Evidence for a common batho-intermediate in the bleaching of rhodopsin and isorhodopsin

Vision Res. Vol. 24, No. 11, pp. 1465-1470, 1984 Printed in Great Britain.All rights reserved

0042-6989/84 $3.00+0.00 Copyright © 1984PergamonPress Ltd

EVIDENCE FOR A COMMON BATHO-INTERMEDIATE IN THE BLEACHING OF RHODOPSIN AND ISORHODOPSIN D . S. KLIGER, J. S. HORWITZ, J. W. LEWIS and C. M. EINTERZ Division of Natural Sciences, University of California, Santa Cruz, CA 95064, U.S.A.

Abstract--Nanosecond transient spectroscopic measurements of the BATHO products formed from photolysis of bovine rhodopsin (RHO) and isorhodopsin (ISO) are discussed. BATHO absorption spectra of RHO and ISO differ slightly,but both pigments exhibit wavelength maxima near 560 nm. An additional transient absorption at 440 nm is observed immediately following excitation of RHO but not ISO. The decay times and Arrhenius activation energies of the two 560 nm absorbing transients are the same. In addition to spectral differences in the photolysis of RHO and 1SO, the bleaching yield as a function of 532 nm laser power is different, with the yield in RHO saturating at lower laser power than ISO. The bleaching yield of the two pigments has been modeled using the known extinction coefficients and quantum yields for the interconversion of RHO, ISO, and a single BATHO species. Agreement between experiment and the model is found if the effects of the laser polarization are considered. The data are Consistent with a common BATHO in the photolysis of RHO and ISO. Rhodopsin

Isorhodopsin

Bathorhodopsin

Visualpigments

INTRODUCTION

In 1958, Yoshizawa and Kito first observed that photolysis of visual pigments produces an intermediate which is stable at liquid nitrogen temperatures and has a spectrum that is red shifted relative to the parent pigment. This intermediate, bathorhodopsin (BATHO, originally called prelumirhodopsin), was assumed to be the primary photochemical product and was thought to involve an isomerization of the pigment chromophore. Another rapidly formed intermediate, hypsorhodopsin, which is stable at liquid helium temperatures, has been observed (Yoshizawa, 1972); but it is unclear whether this intermediate is a precursor to bathorhodopsin (Kobayashi, 1980; Sarai et al., 1980). Thus, intense interest remains in the nature of BATHO formation as this process is commonly viewed as the "trigger" for subsequent reactions involved in visual transduction. Picosecond spectroscopic techniques have been used in most recent studies of BATHO in an attempt to deduce the nature of BATHO by measuring its rate of formation (Applebury and Rentzepis, 1982). From these studies, two different views for the mechanism of BATHO formation have been put forward. Monger et al. (1979) found that BATHO products of both rhodopsin (RHO) and isorhodopsin (ISO) form within 3 psec of excitation. Based on previous quantum yield measurement (Rosenfeld et al., 1977) and similar BATHO spectra from the two pigments, they assumed that RHO and ISO share a common BATHO intermediate. This, combined with the very rapid BATHO formation in the two pigments, led

these workers to conclude that the primary photochemical process involves a simple isomerization. In other picosecond experiments, Peters et al. (1977) came to a different conclusion about the nature of BATHO formation. They found that, while BATHO forms within 6 psec upon excitation of RHO at room temperature, the formation kinetics slow down considerably at temperatures below about 50 K. Furthermore, they found that the kinetics do not follow Arrhenius behavior. The rate initially slows in an Arrhenius fashion with decreasing temperature but then remains essentially constant as the temperature is lowered below 20 K. This is indicative of a tunneling process and, together with a large deuterium effect on BATHO formation kinetics, led these workers to conclude that the primary process involves a proton translocation at the SchilTs base chromophore. One issue arising from this controversy is whether or not RHO and ISO produce common BATHO products upon photolysis. In this paper we address this question using nanosecond laser photolysis techniques. Nanosecond photolysis studies of BATHO products of RHO [batho(ll)] and ISO [batho(9)] allow us to measure spectra of these species as well as the decay kinetics. Combining this information with results on formation kinetics will give us a more complete picture of the nature of these species. Presented here are results from two types of studies. First, we will review recent results (Horwitz et al., 1983) on spectra and decay kinetics of batho(11) and batho(9). Second, experiments will be discussed which measured RHO and ISO bleaching as a function of laser pulse energy. Taken as a whole, these

1465

1466

D.S. KL1GERet al.

two types of experiments show that b a t h o ( l l ) and batho(9) have similar decay kinetics, spectra and photochemical properties. Thus ISO and RHO either have a common BATHO intermediate or have BATHO intermediates which differ only slightly.

