Visual Pigments" (H. Langer, ed.), pp. 69-81. Springer-Verlag, Berlin and New York, 1973.
344
S P E C T R A L RESPONSES O F V I S U A L P I G M E N T S
~
Lumirhodopsin
[49]
warming= LM-rhodopsln
16,]
.
. mensured
~ i.o.
at - 8 5 0 C
I
o5-
o.o-
I ,~.,
.
460
,
56o Wovelength
66o
700
(nm}
FIG. 7. C o u r s e o f c o n v e r s i o n o f squid lumirhodopsin to L M - r h o d o p s i n by warming. Rhod o p s i n / 2 % digitonin/66% glycerol mixture (pH 10.5) w a s irradiated with blue light (437 nm) at - 188 °, t h e n w a r m e d to - 8 5 ° for c o n v e r s i o n o f b a t h o r h o d o p s i n to lumirhodopsin (curve 1). This preparation was s u c c e s s i v e l y w a r m e d to - 75, - 65, - 45, - 35, and - 25 °, and each time w a s recooled to - 85 ° for m e a s u r e m e n t of s p e c t r u m (curves 2 - 6 ) . T h e spectral c h a n g e s from c u r v e 2 to c u r v e 6 r e p r e s e n t s the c o n v e r s i o n s of lumirhodopsin to L M - r h o d o p s i n . [ F r o m Y. Shichida, F. T o k u n a g a , and T. Yoshizawa, Biochim. Biophys. Acta 504, 413 (1978).]
rhodopsin to LM-rhodopsin. ~6 Rhodopsin was irradiated with 437 nm light at a liquid nitrogen temperature and then warmed to - 85 °. The rhodopsin changed to a mixture of rhodopsin, isorhodopsin, and lumirhodopsin. After measurement of the absorption spectrum, the mixture was warmed to a required temperature, followed by cooling to - 8 5 °. The spectrum further shifted to shorter wavelengths with an isosbestic point at 502 nm, indicating that there was no intermediate between lumirhodopsin and LM-rhodopsin. Absorption Properties of Intermediates of Rhodopsin Bleaching sequences of cattle and squid rhodopsins are shown in Fig. 1, where several experimental results of low-temperature spectrophotometry and flash and laser photolyses were summarized. Figure 8 shows the absorption spectra of squid intermediates and the accompanying table shows absorption properties of intermediates of several visual pigments and rhodopsin analogs. Hypso-intermediate. Hypsorhodopsin is an intermediate discovered by irradiating cattle rhodopsin with orange light (> 520 nm) at liquid helium temperatures. 4,29 As its name indicates, hypsorhodopsin has a Xmax shorter than that of rhodopsin (hypsochromic shift). Now the hypso-intermediate is detected in all the animal rhodopsin systems we have tested, for example, in cattle, 4,29chicken, TM frog, 3° and squid 2s rhodopsin systems. 30 S. Horiuchi, F. T o k u n a g a , and T. Yoshizawa, Biochim. Biophys. Acta 503, 402 (1978).
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY 1.5-
t.o
(3
2
._~ -~ 0.5
O0
345
~5
4O0
500
600
Wovelength (nm)
15
RiO
I
6
o _>
_~05
O0
~o
sao
660
Wavelenoth (nm)
FIG. 8. The absorption spectra of squid rhodopsin and its intermediates in 2% digitonin/66% glycerol mixture (pH 10.5). 1, rhodopsin; 2, isorhodopsin; 3, hypsorhodopsin; 4, bathorhodopsin; 5, lumirhodopsin; 6, LM-rhodopsin.
Hypsorhodopsin was also detected at room temperatures by means of picosecond laser photolysis, 3r-3a indicating that it is a physiological intermediate of rhodopsin. However, in chicken iodopsin and bacteriorhodopsin systems it was not detected. 1s'33 Photoconversion of rhodopsin to mainly hypsorhodopsin is shown in Fig. 9, 2s in which squid rhodopsin (curve 1) was irradiated with longer wavelengths light than 480 nm at a liquid helium temperature. The absorption spectrum shifted to shorter wave31 R. Hara, T. Hara, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 33, 883 (1981). 32 O. Muto, F. Tokunaga, T. Yoshizawa, M. Ito, and K. Tsukida, to be published. 33 T. Iwasa, F. Tokunaga, and T. Yoshizawa, FEBS Lett. 101, 127 (1979). 34 T. Iwasa, F. Tokunaga, and T. Yoshizawa, Biophys. Struct. Mech. 6, 253 (1980). 35 T. Iwasa, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 33, 539 (1981). 36 T. Iwasa, F. Tokunaga, T. Yoshizawa, and T. G. Ebrey, Photochem. Photobiol. 31, 83 (1980). 3r y. Shichida, T. Yoshizawa, T. Kobayashi, H. Ohtani, and S. Nagakura, FEBS Lett. 80, 214 (1977). ~8 y. Shichida, T. Kobayashi, H. Ohtani, T. Yoshizawa, and S. Nagakura, Photochem. Photobiol. 27, 335 (1978). 39 T. Kobayashi, FEBS Lett. 106, 313 (1979).
346
[49]
SPECTRAL RESPONSES OF VISUAL PIGMENTS
% ¢) .¢
.¢Z Z a~ O r~ O
e~ Z
Z g]
a
,.J .¢
;>
u~ L~ ¢) [--
[-,
z
!o
a~ ~h Z
_q a~
.¢
"i
"'q ~
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY
1.5 ¸
Rhodapsin
> 4 8 0 nm - Hypsorhodopsin
I.O-~
I
/
.~
0.5
347
I I \ Lm-~.. ~ . : /
Measurement at 4 K
2
: 2
oo
. . . . . . . 400
500 Wavelength (nm)
i 6OO
700
FIG. 9. Course of the conversion of squid rhodopsin to hypsorhodopsin. Rhodopsin/2% digitonin/66% glycerol mixture (pH 10.5, curve 1) was successively irradiated with yellow light (>480 nm) at 4 K for a total of 5, 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 sec (curves 2-11). The final spectrum (curve 11) represents a photosteady state mixture composed of rhodopsin, isorhodopsin, and hypsorhodopsin with a small amount of bathorhodopsin. [From Y. Shichida, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 29, 343 (1979).]
lengths, indicating formation of hypsorhodopsin. Prolonged irradiation formed a photosteady state mixture composed of rhodopsin, isorhodopsin, and hypsorhodopsin with a small amount of bathorhodopsin (curve 11). When isorhodopsin was irradiated with the same light at liquid helium temperatures, the same photosteady state mixture was produced. Therefore, the interconversion between l l-cis (rhodopsin)-and 9-cis (isorhodopsin)-retinylidene chromophores occurs via hypsorhodopsin or bathorhodopsin, suggesting that hypsorhodopsin and bathorhodopsin have twisted all-trans-retinals as their chromophores. Photochemical reactions of rhodopsin and its intermediates at liquid helium temperatures are summarized in Fig. 10a. Hypsorhodopsin converts to bathorhodopsin on warming. When a squid preparation containing mainly hypsorhodopsin was warmed above 35 K, its absorption spectrum shifted to longer wavelengths through sharp isosbestic points at 476 and 366 nm. ~s In the case of cattle hypsorhodopsin, it converted to bathorhodopsin through an isosbestic point near 470 nm above 23 K. 4'29 There is a difference in photochemical behavior at liquid helium temperatures between cattle and squid hypsorhodopsins. When cattle hypsorhodopsin was irradiated, it converted to bathorhodopsin, 29 whereas squid hypsorhodopsin converted mainly to rhodopsin and isorhodopsin. 2s As already stated, hypsorhodopsin as well as bathorhodopsin has been suggested to have a twisted all-trans-retinal as its chromophore. Recently, the low-temperature spectrophotometry of frog retina showed that the transition dipole moment of hypsorhodopsin is almost the same in off-
348
SPECTRAL RESPONSES OF VISUAL PIGMENTS
(a)
Rhodopsin (11-cis) x
x Hypsorhodopsin (trans)
~
[49]
x Isorhodopsin •
(9-cis)
Bathorh!dopsin ~ (trans)
(b)
Rhodopsin
(11--ci$)
x
x
Bathorhodopsin (trans)
x
x
Isorhodopsin (9--cis)
7--cis
(C)
Rhodopsin (I 1--ci$) x
x Lumirhodopsin (trans)
product
~' Isorhodopsin ~
(9--ci$)
product FIG. 10. Schemashowing interconversion among rhodopsin and its photoproducts by light at (a) liquid helium, (b) liquid nitrogen and (c) dry ice/acetone temperatures. 13--cis
entation to the disk plane as that o f bathorhodopsin, 4° indicating that the c o n f o r m a t i o n of retinylidene c h r o m o p h o r e o f h y p s o r h o d o p s i n m a y be similar to that o f bathorhodopsin, that is, a twisted a l l - t r a n s - r e t i n y l i d e n e c h r o m o p h o r e . H o w e v e r , ~max o f h y p s o r h o d o p s i n (cattle, 430 nm) greatly shifts to shorter wavelength than that o f b a t h o r h o d o p s i n (cattle, 543 nm). In o r d e r to explain the blue shift, we has p r o p o s e d three possible models a b o u t conformation o f the retinylidene c h r o m o p h o r e o f h y p s o r h o d o p s i n as follows: I II III
U n p r o t o n a t e d retinylidene Schiff b a s e 4,2a'29 Protonated retinylidene S c h i f f b a s e in which conjugated s y s t e m is dissected at the 7 - 8 or 8 - 9 b o n d 41 Protonated retinylidene Schiff base that has no specific interaction with neighboring groups o f opsin 4"29
40 F. Tokunaga, N. Sasaki, and T. Yoshizawa, Photochem. Photobiol. 32, 447 (1980). 41 M. Ito, K. Hirata, A. Kodama, K. Tsukida, H. Matsumoto, K. Horiuchi, and T. Yoshizawa Chem. Pharm. Bull. 26, 925 (1978).