I

I

I

I

I

1

3.5

3.6

I

o

i00 k " 2 0

~

EXPERIMENTAL Procedures for the measurements of BATHO decay kinetics and spectra have been reported elsewhere (Horwitz et al., 1983). Transient spectra were measured on an apparatus that employed a Molectron DL200 dye laser pumped by a Molectron UV1000 nitrogen laser as an excitation source. This laser produced light at 457nm (6 nsec FWHM) which minimized photoreactions resulting from absorptions of light by BATHO. Transient absorptions were monitored with a xenon flashlamp (2-10/~ sec) filtered by 10 nm bandpass interference filters. Bleaching yield experiments were performed with a similar apparatus operated in a slightly different mode (Horwitz, 1983). Here a Quanta-Ray DCR-1 Nd:YAG laser was used as a pump source (532 nm, 8nsec FWHM) to enhance BATHO photolysis. Bleaching was monitored at 500 nm with the probe pulses delayed so as to monitor pigment absorption 100 msec after excitation. The choice of 500 nm as monitoring wavelength in the power dependence measurements was based on preliminary experiments conducted with an optical multichannel analyzer on loan from Princeton Applied Research. From the spectra obtained with this instrument, it was apparent that isorhodopsin was being formed during rhodopsin photolysis at high laser powers. Thus to measure the amount of pigment bleached, measurements had to be made at the RHO-ISO isosbestic (500 nm). The same apparatus, with no time delay introduced between the pump and probe pulses, was also used to measure transient linear dichroism. In this case, a sheet polarizer was inserted in front of the probe source in order to measure BATHO absorption with light polarized parallel or perpendicular to the laser polarization axis. Laser energies were measured with a Scientech 3600 joulemeter. Bovine rhodopsin, as well as isorhodopsin derived from it, was prepared as described earlier (Horwitz et al., 1983; Lewis et al., 1981). The pigment concentrations used here (2 mg/ml) produce an optical density of approx. 0.4 in a 2 mm cell at the wavelength of maximum absorbance for RHO and ISO.

-2.5

3.0

I

3.2

3.3

34

3

I/T ,, IO 3 Fig. 1. Arrhenius plot of the decay kinetics monitored at 580 nm for rhodopsin (©) and isorhodopsin ( A ) in 2% octyl glucoside.

rhodopsin in sonicated membrane suspensions and samples in which octyl glucoside detergent has been added. The kinetics are also not affected by moderate changes in pH. Figure 2 shows the difference spectra obtained 50 nsec after excitation of RHO and ISO at 20°C. Three features can be seen in these difference spectra. First, excitation of both RHO and ISO results in positive absorption changes near 560 nm. Second, negative absorption changes are found around the parent pigment absorption maxima (500nm for RHO and 485 nm for ISO). Finally, a positive absorption change is seen near 450 nm in the RHO difference spectrum; but no comparable absorption is

(a]

005

-005 ,<] 005

o

/ ~ e -

~

/ •

":\

RESULTS

Kinetics a n d spectra

I

- 005

Figure 1 shows an Arrhenius plot for the decay rates of the transient absorption at 580 nm in the photolysis of RHO and ISO. It is clear that the kinetics and activation energies are the same within experimental error. The kinetics are identical for

400

450

I 500

i 550

600

Wavelength (nm) Fig. 2. Absorption changes in rhodopsin (A) and isorhodopsin (B) 50 nsec following excitation at 457 nm and 20°C.

1467

Batho-intermediate in the bleaching of rhodopsin and isorhodopsin

005

o

j

0

.~

(o)_

J

I

I

"~1

1

(b)

005

2

0 400

450

500

550

600

Wavelength (nm)

Fig. 3. Corrected absorption spectra for the transients in the photolysis of rhodopsin and isorhodopsin 50 nsec following excitation. (A) The initial rhodopsin spectrum (1) and the corrected transient spectrum 50nsec following excitation (2). (B) The initial isorhodopsin spectrum (1) and the corrected transient spectrum 50nsec following excitation (2). evident in the ISO spectrum. Since RHO and ISO have slightly different spectra, these difference spectra do not provide direct information for comparisons of batho(11) and batho(9). For this we must correct the difference spectra by adding in the absorption effects of each parent pigment. The procedure for this has been described earlier (Horwitz et al., 1983) and the resulting 50nsec corrected transient spectra are shown in Fig. 3. The 50nsec corrected transient spectra clearly show a 440nm transition in b a t h o ( l l ) but not in batho(9). This difference does not, however, have direct significance for comparing b a t h o ( l l ) and batho(9). The 440 nm transition has been shown to have different formation kinetics (Applebury et al., 1979) and decay kinetics (Horwitz et al., 1983) from the 560 nm transient identified with BATHO absorption. Thus, while it is interesting that this transition is seen in RHO but not ISO, it is not due to BATHO absorption. The red transition seen on excitation of RHO or ISO clearly has the classical BATHO spectrum. The b a t h o ( l l ) spectrum shows a peak at 560 nm while the absorption maximum of batho(9) lies at 550 nm. This difference will be discussed below.

absorption with a spectrum characteristic of metarhodopsin I. However, at high laser energies, little decay of the transient spectrum was observed. This is an indication that a large amount of ISO was formed during the excitation pulse (the R H O - I S O difference spectrum is similar to that of R H O - L U M I but ISO does not thermally decay). Thus, high laser energies can drastically change the observed bleaching behavior of the pigments due to production of significant amounts of ISO (when exciting RHO) or RHO (when exciting ISO). Modeling this behavior should yield information about the photochemical properties of the pigments and their bleaching intermediates. Figures 4A and 4B show the degree of bleaching of RHO and ISO, respectively, as a function of laser energy. The laser spot size in the sample was about 3 mm in diameter so that an energy of 1 m J/pulse corresponds to a fluence of approx. 14 mJ/cm 2. It can be seen that bleaching monotonically increases with increasing energy, reaching a saturation level above 1 m J/pulse for RHO. The bleaching increases more slowly in ISO and saturates at higher laser energies. To model the saturation behavior of pigment bleaching we first used the simple scheme shown below: ¢ = 0.67

RHO , @ = 0.25

V.R. 24/11

C

~

> ISO

(1)

@ = 0.05

¢

LUM1

"

0~0

005

Ca)
I

I

I

~

l 4

I 6

I 8

I

010

g 005

Bleaching saturation curves

We next investigated the effect of laser energy on the bleaching yield of RHO and ISO. The fact that laser energy affects kinetic and spectral measurements of visual pigment intermediates is dramatically seen in measurements on lumirhodopsin (LUMI). Transient spectra associated with L U M I were obtained at low and high laser energies. At low energies, the transient absorption was observed to decay in detergents with a half-life of approx. 20 #sec to an

qb = 0.20

> BATHO <

t

(bl 0

I 2

Energy

l 10

(mJ/pulse}

Fig. 4. Change in optical density at 500nm, 100msec following excitation of rhodopsin (A) and isorhodopsin (B) at 532 rim, as a function of pulse energy. The dashed line indicates the single population fit to data. The solid line indicates the model which accounts for the lack of rotational reorientations during the laser pulse (see text).

1468

D.S. KL1GERet al.