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY
349
Model I is suggested by the fact that the energy difference of the bathochromic shift in hmax between hypsorhodopsin and bathorhodopsin is similar to that between unprotonated Schiff base and its protonated form in alcohol. Model II is suggested by the fact that the hrnax of hypsorhodopsin is close to that ofrecto-y-rhodopsin (hmax = 425 nm), 41 whose chromophore is dissected at the 7-8 bond. Model III is suggested by the fact that hmax of protonated retinylidene Schiff base in alcohol is close to that of hypsorhodopsin. The most plausible model among them may be the unprotonated Schiff base (model I). Since the oscillator strength of hypsorhodopsin is close to that of bathorhodopsin, ~8 it seems unlikely that the conjugated system of hypsorhodopsin is dissected, because the oscillator strength of a conjugated polyene molecule general increases with length of the conjugated system in a relatively short system. In addition, the fact that the transition dipole moment of hypsorhodopsin is the same in orientation as that of bathorhodopsin4° may not support model II. A hypso-intermediate of retro-3,-rhodopsin was also observed by irradiation at liquid helium temperatures, as Thus the formation of hypsorhodopsin is not due to the breakdown at 7 - 8 - 9 of the conjugated double bond system. A noteworthy point is that we have failed to get hypsorhodopsin without contamination of rhodopsin and isorhodopsin on irradiation at the tail in longer wavelengths region of isorhodopsin at liquid helium temperatures, in spite of the fact that hypsorhodopsin has hmax at a shorter wavelength than those of rhodopsin and isorhodopsin. There are at least two possible reasons for this. First, if hypsorhodopsin has a small absorbance at the wavelength region of irradiation, the quantum yield of rhodopsin (or isorhodopsin) to hypsorhodopsin may be small or that of hypsorhodopsin to rhodopsin, isorhodopsin, and bathorhodopsin may be large. 42 Second, hypsorhodopsin may have relatively larger absorbance at longer wavelengths than rhodopsin or isorhodopsin. We have found that cattle or frog hypsorhodopsin has a small absorption band near 530 nm concomitant with the main absorption band near 430 nm. 4a Squid hypsorhodopsin, however, has a long tail only in the spectrum at the long wavelength region (Fig. 8). Batho-intermediate. Batho-intermediate4,29 (formerly called prelumiintermediate 11) is an intermediate stable at liquid nitrogen temperatures (about - 195°). Its hmax is located at a longer wavelength than its parent 42 A. Sarai, T. Kakitani, Y. Shichida, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 32, 199 (1980). 43 N. Sasaki, T. Yoshizawa, and F. Tokunager, Int. Syrup. Physicochem. Aspects Primary Process Vis. Excitation Photosynth., 1980.
350
SPECTRAL RESPONSES OF VISUAL PIGMENTS
[49]
O.6.
0.4 ¸
~
.
measuredot-175"C
9
7"9.
.
0.2 ¸
0.0
0.0 400
500 Wavelength
600
700
- n rn
FIG. 11. Course of conversion of rhodopsin to bathorhodopsin in (a) frog retina and (b) rhodopsin/2% digitonin extract. (a) Rhodopsin in the retina in 75% glycerol (curve 1) was successively irradiated with blue light (437 nm) at - 175° for a total of S, 10, 20, 40, 80, 160, 320, and 640 sec (curves 2-9). (b) Rhodopsin/2% digitonin/66% glycerol mixture in phosphate buffer (pH 7.0) was successively irradiated with blue light (437 nm) at - 165° for a total ofS, 10, 20, 40, 80, and 160 sec (curves 2-7). The final spectrum [curve 9 in (a) or curve 7 in (b)] represents a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin. [From F. Tokunaga, S. Kawamura, and T. Yoshizawa, Vision Res. 16, 633 (1976).]
visual pigment. All the visual pigments including bacteriorhodopsin 44 that we tested produced batho-intermediates on irradiation at liquid nitrogen temperatures. The only exceptional case is retinochrome, 31 which is a photosensitive pigment in cephalopod retina having all-trans-retinal as its chromophore. Formation of bathorhodopsin is also confirmed in frog retina by irradiation at liquid nitrogen s and liquid helium 3° temperatures (Fig. 11). In the table given earlier the absorption properties of bathorhodopsin are shown. Laser photolytic experiments confirmed that bathorhodopsin is produced at physiological temperatures2 °-32'37-39,45,46 Bathorhodopsin has been thought to have a twisted all-trans-retinal as its chromophore (isomerization model), because it was formed by irradiating both rhodopsin and isorhodopsin at liquid nitrogen temperatures 11 (Fig. 10). Since the first picosecond experiment, many alternative hypotheses have been proposed in which the change of rhodopsin to bathorhodopsin does not contain rotation of the 11-12 double bond of the retinylydene chromophore, but is due only to translocation of a proton on a 44 F. Tokunaga, T. Iwasa, and T. Yoshizawa, F E B S Lett. 72, 33 (1976). 45 R. A. Cone, N a t u r e (London), N e w Biol. 236, 39 (1972). 46 T. Rosenfeld, A. Alkalel, and M. Ottolenghi, N a t u r e (London) 240, 482 (1972).
[49]
LOW-TEMPERATURE
RHODOPSIN
SPECTROPHOTOMETRY
351
0.6
g
~0.3
o I
400
5()0
Wavelength (nm) FIG. 12. Course of conversion of 9-cis-retro-y-rhodopsin to its bathoproduct at - 185 °. A 9-cis-retro-T-rhodopsin/2% digitonin/66% glycerol mixture (pH 7.0) was irradiated at - 185° with 380 nm light for a total of 15, 40, 80, 150, 270, and 510 sec (curves 2-7). The final spect r u m represents a photosteady state mixture composed of original 9-cis pigmen t and bathoproduct with possibly 11-cis pigment. [From S. Kawamura, T. Yoshizawa, K. Horiuchi, M. Ito, A. Kodama, and K. Tsukida, Biochim. Biophys. Acta $48, 147 (1979).]
/~-ionone ring to a Schiff base nitrogen (proton translocation model).47-5° However, the formations of batho-intermediate by irradiation of retro-yrhodopsin (Fig. 12) has completely ruled out the proton translocation model, because a proton on the ring of the retro-y-retinylidene chromophore can not move to the Schiff base nitrogen through the side chain of the chromophore that is dissected at the 7 - 8 bond. Moreover, the following experiments also give some evidence that bathorhodopsin may have a twisted all-trans retinylidene chromophore. First, the CD spectrum of bathorhodopsin at the main absorption band is negative, which presents a striking contrast to the positive CD of rhodopsin.26"27"29The inversion of the positive to the negative CD spectrum in the process of converting rhodopsin to bathorhodopsin may be due to the conformational change of the retinylidene chromophore. 4r K. Van der Meer, J. J. C. Mulder, and J. Lughtenburg, Photochem. Photobiol. 24, 363 (1976). 48 K. Peters, M. L. Applebury, and P. M. Rentzepis, Proc. Natl. Acad. Sci. U.S.A. 74, 3119 (1977). 49 A. Lewis, Proc. Natl. Acad. Sci. U.S.A. 75, 549 (1978). so A. Warshel, Proc. Natl. Acad. Sci. U.S.A. 75, 2558 (1978).
352
S P E C T R A L RESPONSES O F V I S U A L P I G M E N T S
[49]
Second, the transition dipole moment of rhodopsin changes 26 degrees in orientation when it converts to bathorhodopsin. 1° This large orientational change also suggests the cis-trans isomerization of the retinylidene chromophore. The third is that the same bathrohodopsin is produced from 7-cis-rhodopsin by irradiation at liquid nitrogen temperatures 5 as those from rhodopsin and isorhodopsin. Recently, kinetic studies of photoconversion at liquid nitrogen temperatures showed that the bathorhodopsin, which had been produced by irradiating cattle or frog rhodopsin, was not a single product but a mixture of two components. 51"~2It was also confirmed that all the intermediates of rhodopsin, including hypsorhodopsin, are the mixture of two components .53 Lumi-intermediates. When batho-intermediate is warmed from a liquid nitrogen temperature to above - 140°, the spectrum shifts to shorter wavelengths, indicating formation of lumi-intermediate. The only exceptional case is bathoiodopsin, which converts to the original iodopsin above 180°.17 Laser photolytic experiments showed that bathorhodopsin converts to lumirhodopsin at room temperature with a time constant of about 40 nsec in the cattle rhodopsin system46 and about a few hundreds nanoseconds in frog45 and squid aT'a8rhodopsin systems. The absorption spectrum and spectroscopic properties of lumirhodopsin are shown in Fig. 8 and the table, respectively. Lumirhodopsin has a large/3-band in comparison with those of rhodopsin and bathorhodopsin, z6 The fact that the/3-band of bathorhodopsin is smaller than that of rhodopsin is consistent with the viewpoint that bathorhodopsin has an all-trans-retinylidene chromophore. On the contrary, the fact that lumirhodopsin has a larger E-band than rhodopsin seems to be inconsistent with the familiar view that lumirhodopsin has an all-trans-retinylidene chromophore. Probably there exists some special interaction between retinylidene chromophore and opsin in lumirhodopsin. In fact, we found that the conversion of rhodopsin to bathorhodopsin caused no CD change in the range between 280 nm and 300 nm, whereas the conversion of bathorhodopsin to lumirhodopsin showed a remarkable increase of the CD signal. 26"~4These results may support the idea that the photoconversion of rhodopsin to bathorhodopsin is mainly due to the isomerization of the retinylidene chromophore, whereas the process of conversion of bathorhodopsin to lumirhodopsin is due to some conforma-
sl N. 52 N. 53 N. 54 T.