Here it is assumed that RHO, BATHO and ISO can all be formed within the lifetime of the laser pulse and that each can absorb light and photoreact with known quantum yields as indicated (Hurley et al., 1977; Suzuki and Callender, 1981). In this model the laser pulse is considered to consist of a number of segments. Each segment (corresponding to one photon for every four molecules) of the pulse interacts with the sample resulting in changes due to the photolysis reactions as indicated above. After interaction with a particular segment, new concentrations of each species are calculated. The sample is then allowed to interact with the next segment. The number of segments that interact with the sample is proportional to the energy per pulse of the laser. Literature values for the extinction coefficients for each species at 532nm [ e = 2 0 0 0 0 M - ~ c m -~ for RHO, e = 1 6 0 0 0 M - l c m ~ for ISO and e = 4 0 0 0 0 M -~ cm -j for BATHO (Hubbard, 1969; Applebury et al., 1974; Yoshizawa, 1972)] were used. The most important feature of this model is that it assumes a common BATHO intermediate in the photolysis of RHO and ISO. The fit of the predicted saturation behavior from this model are the dashed lines in Figs 4A and 4B. The model was fit to the data by varying two parameters, the absorbance change at high powers and the relative energy of the laser. These parameters essentially are adjustments of the effective path length and the laser spot size, two experimental parameters which are difficult to determine accurately due to laser beam shape and pump-probe beam overlap effects (the pump laser operates in the TEM0~ mode and the pump and probe beams intersect at an angle of approx. 30°). Since photolysis experiments for RHO and ISO were performed under identical conditions, the same relative energy scale is used for both pigments. As can be seen, the best fit, particularly for the RHO saturation behavior, is less than satisfactory. The most obviously incorrect prediction of this simple model is the pronounced peaking at 1 mJ per pulse of BATHO production starting from RHO. Although at first this seems strange, the explanation is quite simple. Because the quantum yield for BATHO formation from RHO is ten times as great as for ISO formation from BATHO, the photoequilibrium on the left side of relation 1 is reached at lower pulse energies than the photoequilibrium on the right hand side. BATHO concentration reaches a maximum under these conditions. As the slower B A T H O = I S O photoequilibrium is approached at higher energies, ISO production comes at the expense of BATHO and RHO. Thus the peaking behavior is caused principally by the fact, on which there is general agreement in the literature (Hurley et al., 1977; Suzuki and Callender, 1981), that the quantum yield for BATHO formation from RHO is much greater than the quantum yield for ISO formation from BATHO. Although the quantum yields on the right hand side of relation 1 seem to make ISO

formation unimportant, the extinction coefficients of BATHO and ISO at the 532 nm actinic wavelength favor ISO production. To improve the above model, a minor modification was made. In this second model (Horwitz, 1983) recognition was made of the fact that the N d : Y A G laser used in the experiments is polarized and that detergent solubilized rhodopsin does not undergo rotational reorientation during the 8 nsec laser pulse. Rhodopsin solubilized in various detergents has been shown to rotate on a time scale of 200 nsec (T. P. Williams, private communication). Thus, during the laser pulse each molecule, as well as its photoproducts, remains in a fixed orientation relative to the laser polarization. This is important because under these conditions one cannot simply use the literature values for the extinction coefficients of RHO, BATHO and ISO. Molecules oriented such that their transition dipoles lie parallel to the laser polarization have extinction coefficients three times the average, while molecules oriented perpendicular to this will not absorb any light. Although experimental evidence has indicated a slight change in orientation of the transition dipole upon photolysis of RHO to BATHO (Michel-Villaz et al., 1982; Kawamura et al., 1979), this change produces negligible effects in the model presented here. Thus, to simplify calculations it is assumed that the orientation of the transition dipoles of RHO, BATHO and ISO are similar. In general, the extinction coefficient will vary as cos 2 0 where 0 is the angle between the laser polarization axis and the axis of the transition moment. The sample is taken to be comprised of several subsets of molecules each oriented in different directions. The sum of these subsets represents a randomly oriented collection of molecules. By use of this model the saturation data in Fig. 4 can be fit as shown by the solid lines. While the fits of the second model are excellent, we sought additional proof to show that the polarization effect is indeed important under our experimental conditions and that the saturation behavior can be accurately modeled using a common BATHO intermediate. To show this, we measured the linear dichroism of batho(11) at various laser energies. From the above model, one would predict that at low laser energies BATHO products would be found whose transition moments would preferentially lie along the axis of laser polarization. This would produce a large dichroism in the BATHO absorption. At high laser energy one would predict that molecules in all orientations would be excited. Even though extinction coefficients of off-axis molecules are lower, there are enough photons in this case to make it likely that these molecules will be excited. Thus one would expect a decrease in the observed dichroism at high laser powers. These predictions are dramatically born out by the data shown in Fig. 5, where a large dichroism is found at a laser energy of 200 #J/pulse (where the saturation curve is still rising linearly); but

Batho-intermediate in the bleaching of rhodopsin and isorhodopsin

,03

0 o 0

o

.03

o 0

I00 200 Time(ns)

300

Fig. 5. Transient dichroism in rhodopsin measured at 580 nm resulting from an excitation pulse at 532 nm. Absorptions are shown for the monitoring light polarized parallel and perpendicular to the excitation pulse polarization where the excitation pulse energy is 1 mJ/pulse (A) and 200 #J/pulse (B). no dichroism is seen at an energy of 1 m J/pulse (where the bleaching signal has reached the saturation level). Recently, similar polarization effects have been observed and modeled in the photolysis of bacteriorhodopsin (bR) (Nagle et al., 1983). There, the saturation behavior in the interconversion between bR and the K intermediate was modeled. First, saturation curves were calculated for the simple two state model. Then saturation curves were calculated for a model that includes photoselection due to incomplete reorientation of the chromophore during the excitation flash. The observation of a large dichroic ratio at low excitation energies, as well as excellent agreement between the predicted and observed dichroic ratio as a function of excitation energy, led to authors to conclude that this was an important effect in their experiments as well as any other flash photolysis experiments on large molecules. DISCUSSION

The data presented above gives us three probes to judge the differences or similarities of batho(11) and batho(9). The kinetic decays of these two species are identical and indicate that their thermal deactivations are the same. The results of the spectral measurements are more equivocal. At 50 nsec following excitation one can see a 440 nm absorbing species in RHO but not in ISO. The reason for this difference is not clear. However, one could imagine that RHO

1469

might exist in two energetically distinct forms while ISO appears to exist in only one. For example, 11-cis retinal has been shown to exist in both 12-s-trans and 12-s-cis conformations (Honig and Karplus, 1971). If two conformers were possible in RHO they might yield pigments giving different photoproducts. In 9-cis retinal this type of dual conformation may not be found due to the absence of chromophore-based steric interactions such as those found in the 11-cis chromophore. Thus, there may be a local minimum in the potential surface for the rotation of the 11-cis chromophore not present in that of the 9-cis chromophore. This phenomenon could reflect differences in ground state properties of the pigments, in excited state properties, or both. Clearly more work is needed to understand the nature of the potential surfaces and how they relate to the 440 nm transition. The spectra shown in Fig. 3 indicate spectral maxima at 560nm for b a t h o ( l l ) and 550nm for batho(9). This is a significant shift for pigment spectra. However, transient spectra shown here were obtained at 10 nm intervals. Thus, our spectral resolution does not warrant drawing significant conclusions about the differences in batho(11) and batho(9) spectra. It should be noted that picosecond spectra have also shown b a t h o ( l l ) and batho(9) spectral maxima to be at 560 and 550 nm, respectively (Spalink et al., 1983). Thus the 10 nm shift in the spectra may be real. Nevertheless, this may not indicate significant structural differences in b a t h o ( l l ) and batho(9). In the parent pigments the proteins would create pockets around the pigment chromophores which would represent some mimimum energy configurations. Isomerization to form a transoid BATHO could produce different protein conformations in RHO and ISO which would yield different BATHO spectra. Small changes in charge locations could affect spectra (Honig et al., 1979) without having significant effects on subsequent thermal or photochemical reactions. This conclusion seems well borne out by our saturation data and modeling. If polarization effects are included, one can accurately model the saturation behavior of RHO and ISO bleaching using only a single BATHO intermediate and using standard values for extinction coefficients and quantum yields for each species and its photoreactions. While ISO bleaching saturates more slowly than that of RHO, as seen here and in picosecond experiments (Spalink et al., 1983), this seems to reflect lower quantum yields in ISO photoreactions rather than the production of different photolysis intermediates. CONCLUSIONS

The evidence presented here lead us to conclude that there are probably only minor differences between b a t h o ( l l ) and batho(9). Both species thermally decay at the same rates and with the same activation energies. The photochemical reactions of

1470

D.S. KLIGER et al.

b o t h are similar e n o u g h t h a t we can accurately describe o u r s a t u r a t i o n curves assuming a single form of B A T H O . While the spectra o f b a t h o ( l l ) a n d b a t h o ( 9 ) are s o m e w h a t different, the properties o f these two B A T H O p r o d u c t s which are m o s t imp o r t a n t for visual transduction, i.e. t h e r m a l a n d p h o t o c h e m i c a l reaction rates, are the same. Acknowledgements--We thank Meridithe Applebury and Ted Williams for useful discussions. We also thank Princeton Applied Research Corporation for the loan of an OMA and the National Eye Institute for support of this work under grant EY00983.