-
Sasaki, F. T o k u n a g a , and T. Yoshizawa, FEBS Lett. 114, 1 (1980). Sasaki, F. T o k u n a g a , and T. Yoshizawa, Photochem. Photobiol. 32, 433 (1980). Sasaki, F. T o k u n a g a , and T. Yoshizawa, Annu. Meet. Biophys. Soc. Jpn., p. 222 (1980). G. E b r e y and T. Yoshizawa, Exp. Eye Res. 17, 545 (1973).
[49]
LOW-TEMPERATURERHODOPSIN SPECTROPHOTOMETRY
353
tional change of protein moiety, by which the highly twisted state of the retinylidene chromophore of bathorhodopsin is concurrently relaxed. As already stated, lumirhodopsin is found in a photosteady state mixture formed by irradiating rhodopsin at about - 8 0 °. However, the lumirhodopsin produced is a mixture of isomers having all-trans, 7-cis-, and 13-cis-retinals as their chromophores (Fig. 10c). Thus the protein moiety of lumirhodopsin may be flexible enough to accommodate these isomers, whereas that of bathorhodopsin may not. This may provide additional evidence that the conversion of bathorhodopsin to lumirhodopsin is due to the opening up of the conformation of opsin. M e t a - i n t e r m e d i a t e . When cattle lumirhodopsin is warmed to - 4 0 °, it converts to metarhodopsin I, which changes to metarhodopsin II above 15°.~ Flash photolytic measurements showed that a large increase of entropy occurred in these processes, 56 indicating large conformational changes of the protein moiety. Moreover, the rates of formations of these intermediates depend on the lipid content in the parent rhodopsin molecule ?7 The conversion between metarhodopsin I and II is a tautomeric equilibrium, in which metarhodopsin II is favored by an increase in temperature, or ionic strength, and addition of glycerol or methanol. 55 In the case of cephalopod rhodopsin, lumirhodopsin converts to LMrhodopsin above - 6 0 ° and then converts to acid and alkaline metarhodopsins, 26 -
Photochemical Reactions of Visual Pigment Analogs at Low Temperatures Various kinds of visual pigment analogs have been prepared from cattle opsin and chemically synthesized retinal analogs. For elucidation of the changes of interaction between retinylidene chromophore and opsin in the photobleaching process of rhodopsin and analysis of the structure of retinylidene chromophore of its intermediates, photochemical reactions of visual pigment analogs are investigated at low temperatures. Figure 12 shows a formation of bathoproduct on irradiation of retro-7rhodopsin with 380-nm light at a liquid nitrogen temperature2 This result may be one of the definite evidences by which the proton translocation model of bathorhodopsin was ruled out, as already stated. 55R. G. Matthews,R. Hubbard, P. K. Brown,and G. Wald,J. Gen. Physiol. 47, 215 (1963). 56R. Hubbard, D. Bownds,and T. Yoshizawa,Cold Spring Harbor Symp. Quant. Biol. 30, 301 (1965). 57H. Shichi, S. Kawamura,C. G. Muellenberg,and T. Yoshizawa,Biochemistry 16, 5376 (1977).
354
SPECTRAL
RESPONSES
OF
VISUAL
PIGMENTS
[50]
Almost all the visual pigment analogs formed their own bathoproducts on irradiation at liquid nitrogen temperatures. However, in contrast with batho-intermediates of the native visual pigments, those of visual pigment analogs have small extinction coefficients. In the table, absorption properties of bathoproducts of visual pigment analogs we have investigated are listed. A 13-dm pigment which has 13-dm retinal as its chromophore has a new intermediate between batho- and lumi-intermediates, r The appearance of the new intermediates may provide a new insight into the chromophore-protein interaction in visual pigment. Transition temperature from one intermediate to another may also give a valuable information about the chromphore-protein interaction in visual pigment. For example, batho-intermediate of 13-dm pigment is more unstable than bathorhodopsin, r suggesting that only a small conformational change of opsin near the 13-position may relax the strained chromophore of bathointermediate to form BL-intermediate of 13-dm pigment. 58 S. E. Ostroy,Biochim. Biophys. Acta 463, 91 (1977). 59S. E. Ostroy, F. Erhardt, and E. W. Abrahamson,Biochim. Biophys. Acta 112, 265 (1966). 60y. Ebina, N. Nagasawa, and Y. Tsukahara,Jpn. J. Physiol. 25, 217 (1975). 61F. Tokunaga, Y. Shichida, and T. Yoshizawa,FEBS Lett. 55, 229 (1975).
[50] P i c o s e c o n d
Spectroscopy
of Visual Pigments
B y M. L. APPLEBURY and P. M. RENTZEPIS
Technological developments in picosecond spectroscopic instrumentation have made it possible to investigate early dynamic events in photo° biological systems. Application of these picosecond techniques over the last eight years to the study of visual pigments and analogous systems, for example, has led to a description of the dynamic events involved in the production of initial photoproducts following the absorption of a photon, i.e., bathorhodopsin formed from rhodopsin or the bK intermediate formed from bacteriorhodopsin. The following text describes the instrumentation used for picosecond kinetics, data collection, analysis, and methods of sample preparation as they have been applied to experimental study of visual pigments. Instrumentation for a Double-Beam Picosecond Spectroscopic Apparatus Picosecond kinetics and spectroscopy are practiced by a large number of investigators emphasizing either absorption, emission, or, lately, resoMETHODS IN ENZYMOLOGY, VOL. 81
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181981-7
LOW-TEMPERATURE
RHODOPSIN
[49] L o w - T e m p e r a t u r e Intermediates
SPECTROPHOTOMETRY
Spectrophotometry of Rhodopsin
333
of
By T6RU YOSHIZAWA and YOSHINOR1 S H I C H I D A It is well known that the absorption spectrum of rhodopsin in the visible range is in good agreement with the scotopic luminosity curve. 1 This fact gives us a basis for the proposition that the photochemical reaction of rhodopsin triggers the excitation of the visual cell. Since it takes a few milliseconds to generate the receptor potential of the visual cell after capture of a photon,2 the photoreaction of rhodopsin in less than a few milliseconds is important for elucidating the function of rhodopsin in visual transduction. Because such a fast photoreaction was so difficult to study at room temperature, most investigations had been carried out at low temperatures, 3'4 where the thermal reaction rate of intermediates of rhodopsin is remarkably reduced. The application of low-temperature spectrophotometry to the rhodopsin system has led to discovery of several •intermediates in the process of photobleaching of rhodopsin, as shown in Fig. 1. Thus it may safely be said that one of the most powerful tools for detection and identification of the intermediates of bleaching of rhodopsin is low-temperature spectrophotometry. In combination with this method, other spectroscopic measurements, such as CD, resonance Raman, and laser photolysis, also provide reliable information for analysis of the structure and kinetics of the intermediates of rhodopsin. Recently, lowtemperature spectrophotmetry has been applied to rhodopsin analogs prepared from cattle opsin and retinal analogs synthesized chemically. The studies on the photoreactions of rhodopsin analogs have given us invaluable information on structure of the intermediates of rhodopsin and its photoreceptive mechanism. 5-7 In the following, procedures for preparation of samples, techniques for low-temperature spectrophotometry, and absorption characteristics of the intermediates of rhodopsin will be described. i G. Wald and P. K. Brown, Science 127, 222 (1958). 2 T. E. Ogden and K. T. Brown, unpublished results, cited in K. T. Brown, Nature (London) 193, 958 (1962). 3 G. Wald, Science 162, 230 (1968). 4 T. Yoshizawa, in " H a n d b o o k of Sensory P h y s i o l o g y " (H. J. A. Dartnail, ed.), Vol. 7, Part 1, pp. 146-179. Springer-Verlag, Berlin and N e w York, 1972. 5 S. K a w a m u r a , S. Miyatani, H. Matsumoto, T. Yoshizawa, and R. S. H. Liu, Biochemistry 19, 1549 (1980). 6 S. K a w a m u r a , T. Yoshizawa, K. Horiuchi, M. Ito, A. K o d a m a , and K. Tsukida, Biochim. Biophys. Acta 548, 147 (1979). r y . Shichida, A. Kropf, and T. Yoshizawa, Biochemistry 20, 1962 (1981). Copyright © 1982by AcademicPress, Inc. METHODS IN ENZYMOLOGY, VOL. 81 All rights of reproduction in any form reserved. ISBN 0-12-181981-7
334
SPECTRAL RESPONSES OF VISUAL PIGMENTS
cattle rhodopsin (498 nm)* 3
squid rhodopsin (480 nm)"15
Zve < 15 psec 39 }
T1/e
hv
[49]
< 19 psec37 l hv
T
hypsorhodopsin (430 nm) 4 1"1~e
--- 50 psec39 I
> -251° C4
bathorhodopsin (543 nm) T1/e
-~ 40 nsec46 I
hypsorhodopsin (446 nm) 28
11
7"1/e
~- 50 psec37 I > - 2 3 8 ° C28
bathorhodopsin (534 nm)
> - 1 4 0 ° Cll
lumirhodopsin (497 nm) 23
7"1/e
26
300 nsec37 J > - 1 6 0 ° C 26
lumirhodopsin (515 nm) 26
/
T1/e
--~16sec 58 ]
> - 4 0 °C 11
metarhodopsin I (478 nm)
55
° C 26
LM-rhodopsin (486 nm)
/
!
T1/~-~ 200 msecS4l
>-15 °
C 11
metarhodopsin !1 (380 nrn) 55 T1~-~1 hr
>-65
!
/
l
> 0 °C55 /
¥
pararhodopsin (465 nm)
TI~ ~-10msec 6° [ > - 2 0 ° C26 acid metarhodopsin (482 nml ~ 61 +H+
11 iT
- H+
alkaline metarhodopsin (367 nm)* 61
> 5° C 55 all-trans-retinal (387 nm)* +
opsin FIG. 1. Photobleaching processes of cattle and squid rhodopsins. Photochemical reactions are denoted by wavy lines and thermal (dark) reactions by straight lines. Transition temperatures from one intermediate to another measured by low-temperature spectrophotometry are shown at the right sides of the straight lines and decay times of intermediates measured by flash photolysis at room temperatures are shown at the left sides of the straight lines. Absorption maxima are shown in parentheses. The absorption maxima indicated * are measured at room temperatures. S a m p l e for L o w - T e m p e r a t u r e
Spectrophotometry
Three kinds of rhodopsin preparations have been used, i.e., a whole r e t i n a , s-~° a s u s p e n s i o n o f r o d o u t e r s e g m e n t s TM ( R O S ) , a n d an e x t r a c t o f r h o d o p s i n . 11 W h o l e R e t i n a . A r e t i n a i s o l a t e d f r o m a d a r k - a d a p t e d f r o g e y e h as b e e n u s e d b e c a u s e o f e a s e in i s o l a t i o n o f an i n t a c t r e t i n a a n d t h e l a r g e a m o u n t
s F. 9 S. 10 S. 11 T.
Tokunaga, S. Kawamura, and T. Kawamura, F. Tokunaga, and T. Kawamura, F. Tokunaga, and T. Yoshizawa and G. Wald, Nature
Yoshizawa, Vision Res. 16, 633 (1976). Yoshizawa, Vision Res. 17, 991 (1977). Yoshizawa, Vision Res. 19, 879 (1979). (London) 197, 1279 (1963).
[49]
LOW-TEMPERATURE
RHODOPSIN
SPECTROPHOTOMETRY
335
of rhodopsin in the retina. After isolation of the retina in Ringer's solution, it is put between two sheets of filter paper with a hole (about 7 mm in diameter) at the center and then a small amount of glycerol is dropped on it. Afterward it is set in a sample cell of an optical cryostat described below. Suspension of Rod Outer Segments. In the case of the frog, two or three retinas isolated from dark-adapted frog eyes are gently shaken in Ringer's several times. ROS suspension thus obtained is centrifuged at 3000 g for 5 min in order to collect ROS as a precipitate. Afterward it is resuspended in a small amount of Ringer's containing 0.1 M hydroxylamine (pH 7.0) and then glycerol is added to give a final concentration of 75% or more. This mixture is used as a sample. Cattle ROS and squid microvilli both are prepared by a conventional sucrose flotation method 12-14 with or without lyophilization and extraction of lipid and retinal derivatives with petroleum ether. Glycerol is added to give a final concentration of 75% or more. Rhodopsin Extract. Vertebrate rhodopsin is extracted from ROS that have been purified by a conventional sucrose flotation method lz-14 followed by lyophilization and then extraction of lipid and retinal derivatives with petroleum ether. In the case of isolation of microvilli from squid retina, particular attention must be paid not to contaminate the inner segment fraction containing retinochrome. Here a procedure for preparation of squid rhodopsin is described as an example. Squid eyes are bisected and the rears are shaken in 0.1 M phosphate buffer (pH 6.8) or 0.1 M HEPES buffer (pH 7.0) to separate the outer limbs (microvilli) of the photoreceptor cells. The microvilli suspension thus obtained is filtrated through double sheets of gauze to remove the retinal debris, mixed with an equal volume of 80% sucrose solution (w/v in distilled water), and then centrifuged at 14,000 g for 1 hr. The precipitate is suspended in 38% sucrose solution (w/v in 0.1 M phosphate buffer, pH 6.8), followed by the centrifugation. The supernatant is diluted with more than 3 vol of phosphate buffer and centrifuged in order to get a precipitate composed of microvilli. The 38% sucrose flotation is repeated. The precipitate thus obtained is frozen and thawed several times, and is then layered on a sucrose linear gradient (25-50% in distilled water) followed by centrifugation at 100,000 g for more than 4 hr. The microvilli fraction containing rhodopsin floats at about the 29% sucrose density region, whereas the retinochrome fraction, if contaminated, floats at about the 36% sucrose density region with a small amount of micro12 E. K i m u r a , Jpn. J. Physiol. 3, 250 (1952). 13 D. S. P a p e r m a s t e r and W. J. Dreyer, Biochemistry 13, 2438 (1974). 14 R. H u b b a r d , P. K. Brown, and D. B o w n d s , this series, Vol. 18, Part C, pp. 615-653.
336
SPECTRAL RESPONSES OF VISUAL PIGMENTS
[49]
villi fraction. Addition of neutralized hydroxylamine (pH 7.0) to the microvilli fraction is recommended as a test of whether or not they contain retinochrome. Since rhodopsin is stable in the presence of hydroxylamine (0.1 M) but retinochrome is not, 1~one can check the contamination of retinochrome by measuring the descrease of absorbance at 480 nm. Digitonin solution (2%) is usually used for extraction of rhodopsin. For low-temperature spectrophotometry, the extract is mixed with glycerol in a final concentration of6611 or 75%, s by which the absorption spectrum of rhodopsin is not distorted. The mixture can be frozen to a clear glass without cracks above about - 100°, but below this temperature some cracks are usually formed in the mixture. Since the absorbance of rhodopsin at hmax should be more than 0.5 in a 2-mm light path for low-temperature spectrophotometry, the rhodopsin extract is concentrated by means of ultracentrifuge at 105,000 g for more than 12 hr or a millipore filter such as Amicon, if necessary. When a sample cannot be concentrated to such a high absorbance, it is recommended that the glycerol concentration in the sample be lowered to about 50%. The reason is that a 50% glycerol mixture forms a large amount of microcrystals on warming from liquid nitrogen temperatures (about - 195°, 77 K) to about - 500,TM so that the absorbance of rhodopsin in the mixture increases about 10 times because of multiple reflection of the measuring light by the microcrystals. Since the rnicrocrystals is stable at any temperature below - 5 0 °, one can measure the absorption spectrum at these temperatures using an opal glass. On application of this technique, photochemical reactions of iodopsin were studied at liquid nitrogen 17 and liquid helium temperatures TM (about - 269°, 4 K). The reversible photochemical process between iodopsin and bathoiodopsin at 4 K is shown in Fig. 2. Optical Cryostats For measurement of the absorption spectrum at low temperatures an optical cryostat must be used. 11 As an example, a cryostat we have used at liquid nitrogen temperature or above is shown in Fig. 3. The sample cell consists of three parts: a front quartz window, a middle silicone rubber ring, (1-5 mm thick) and a back opal glass. A visual pigment sample is filled into the sample cell, which is fixed in a sample cell holder (made of unoxygenated copper) by a screw-on ring. Then the cell holder is screwed to a copper tube at the bottom of the cold finger made of Pyrex glass. The 15 T. 16 T. 17 T. 18 y .
Hara and R. Hara, Proc. 1SCERG Syrup. Jpn. Ophthalmol. Vol. 10, Suppl. 22 (1966). Yoshizawa and G. Wald, Nature (London) 212, 483 (1966). Yoshizawa and G. Wald, Nature (London) 214, 566 (1967). Tsukamoto, S. Horiuchi, and T. Yoshizawa, Vision Res. 15, 819 (1975).
[49]
LOW-TEMPERATURE RHODOPS1N SPECTROPHOTOMETRY
337
Iodop#ln540nma 4 K batholoc;opBIn
1.0" 0.8" 0.6"
4 2'
7.1
0.4
o.z a
o
o 1.0
i
i
Bathoiodopsln )64Ohm, 4 K Iodopsln
0.8 0.6 0.4 0.2
b 0
400
500
600
700
Wavelength (nrn)
FIG. 2. Course of reversible photoconversion between iodopsin and bathoiodopsin at 4 K. Iodopsin/2% digltonin/50% glycerol mixture was cooled to 77 K and gradually warmed to 215 K for formation of microcrystals in the mixture, followed by cooling to 4 K for measuring the spectrum [curve 1 in (a)]. The absorbance of iodopsin in the mixture (Amax: 0.06) was intensified about 12 times (Amax: 0.7). (a) Iodopsin/2% digitonin/50% glycerol mixture (curve 1) was successively irradiated with green light at 540 nm for a total of 4, 8, 16, 32, 64, 128, and 256 sec (curves 2-8). In these irradiations, iodopsin changed to bathoiodopsin. The final curve (curve 8) represents a photosteady state mixture composed of iodopsin and bathoiodopsin. (b) Curve 8 in (a) was redrawn as curve 1, which was successively irradiated with red light at wavelengths longer than 640 nm for a total of 0.2, 0.5, 1, 2, 4, and 8 sec (curves 2-7). Bathoiodopsin converted to mainly iodopsin with a small amount of isoiodopsin. [From Y. Tsukamoto, S. Horiuchi, and T. Yoshizawa, Vision Res. 15, 819 (1975).]
cold finger with the sample cell holder is put into the glass jacket and then filled with liquid nitrogen. Immediately after that, the space between the sample cell holder and the glass jacket is evacuated by a rotary pump. The temperature of the sample is monitored by a copper-Constantan thermocouple attached to the sample cell holder. When the experiments are carded out at a desired temperature above 190°, a small volume of liquid nitrogen is dropped into the cold finger. The temperature can be kept at a constant temperature within _-_2° if one manages carefully. When dry ice/acetone is used instead of liquid nitrogen, the sample can be kept at about - 7 3 ° or above. Figure 4 shows a double vacuum glass cryostat for spectrophotmetry at liquid helium temperatures. The container of liquid helium is surrounded by that of liquid nitrogen through the vacuum space in order to -
338
[49]
SPECTRAL RESPONSES O F V I S U A L PIGMENTS
Thermocouple (copper vs. constantan) Flanges ~ E .....