REFERENCES

Applebury M. L., Peters K. S., Kobayashi T. and Rentzepis P. M. (1979) Picosecond studies of an early "blue-shifted" intermediate in the photochemistry of rhodopsin. Biophys. J. 25, 317a. Applebury M. L. and Rentzepis P. M. (1982) Picosecond spectroscopy of visual pigments. Meth. Enzym. 81, 354-368. Applebury M. L., Zuckerman D. M., Lamola A. A. and Jovin T. M. (1974) Rhodopsin purification and recombination with phospholipids assayed by the metarhodopsin I-,metarhodopsin II transition. Biochemistry 13, 3448-3458. Honig B., Dinur U., Nakanishi K., Balogh-Nair V., Gawinowicz M. A., Arnaboldi M. M. and Motto M. G. (1979) An external point-charge model for wavelength regulation in visual pigments. J. Am. chem. Soc. 101, 7084-7086. Honig B. and Karplus M. (1971) Implications of torsional potential of retinal for visual excitation. Nature 193, 55~560. Horwitz J. S. (1983) Nanosecond laser photolysis of polyenes and visual pigments. Ph.D. Thesis, University of California, Santa Cruz. Horwitz J. S., Lewis J. W., Powers M. A. and Kliger D. S. (1983) Nanosecond laser photolysis of rhodopsin and isorhodopsin. Photochem. Photobiol. 37, 181-188. Hubbard R. (1969) Absorption spectrum of rhodopsin: 500 nm absorption band. Nature, Lond. 221, 432-435. Hurley J. B., Ebrey T. G., Honig B. and Ottolenghi M. (1977) Temperature and wavelength effects on the photo-

chemistry of rhodopsin, isorhodopsin, bacteriorhodopsin and their photoproducts. Nature 270, 540 542. Kawamura S., Tokunaga F., Yoshizawa T., Sarai A. and Kakitani T. (1979) Orientational changes of the transition dipole moment of retinal chromophore on the disk membrane due to the conversion of rhodopsin to bathorhodopsin and to isorhodopsin. Vision Res. 19, 879-884. Kobayashi T. (1980) Existence of hypsorhodopsin as the first intermediate in the primary photochemical process of cattle rhodopsin. Photochem. Photobiol. 32, 207-215. Lewis J. W., Winterle J., Powers M. A., Kliger D. S. and Dratz E. A. (1981) Kinetics of rhodopsin photolysis intermediates in retinal rod disk membranes. I. Temperature dependence of lumirhodopsin and metarhodopsin I kinetics. Photochem. Photobiol. 34, 375-384. Michel-Villaz M., Roche C. and Chabre M. (1982) Orientational changes of the absorbing dipole of retinal upon the conversion of rhodopsin to bathorhodopsin, lumirhodopsin and isorhodopsin. Biophys. J. 37, 603-616. Monger T. G., Alfano R. R. and Callender R. H. (1979) Photochemistry of rhodopsin and isorhodopsin investigated on a picosecond time scale. Biophys. J. 27, 105-116. Nagle J. F., Bhattacharjee S. M., Parodi L. A. and Lozier R. H. (1983) Effect of photoselection upon saturation and the dichroic ratio in flash experiments upon effectively immobilized systems. Photochem. Photobiol. 38, 331-339. Peter K., Applebury M. L. and Rentzepis P. M. (1977) Primary photochemical event in vision: proton translocation. Proc. natn. Acad. Sci., U.S.A. 74, 3119-3123. Rosenfeld T. B., Honig B. and Ottolenghi M. (1977) Cis-trans isomerization in the photochemistry of vision. Pure appl. Chem. 49, 341-351. Sarai A., Kakitani T., Shichida Y., Tokunaga F. and Yoshizawa T. (1980) Simulation analysis of the photoconversion process in squid rhodopsin at liquid helium temperature. Photochem. Photobiol. 32, 199-206. Spalink J. D., Reynolds A. H., Rentzepis P. M., Sperling W. and Applebury M. L. (1983) Bathorhodopsin intermediates from l l-cis-rhodopsin and 9-cis-rhodopsin. Proc. natn. Acad. Sci., U.S.A. 80, 1887-1891. Suzuki T. and Callender R. H. (1981) Primary photochemistry and photoisomerization of retinal at 77°K in cattle and squid rhodopsins. Biaphys. J. 34, 261-265. Yoshizawa T. (1972) The behavior of visual pigments at low temperatures. In Handbook of Sensory Physiology Vol. VII, Part 1 The Photochemistry of Vision (Edited by Dartnall H. J. A.), pp. 146-179. Springer-Verlag, Berlin. Yoshizawa T. and Kito T. (1958) Chemistry of the rhodopsin cycle. Nature, Lond. 182, 1604-1605.