3
:_--_ --.
Liquid nitrogen-..
, Vacuum
X 7-_" ~'-: _%
Glassjacket
,t..J,
---S
~ Cold finger
-=
.-_-
Cobar \
Coppe
__-~': ~.~. Screw-on ring .~. % =
Copper
y_-
- tube
\I ,I~ I
. Screw-on ring
I
Light
Cell holder
Quartz window J
Thermocouple
k ~ I
. . . .
5 cm
=
Opal glass Silicone rubber Sample Quartz plate
t
Samplecell
FIG. 3. Diagram of an optical cryostat for measuring the absorption spectrum at liquid nitrogen temperature or above. Details are given in the text. [From T. Yoshizawa and S. Horiuchi., Bunko Kenkyu 20, 206 (1971).]
insulate the heat radiation from the outside. The sample cell and its holder are the same as those of Fig. 3. Monitoring light from a spectrophotometer falls on the sample cell through the outer quartz window and the vacuum space. A volume of the container of liquid helium is about 1.2 liter. The sample can be kept at about 7 K for more than 12 hr if the cryostat is evacuated to about 10 -s mm Hg by a diffusion pump. Calculations of Absorption Spectra of I n t e r m e d i a t e s When a visual pigment is irradiated at a low temperature, a photosteady state mixture composed of the original pigment, its isopigment and intermediates is formed because of overlapping of their absorption spectra at a wavelength of irradiation. Accordingly the absorption spectra of
[49]
LOW-TEMPERATURERHODOPSIN SPECTROPHOTOMETRY
he,iuil
Cupro-nickelpipe
Liquid
339
~1
~
Balloon
~
Liquidnitrogen,~
~
Vacuum
~, Liquidnitrogen
llJ J:}l r
/
0o,0r. mp,e
Ce,, ho,der
FIG. 4. Diagram of an optical cryostat for measuring the absorption spectrum at liquid helium temperature or above. Details are given in the text. [From F. Tokunaga, N. Sasaki, and T. Yoshizawa, Photochem. Photobiol. 32, 447 (1980).]
intermediates can be estimated by subtracting the spectra of the original pigment and its isopigment from the photosteady state mixture. Absorption Spectrum of lsopigment. Isopigment can be prepared by adding 9-cis-retinal to an opsin TM and then incubating in the dark, or by irradiating rhodopsin at low temperatures. 2°'21 In the following a typical experiment by the latter method will be described. 2~ Frog ROS containing rhodopsin was irradiated with light at wavelengths longer than 540 nm at a liquid nitrogen temperature (about 77 K) to produce a photosteady state mixture composed mainly of isorhodopsin 19R. Hubbard and G. Wald, J. Gen. Physiol. 36, 269 (1952). s0 y . Kito, M. Ishigami, and T. Yoshizawa, Biochim. Biophys. Acta 48, 287 (1961). zl T. Yoshizawa and G. Wald, Nature (London) 201, 340 (1964). z~ S. Kawamura, S. Wakabayashi, A. Maeda, and T. Yoshizawa, Vision Res. 18, 457 (1978).
340
[49]
SPECTRAL RESPONSES OF VISUAL PIGMENTS
with small amounts of rhodopsin and bathorhodopsin. Then the photosteady mixture w a s irradiated with light at wavelengths longer than 610 nm to convert the bathorhodopsin to rhodopsin and isorhodopsin. After warming the mixture to room temperature, the spectrum was compared with that of the original rhodopsin. The intersection point between them is an isosbestic point (508 nm) between rhodopsin and isorhodopsin because the preparation contains only rhodopsin and isorhodopsin. In order to estimate the amount of isorhodopsin in the preparation, it was successively irradiated at room temperature with orange light at wavelengths longer than 540 nm until completely bleached and each time the spectrum was recorded (Fig. 5a). Under this irradiation, rhodopsin bleached much more rapidly than isorhodopsin. Then the difference in absorbance at wavelength of the isosbestic point (508 nm) between each spectrum (curves 1-7) and the final spectrum completely bleached (curve 8) was plotted on the semilogarithmic scale against the time of irradiation as shown in Fig. 5b. A straight line in the later stage of the irradiation is due to the bleaching of isorhodopsin. Extrapolating the straight line to zero time yields a measure of the proportion of isorhodopsin present in i
/
0.4
/+x
I
\
'
Rh. ~ 5~ , , = , o ~,Ret.oxime
I00
i
i
i
I
I
I
20
,0
i
i
i
i
i
50
O3
"6 I0 t)
~t o.I
'6 4~
..~ ~o
~bo''~
Wavelength (nm)
b
I
I
80
,00
,b0
Time ( s e c )
FIo. 5. (a) Successive irradiation of a mixture of rhodopsin and isorhodopsin in frog ROS at room temperature. The mixture (curve 1) of rhodopsin and isorhodopsin was prepared by irradiation of dark-adapted frog ROS suspension in 75% glycerol/0.1 M phosphate buffer (pH 7.0) with orange light (>540 nm) for 30 min at 77 K. After warming to room temperature, the mixture was successively irradiated with orange light ( > 540 nm) for 5, 5, 10, 20, 40 and 80 sec (curves 2-7) until finally the residual pigments were completely decomposed (curve 8). (b) Kinetics of bleaching of the mixture of rhodopsin and isorhodopsin. Difference absorbances at 508 nm (an isosbestic point between rhodopsin and isorhodopsin) between curves 1-7 and curve 8 in (a) are calculated and then normalized by the difference absorbance between curves 1 and 8. Filled circles represent the experimental values. A solid line shows a slow component of the kinetics that has been calculated by the least-squares method. The value obtained by extrapolating the solid line to zero gives the molar percentage of isorhodopsin. [From S. Kawamura, S. Wakabayashi, A. Maeda, and T. Yoshizawa, Vision Res. 18, 457 (1978).]
[49]
LOW-TEMPERATURE
RHODOPSIN
SPECTROPHOTOMETRY
341
[5-
1.0-
•
I
3
o
2
5 0.5-
0.0-
460
~6o
660
700
Wovelength (rim)
FIG. 6. A n experiment for calculation of absorption spectrum of squid lumirhodopsin. Absorption spectrum of rhodopsin/2% digitonin/66% glycerol mixture ( p H 10.5) was measured at - 85 ° ( c u r v e l). After cooling to - 188 °, the preparation was irridiated with blue light (437 nm) for 40 min, and then warmed to - 85 ° for measurement of spectrum (curve 2). After incubation at 2 ° for 30 min, the spectrum was measured at - 85 ° ( c u r v e 3). Finally, the preparation was irradiated with light at wavelengths longer than 510 nm at 2 °, and then recooled to -85°to measure the spectrum (curve 4). [From Y. Shichida, F. Tokunaga, and T. Y o s h i z a w a , Biochim. Biophys. Acta 504, 413 (1978).]
the preparation. Thus the absorption spectrum of isorhodopsin can be obtained by subtracting the absorption spectrum of the rhodopsin in the preparation from that of the mixture. Absorption Spectra of Lumi- and Meta-intermediates. W h e n a visual pigment is irradiated at about - 80 °, it changes to a photosteady state mixture composed of visual pigment, isopigment and an intermediate, the latter of which has been called lumi-intermediate.23 However, the lumi-intermediate is not always a single isomer, but may be a mixture of isomers that have all-trans-, 13-cis- and 7-cis-retinals as their chromophores according to analysis by high-performance liquid chromatography of cattle and squid lumirhodopsins 24,25 (Fig. 10c). In order to get the absorption spectrum of lumirhodopsin (all-trans), rhodopsin must be irradiated at liquid nitrogen temperatures, followed by warming to about - 8 0 °, because the irradiation of rhodopsin at liquid nitrogen temperatures does not yield other isomers than all-trans-bathorhodopsin (Fig. 10b), which converts to all-trans-lumirhodopsin on the warming. Figure 6 shows a typical experiment from which the absorption spectrum of squid lumirhodopsin was calculated, z6 All the spectra were mea23 R H u b b a r d , P. K. B r o w n , a n d A. K r o p f , Nature (London) 183, 442 (1959). 24 A. M a e d a , T. O g u r u s u , Y. S h i c h i d a , F. Tokunaga, and T. Yoshizawa, FEBS Lett. 92, 77 (1978). z5 A. M a e d a , Y. Shichida, and T. Yoshizawa, Biochemistry 18, 1499 (1979). 26 y . S h i c h i d a , F. Tokunaga, and T. Yoshizawa, Biochim. Biophys. Acta 504, 413 (1978).
342
SPECTRAL RESPONSES OF VISUAL PIGMENTS
[49]
sured at - 85° to counteract the effects of temperature on absorption spectra. To begin with, the spectrum of rhodopsin was recorded at - 8 5 ° (curve 1). Then the preparation was cooled to a liquid nitrogen temperature and irradiated with 437 nm light until the photosteady state mixture was formed. Warming to - 8 5 ° converted bathorhodopsin in the mixture to lumirhodopsin (curve 2). After incubation at 2° for 30 min for bleaching of lumirhodopsin to alkaline metarhodopsin, the spectrum was measured at - 85° (curve 3). Then it was irradiated at 2° with yellow light at wavelengths longer than 510 nm to bleach the residual rhodopsin and isorhodopsin. The final spectrum was measured at - 8 5 ° (curve 4). The difference spectrum between lumirhodopsin and alkaline metarhodopsin was calculated by subtracting curve 3 from curve 2. The amount of lumirhodopsin can be estimated by the formula (A1 - An)/(A1 - A4), where A is the absorbance at the isosbestic point (490 nm) between rhodopsin and isorhodopsin and the suffixes are the curve numbers. Thus the absorption spectrum of lumirhodopsin (curve 5 in Fig. 8) can be obtained by adding curve 4 to the difference spectrum corrected to 100% conversion. Absorption spectrum of metarhodopsin I can be obtained by the same procedures except that all the spectra are measured at about - 40° where metarhodopsin I is stable. Absorption Spectrum o f Batho-intermediate. Batho-intermediate is produced by irradiating visual pigment or isopigment at liquid nitrogen temperature or below. Since the glycerol mixture containing visual pigment forms a clear glass with some cracks on cooling to a liquid nitrogen temperature, the spectrum of batho-intermediate cannot be calculated by the same procedure as that of lumi-intermediate, because the second cooling from room temperature to the liquid nitrogen temperature freezes to a glass having a different number of cracks than the first one. Thus the spectrum of batho-intermediate must be calculated by either of the methods described below. The first is the application of the rapid cooling technique described in Article [85], this volume, by which a clear glass without any cracks can be prepared at liquid nitrogen temperature or below. Experimental method and calculation for the absorption spectrum of batho-intermediate are almost the same as those for lumi-intermediate except that the spectral measurement is carried out at liqui d nitrogen temperatures, z7 The second precedure can be applied when the absorption spectra of visual pigment and its isopigment at liquid nitrogen temperatures are already known, z6"28The photosteady state mixture composed of visual pig27 S. Horiuchi, F. Tokunaga, and T. Yoshizawa, Biochim. Biophys. Acta 591, 445 (1980). 2a y . Shichida, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 29, 343 (1979).
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY
343
ment, isopigment, and batho-intermediate is produced by irradiating the visual pigment with light at liquid nitrogen temperatures, and then warmed to room temperatures for bleaching of the batho-intermediate. The amounts of residual visual pigment and isopigment in the sample can be estimated by the successive irradiation of the sample with light at wavelengths longer than 540 nm, followed by plotting the decrease of absorbance at an isosbestic point between the original pigment and its isopigment on the semilogarithmic scale against the time of irradiation as described previously (Fig. 5). The amount of batho-intermediate is estimated from the difference in absorbance at the isosbestic point between the original sample and the sample after warming. Thus the absorption spectrum of batho-intermediate can be calculated by subtracting the absorption spectrum of the sum of the residual pigment and its isopigment from that of the photosteady state mixture. Absorption Spectrum of Hypsorhodopsin. Hypso-intermediate has been detected only in the rhodopsin system. 4'29 Irradiation of rhodopsin with yellow light at liquid helium temperatures (about 4 K) yields hypsorhodopsin. The experimental procedure for calculating the absorption spectrum is almost the same as that for bathorhodopsin except for the measuring temperature. However, one must pay attention to the contamination of bathorhodopsin in a photosteady state mixture, which is produced by irradiating rhodopsin at liquid helium temperatures. By irradiating the photosteady state mixture with longer wavelengths of light than 610 nm, the bathorhodopsin can be removed from the mixture, because the bathorhodopsin converts to rhodopsin and isorhodopsin. Thus, a mixture composed of only rhodopsin, isorhodopsin, and hypsorhodopsin can be prepared. Using this mixture, one can calculate the spectrum of hypsorhodopsin. Spectral Changes in Conversion of One Intermediate to Another. In order to solve the question of how many intermediates exist in the photobleaching process for rhodopsin, the spectral change in the thermal conversion of one intermediate to another must be measured, because if an isosbestic point exists in the conversion, it may safely be said that no intermediate exists between them. For measuring the spectral change, all the absorption spectra must be recorded at the same temperature to eliminate the effect of temperature on spectrum. In fact, the cooling from room temperature to liquid nitrogen temperature causes a shift of the hma x toward the longer wavelengths (cattle rhodopsin: about 8 nm) and increases its extinction coefficient about 1.1 times. 4 Figure 7 shows a spectral change in thermal conversion of squid lumi29 T. Yoshizawa and S. Horiuchi, in "Biochemistry and Physiology of Visual Pigments" (H. Langer, ed.), pp. 69-81. Springer-Verlag, Berlin and New York, 1973.
344
S P E C T R A L RESPONSES O F V I S U A L P I G M E N T S
~
Lumirhodopsin
[49]
warming= LM-rhodopsln
16,]
.
. mensured
~ i.o.
at - 8 5 0 C
I
o5-
o.o-
I ,~.,
.
460
,
56o Wovelength
66o
700
(nm}
FIG. 7. C o u r s e o f c o n v e r s i o n o f squid lumirhodopsin to L M - r h o d o p s i n by warming. Rhod o p s i n / 2 % digitonin/66% glycerol mixture (pH 10.5) w a s irradiated with blue light (437 nm) at - 188 °, t h e n w a r m e d to - 8 5 ° for c o n v e r s i o n o f b a t h o r h o d o p s i n to lumirhodopsin (curve 1). This preparation was s u c c e s s i v e l y w a r m e d to - 75, - 65, - 45, - 35, and - 25 °, and each time w a s recooled to - 85 ° for m e a s u r e m e n t of s p e c t r u m (curves 2 - 6 ) . T h e spectral c h a n g e s from c u r v e 2 to c u r v e 6 r e p r e s e n t s the c o n v e r s i o n s of lumirhodopsin to L M - r h o d o p s i n . [ F r o m Y. Shichida, F. T o k u n a g a , and T. Yoshizawa, Biochim. Biophys. Acta 504, 413 (1978).]
rhodopsin to LM-rhodopsin. ~6 Rhodopsin was irradiated with 437 nm light at a liquid nitrogen temperature and then warmed to - 85 °. The rhodopsin changed to a mixture of rhodopsin, isorhodopsin, and lumirhodopsin. After measurement of the absorption spectrum, the mixture was warmed to a required temperature, followed by cooling to - 8 5 °. The spectrum further shifted to shorter wavelengths with an isosbestic point at 502 nm, indicating that there was no intermediate between lumirhodopsin and LM-rhodopsin. Absorption Properties of Intermediates of Rhodopsin Bleaching sequences of cattle and squid rhodopsins are shown in Fig. 1, where several experimental results of low-temperature spectrophotometry and flash and laser photolyses were summarized. Figure 8 shows the absorption spectra of squid intermediates and the accompanying table shows absorption properties of intermediates of several visual pigments and rhodopsin analogs. Hypso-intermediate. Hypsorhodopsin is an intermediate discovered by irradiating cattle rhodopsin with orange light (> 520 nm) at liquid helium temperatures. 4,29 As its name indicates, hypsorhodopsin has a Xmax shorter than that of rhodopsin (hypsochromic shift). Now the hypso-intermediate is detected in all the animal rhodopsin systems we have tested, for example, in cattle, 4,29chicken, TM frog, 3° and squid 2s rhodopsin systems. 30 S. Horiuchi, F. T o k u n a g a , and T. Yoshizawa, Biochim. Biophys. Acta 503, 402 (1978).
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY 1.5-
t.o
(3
2
._~ -~ 0.5
O0
345
~5
4O0
500
600
Wovelength (nm)
15
RiO
I
6
o _>
_~05
O0
~o
sao
660
Wavelenoth (nm)
FIG. 8. The absorption spectra of squid rhodopsin and its intermediates in 2% digitonin/66% glycerol mixture (pH 10.5). 1, rhodopsin; 2, isorhodopsin; 3, hypsorhodopsin; 4, bathorhodopsin; 5, lumirhodopsin; 6, LM-rhodopsin.
Hypsorhodopsin was also detected at room temperatures by means of picosecond laser photolysis, 3r-3a indicating that it is a physiological intermediate of rhodopsin. However, in chicken iodopsin and bacteriorhodopsin systems it was not detected. 1s'33 Photoconversion of rhodopsin to mainly hypsorhodopsin is shown in Fig. 9, 2s in which squid rhodopsin (curve 1) was irradiated with longer wavelengths light than 480 nm at a liquid helium temperature. The absorption spectrum shifted to shorter wave31 R. Hara, T. Hara, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 33, 883 (1981). 32 O. Muto, F. Tokunaga, T. Yoshizawa, M. Ito, and K. Tsukida, to be published. 33 T. Iwasa, F. Tokunaga, and T. Yoshizawa, FEBS Lett. 101, 127 (1979). 34 T. Iwasa, F. Tokunaga, and T. Yoshizawa, Biophys. Struct. Mech. 6, 253 (1980). 35 T. Iwasa, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 33, 539 (1981). 36 T. Iwasa, F. Tokunaga, T. Yoshizawa, and T. G. Ebrey, Photochem. Photobiol. 31, 83 (1980). 3r y. Shichida, T. Yoshizawa, T. Kobayashi, H. Ohtani, and S. Nagakura, FEBS Lett. 80, 214 (1977). ~8 y. Shichida, T. Kobayashi, H. Ohtani, T. Yoshizawa, and S. Nagakura, Photochem. Photobiol. 27, 335 (1978). 39 T. Kobayashi, FEBS Lett. 106, 313 (1979).
346
[49]
SPECTRAL RESPONSES OF VISUAL PIGMENTS
% ¢) .¢
.¢Z Z a~ O r~ O
e~ Z
Z g]
a
,.J .¢
;>
u~ L~ ¢) [--
[-,
z
!o
a~ ~h Z
_q a~
.¢
"i
"'q ~
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY
1.5 ¸
Rhodapsin
> 4 8 0 nm - Hypsorhodopsin
I.O-~
I
/
.~
0.5
347
I I \ Lm-~.. ~ . : /
Measurement at 4 K
2
: 2
oo
. . . . . . . 400
500 Wavelength (nm)
i 6OO
700
FIG. 9. Course of the conversion of squid rhodopsin to hypsorhodopsin. Rhodopsin/2% digitonin/66% glycerol mixture (pH 10.5, curve 1) was successively irradiated with yellow light (>480 nm) at 4 K for a total of 5, 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 sec (curves 2-11). The final spectrum (curve 11) represents a photosteady state mixture composed of rhodopsin, isorhodopsin, and hypsorhodopsin with a small amount of bathorhodopsin. [From Y. Shichida, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 29, 343 (1979).]
lengths, indicating formation of hypsorhodopsin. Prolonged irradiation formed a photosteady state mixture composed of rhodopsin, isorhodopsin, and hypsorhodopsin with a small amount of bathorhodopsin (curve 11). When isorhodopsin was irradiated with the same light at liquid helium temperatures, the same photosteady state mixture was produced. Therefore, the interconversion between l l-cis (rhodopsin)-and 9-cis (isorhodopsin)-retinylidene chromophores occurs via hypsorhodopsin or bathorhodopsin, suggesting that hypsorhodopsin and bathorhodopsin have twisted all-trans-retinals as their chromophores. Photochemical reactions of rhodopsin and its intermediates at liquid helium temperatures are summarized in Fig. 10a. Hypsorhodopsin converts to bathorhodopsin on warming. When a squid preparation containing mainly hypsorhodopsin was warmed above 35 K, its absorption spectrum shifted to longer wavelengths through sharp isosbestic points at 476 and 366 nm. ~s In the case of cattle hypsorhodopsin, it converted to bathorhodopsin through an isosbestic point near 470 nm above 23 K. 4'29 There is a difference in photochemical behavior at liquid helium temperatures between cattle and squid hypsorhodopsins. When cattle hypsorhodopsin was irradiated, it converted to bathorhodopsin, 29 whereas squid hypsorhodopsin converted mainly to rhodopsin and isorhodopsin. 2s As already stated, hypsorhodopsin as well as bathorhodopsin has been suggested to have a twisted all-trans-retinal as its chromophore. Recently, the low-temperature spectrophotometry of frog retina showed that the transition dipole moment of hypsorhodopsin is almost the same in off-
348
SPECTRAL RESPONSES OF VISUAL PIGMENTS
(a)
Rhodopsin (11-cis) x
x Hypsorhodopsin (trans)
~
[49]
x Isorhodopsin •
(9-cis)
Bathorh!dopsin ~ (trans)
(b)
Rhodopsin
(11--ci$)
x
x
Bathorhodopsin (trans)
x
x
Isorhodopsin (9--cis)
7--cis
(C)
Rhodopsin (I 1--ci$) x
x Lumirhodopsin (trans)
product
~' Isorhodopsin ~
(9--ci$)
product FIG. 10. Schemashowing interconversion among rhodopsin and its photoproducts by light at (a) liquid helium, (b) liquid nitrogen and (c) dry ice/acetone temperatures. 13--cis
entation to the disk plane as that o f bathorhodopsin, 4° indicating that the c o n f o r m a t i o n of retinylidene c h r o m o p h o r e o f h y p s o r h o d o p s i n m a y be similar to that o f bathorhodopsin, that is, a twisted a l l - t r a n s - r e t i n y l i d e n e c h r o m o p h o r e . H o w e v e r , ~max o f h y p s o r h o d o p s i n (cattle, 430 nm) greatly shifts to shorter wavelength than that o f b a t h o r h o d o p s i n (cattle, 543 nm). In o r d e r to explain the blue shift, we has p r o p o s e d three possible models a b o u t conformation o f the retinylidene c h r o m o p h o r e o f h y p s o r h o d o p s i n as follows: I II III
U n p r o t o n a t e d retinylidene Schiff b a s e 4,2a'29 Protonated retinylidene S c h i f f b a s e in which conjugated s y s t e m is dissected at the 7 - 8 or 8 - 9 b o n d 41 Protonated retinylidene Schiff base that has no specific interaction with neighboring groups o f opsin 4"29
40 F. Tokunaga, N. Sasaki, and T. Yoshizawa, Photochem. Photobiol. 32, 447 (1980). 41 M. Ito, K. Hirata, A. Kodama, K. Tsukida, H. Matsumoto, K. Horiuchi, and T. Yoshizawa Chem. Pharm. Bull. 26, 925 (1978).
[49]
LOW-TEMPERATURE RHODOPSIN SPECTROPHOTOMETRY
349
Model I is suggested by the fact that the energy difference of the bathochromic shift in hmax between hypsorhodopsin and bathorhodopsin is similar to that between unprotonated Schiff base and its protonated form in alcohol. Model II is suggested by the fact that the hrnax of hypsorhodopsin is close to that ofrecto-y-rhodopsin (hmax = 425 nm), 41 whose chromophore is dissected at the 7-8 bond. Model III is suggested by the fact that hmax of protonated retinylidene Schiff base in alcohol is close to that of hypsorhodopsin. The most plausible model among them may be the unprotonated Schiff base (model I). Since the oscillator strength of hypsorhodopsin is close to that of bathorhodopsin, ~8 it seems unlikely that the conjugated system of hypsorhodopsin is dissected, because the oscillator strength of a conjugated polyene molecule general increases with length of the conjugated system in a relatively short system. In addition, the fact that the transition dipole moment of hypsorhodopsin is the same in orientation as that of bathorhodopsin4° may not support model II. A hypso-intermediate of retro-3,-rhodopsin was also observed by irradiation at liquid helium temperatures, as Thus the formation of hypsorhodopsin is not due to the breakdown at 7 - 8 - 9 of the conjugated double bond system. A noteworthy point is that we have failed to get hypsorhodopsin without contamination of rhodopsin and isorhodopsin on irradiation at the tail in longer wavelengths region of isorhodopsin at liquid helium temperatures, in spite of the fact that hypsorhodopsin has hmax at a shorter wavelength than those of rhodopsin and isorhodopsin. There are at least two possible reasons for this. First, if hypsorhodopsin has a small absorbance at the wavelength region of irradiation, the quantum yield of rhodopsin (or isorhodopsin) to hypsorhodopsin may be small or that of hypsorhodopsin to rhodopsin, isorhodopsin, and bathorhodopsin may be large. 42 Second, hypsorhodopsin may have relatively larger absorbance at longer wavelengths than rhodopsin or isorhodopsin. We have found that cattle or frog hypsorhodopsin has a small absorption band near 530 nm concomitant with the main absorption band near 430 nm. 4a Squid hypsorhodopsin, however, has a long tail only in the spectrum at the long wavelength region (Fig. 8). Batho-intermediate. Batho-intermediate4,29 (formerly called prelumiintermediate 11) is an intermediate stable at liquid nitrogen temperatures (about - 195°). Its hmax is located at a longer wavelength than its parent 42 A. Sarai, T. Kakitani, Y. Shichida, F. Tokunaga, and T. Yoshizawa, Photochem. Photobiol. 32, 199 (1980). 43 N. Sasaki, T. Yoshizawa, and F. Tokunager, Int. Syrup. Physicochem. Aspects Primary Process Vis. Excitation Photosynth., 1980.
350
SPECTRAL RESPONSES OF VISUAL PIGMENTS
[49]
O.6.
0.4 ¸
~
.
measuredot-175"C
9
7"9.
.
0.2 ¸
0.0
0.0 400
500 Wavelength
600
700
- n rn
FIG. 11. Course of conversion of rhodopsin to bathorhodopsin in (a) frog retina and (b) rhodopsin/2% digitonin extract. (a) Rhodopsin in the retina in 75% glycerol (curve 1) was successively irradiated with blue light (437 nm) at - 175° for a total of S, 10, 20, 40, 80, 160, 320, and 640 sec (curves 2-9). (b) Rhodopsin/2% digitonin/66% glycerol mixture in phosphate buffer (pH 7.0) was successively irradiated with blue light (437 nm) at - 165° for a total ofS, 10, 20, 40, 80, and 160 sec (curves 2-7). The final spectrum [curve 9 in (a) or curve 7 in (b)] represents a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin. [From F. Tokunaga, S. Kawamura, and T. Yoshizawa, Vision Res. 16, 633 (1976).]
visual pigment. All the visual pigments including bacteriorhodopsin 44 that we tested produced batho-intermediates on irradiation at liquid nitrogen temperatures. The only exceptional case is retinochrome, 31 which is a photosensitive pigment in cephalopod retina having all-trans-retinal as its chromophore. Formation of bathorhodopsin is also confirmed in frog retina by irradiation at liquid nitrogen s and liquid helium 3° temperatures (Fig. 11). In the table given earlier the absorption properties of bathorhodopsin are shown. Laser photolytic experiments confirmed that bathorhodopsin is produced at physiological temperatures2 °-32'37-39,45,46 Bathorhodopsin has been thought to have a twisted all-trans-retinal as its chromophore (isomerization model), because it was formed by irradiating both rhodopsin and isorhodopsin at liquid nitrogen temperatures 11 (Fig. 10). Since the first picosecond experiment, many alternative hypotheses have been proposed in which the change of rhodopsin to bathorhodopsin does not contain rotation of the 11-12 double bond of the retinylydene chromophore, but is due only to translocation of a proton on a 44 F. Tokunaga, T. Iwasa, and T. Yoshizawa, F E B S Lett. 72, 33 (1976). 45 R. A. Cone, N a t u r e (London), N e w Biol. 236, 39 (1972). 46 T. Rosenfeld, A. Alkalel, and M. Ottolenghi, N a t u r e (London) 240, 482 (1972).
[49]
LOW-TEMPERATURE
RHODOPSIN
SPECTROPHOTOMETRY
351
0.6
g
~0.3
o I
400
5()0
Wavelength (nm) FIG. 12. Course of conversion of 9-cis-retro-y-rhodopsin to its bathoproduct at - 185 °. A 9-cis-retro-T-rhodopsin/2% digitonin/66% glycerol mixture (pH 7.0) was irradiated at - 185° with 380 nm light for a total of 15, 40, 80, 150, 270, and 510 sec (curves 2-7). The final spect r u m represents a photosteady state mixture composed of original 9-cis pigmen t and bathoproduct with possibly 11-cis pigment. [From S. Kawamura, T. Yoshizawa, K. Horiuchi, M. Ito, A. Kodama, and K. Tsukida, Biochim. Biophys. Acta $48, 147 (1979).]
/~-ionone ring to a Schiff base nitrogen (proton translocation model).47-5° However, the formations of batho-intermediate by irradiation of retro-yrhodopsin (Fig. 12) has completely ruled out the proton translocation model, because a proton on the ring of the retro-y-retinylidene chromophore can not move to the Schiff base nitrogen through the side chain of the chromophore that is dissected at the 7 - 8 bond. Moreover, the following experiments also give some evidence that bathorhodopsin may have a twisted all-trans retinylidene chromophore. First, the CD spectrum of bathorhodopsin at the main absorption band is negative, which presents a striking contrast to the positive CD of rhodopsin.26"27"29The inversion of the positive to the negative CD spectrum in the process of converting rhodopsin to bathorhodopsin may be due to the conformational change of the retinylidene chromophore. 4r K. Van der Meer, J. J. C. Mulder, and J. Lughtenburg, Photochem. Photobiol. 24, 363 (1976). 48 K. Peters, M. L. Applebury, and P. M. Rentzepis, Proc. Natl. Acad. Sci. U.S.A. 74, 3119 (1977). 49 A. Lewis, Proc. Natl. Acad. Sci. U.S.A. 75, 549 (1978). so A. Warshel, Proc. Natl. Acad. Sci. U.S.A. 75, 2558 (1978).
352
S P E C T R A L RESPONSES O F V I S U A L P I G M E N T S
[49]
Second, the transition dipole moment of rhodopsin changes 26 degrees in orientation when it converts to bathorhodopsin. 1° This large orientational change also suggests the cis-trans isomerization of the retinylidene chromophore. The third is that the same bathrohodopsin is produced from 7-cis-rhodopsin by irradiation at liquid nitrogen temperatures 5 as those from rhodopsin and isorhodopsin. Recently, kinetic studies of photoconversion at liquid nitrogen temperatures showed that the bathorhodopsin, which had been produced by irradiating cattle or frog rhodopsin, was not a single product but a mixture of two components. 51"~2It was also confirmed that all the intermediates of rhodopsin, including hypsorhodopsin, are the mixture of two components .53 Lumi-intermediates. When batho-intermediate is warmed from a liquid nitrogen temperature to above - 140°, the spectrum shifts to shorter wavelengths, indicating formation of lumi-intermediate. The only exceptional case is bathoiodopsin, which converts to the original iodopsin above 180°.17 Laser photolytic experiments showed that bathorhodopsin converts to lumirhodopsin at room temperature with a time constant of about 40 nsec in the cattle rhodopsin system46 and about a few hundreds nanoseconds in frog45 and squid aT'a8rhodopsin systems. The absorption spectrum and spectroscopic properties of lumirhodopsin are shown in Fig. 8 and the table, respectively. Lumirhodopsin has a large/3-band in comparison with those of rhodopsin and bathorhodopsin, z6 The fact that the/3-band of bathorhodopsin is smaller than that of rhodopsin is consistent with the viewpoint that bathorhodopsin has an all-trans-retinylidene chromophore. On the contrary, the fact that lumirhodopsin has a larger E-band than rhodopsin seems to be inconsistent with the familiar view that lumirhodopsin has an all-trans-retinylidene chromophore. Probably there exists some special interaction between retinylidene chromophore and opsin in lumirhodopsin. In fact, we found that the conversion of rhodopsin to bathorhodopsin caused no CD change in the range between 280 nm and 300 nm, whereas the conversion of bathorhodopsin to lumirhodopsin showed a remarkable increase of the CD signal. 26"~4These results may support the idea that the photoconversion of rhodopsin to bathorhodopsin is mainly due to the isomerization of the retinylidene chromophore, whereas the process of conversion of bathorhodopsin to lumirhodopsin is due to some conforma-
sl N. 52 N. 53 N. 54 T.
-
Sasaki, F. T o k u n a g a , and T. Yoshizawa, FEBS Lett. 114, 1 (1980). Sasaki, F. T o k u n a g a , and T. Yoshizawa, Photochem. Photobiol. 32, 433 (1980). Sasaki, F. T o k u n a g a , and T. Yoshizawa, Annu. Meet. Biophys. Soc. Jpn., p. 222 (1980). G. E b r e y and T. Yoshizawa, Exp. Eye Res. 17, 545 (1973).
[49]
LOW-TEMPERATURERHODOPSIN SPECTROPHOTOMETRY
353
tional change of protein moiety, by which the highly twisted state of the retinylidene chromophore of bathorhodopsin is concurrently relaxed. As already stated, lumirhodopsin is found in a photosteady state mixture formed by irradiating rhodopsin at about - 8 0 °. However, the lumirhodopsin produced is a mixture of isomers having all-trans, 7-cis-, and 13-cis-retinals as their chromophores (Fig. 10c). Thus the protein moiety of lumirhodopsin may be flexible enough to accommodate these isomers, whereas that of bathorhodopsin may not. This may provide additional evidence that the conversion of bathorhodopsin to lumirhodopsin is due to the opening up of the conformation of opsin. M e t a - i n t e r m e d i a t e . When cattle lumirhodopsin is warmed to - 4 0 °, it converts to metarhodopsin I, which changes to metarhodopsin II above 15°.~ Flash photolytic measurements showed that a large increase of entropy occurred in these processes, 56 indicating large conformational changes of the protein moiety. Moreover, the rates of formations of these intermediates depend on the lipid content in the parent rhodopsin molecule ?7 The conversion between metarhodopsin I and II is a tautomeric equilibrium, in which metarhodopsin II is favored by an increase in temperature, or ionic strength, and addition of glycerol or methanol. 55 In the case of cephalopod rhodopsin, lumirhodopsin converts to LMrhodopsin above - 6 0 ° and then converts to acid and alkaline metarhodopsins, 26 -
Photochemical Reactions of Visual Pigment Analogs at Low Temperatures Various kinds of visual pigment analogs have been prepared from cattle opsin and chemically synthesized retinal analogs. For elucidation of the changes of interaction between retinylidene chromophore and opsin in the photobleaching process of rhodopsin and analysis of the structure of retinylidene chromophore of its intermediates, photochemical reactions of visual pigment analogs are investigated at low temperatures. Figure 12 shows a formation of bathoproduct on irradiation of retro-7rhodopsin with 380-nm light at a liquid nitrogen temperature2 This result may be one of the definite evidences by which the proton translocation model of bathorhodopsin was ruled out, as already stated. 55R. G. Matthews,R. Hubbard, P. K. Brown,and G. Wald,J. Gen. Physiol. 47, 215 (1963). 56R. Hubbard, D. Bownds,and T. Yoshizawa,Cold Spring Harbor Symp. Quant. Biol. 30, 301 (1965). 57H. Shichi, S. Kawamura,C. G. Muellenberg,and T. Yoshizawa,Biochemistry 16, 5376 (1977).
354
SPECTRAL
RESPONSES
OF
VISUAL
PIGMENTS
[50]
Almost all the visual pigment analogs formed their own bathoproducts on irradiation at liquid nitrogen temperatures. However, in contrast with batho-intermediates of the native visual pigments, those of visual pigment analogs have small extinction coefficients. In the table, absorption properties of bathoproducts of visual pigment analogs we have investigated are listed. A 13-dm pigment which has 13-dm retinal as its chromophore has a new intermediate between batho- and lumi-intermediates, r The appearance of the new intermediates may provide a new insight into the chromophore-protein interaction in visual pigment. Transition temperature from one intermediate to another may also give a valuable information about the chromphore-protein interaction in visual pigment. For example, batho-intermediate of 13-dm pigment is more unstable than bathorhodopsin, r suggesting that only a small conformational change of opsin near the 13-position may relax the strained chromophore of bathointermediate to form BL-intermediate of 13-dm pigment. 58 S. E. Ostroy,Biochim. Biophys. Acta 463, 91 (1977). 59S. E. Ostroy, F. Erhardt, and E. W. Abrahamson,Biochim. Biophys. Acta 112, 265 (1966). 60y. Ebina, N. Nagasawa, and Y. Tsukahara,Jpn. J. Physiol. 25, 217 (1975). 61F. Tokunaga, Y. Shichida, and T. Yoshizawa,FEBS Lett. 55, 229 (1975).
[50] P i c o s e c o n d
Spectroscopy
of Visual Pigments
B y M. L. APPLEBURY and P. M. RENTZEPIS
Technological developments in picosecond spectroscopic instrumentation have made it possible to investigate early dynamic events in photo° biological systems. Application of these picosecond techniques over the last eight years to the study of visual pigments and analogous systems, for example, has led to a description of the dynamic events involved in the production of initial photoproducts following the absorption of a photon, i.e., bathorhodopsin formed from rhodopsin or the bK intermediate formed from bacteriorhodopsin. The following text describes the instrumentation used for picosecond kinetics, data collection, analysis, and methods of sample preparation as they have been applied to experimental study of visual pigments. Instrumentation for a Double-Beam Picosecond Spectroscopic Apparatus Picosecond kinetics and spectroscopy are practiced by a large number of investigators emphasizing either absorption, emission, or, lately, resoMETHODS IN ENZYMOLOGY, VOL. 81
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181981-7