energy storage in the primary photoreaction of bovine rhodopsin. a photoacoustic study

Pliorochernistry and Photobiology Vol. 41, No. 4. pp. 459 - 465, 1985 Printed in Great Britain. All rights reserved

003 1-8655/85 $03.00 t O . 0 0 Copyright @ 1985 Pergamon Press Ltd

ENERGY STORAGE IN THE PRIMARY PHOTOREACTION OF BOVINE RHODOPSIN. A PHOTOACOUSTIC STUDY

FRANCOIS BOUCHER* and ROGER M. LEBLANC Centre de recherche en photobiophysique, UniversitC du Quebec A Trois-RiviBres, Trois-Rivikres,

Quebec, Canada, G9A 5H7

(Received 8 June 1984; accepted 20 November 1984)

Abstract-Photoacoustic spectroscopy is used to investi ate the uptake of energy in the primary photoreaction of bovine visual pigment (Rhodopsin + h f Bathorhodopsin). It is shown that very concentrated dried rod outer segment membranes have a sufficient thermal diffusivity to be analyzed by this technique, From the photoacoustic and absorption spectra of these membranes, the low temperature dissipation spectrum has been obtained and the results are consistent with the storage of 145 kJ mol-' in the primary event of vision having a quantum yield of 0.67. By photoacoustic spectroscopy, this process is continuously monitored from 350 to 550 nm and its efficiency is found to vary by less than lo%, even in the spectral region of the p-band of rhodopsin.

INTRODUCTION

The first step of the visual process involves absorption of light by the visual pigment rhodopsin and its fast and efficient conversion to the metastable intermediate bathorhodopsin. Experimental evidence together with theoretical investigations favor a mechanism involving isomerization of the retinyl chromophore followed by a fast proton translocation within the ground state manifold (Honig, 1982).

One can easily conceive that to trigger visual excitation, some fraction of the energy absorbed by rhodopsin has to be stored in bathorhodopsin. In the case of bovine rhodopsin, Cooper (1979a) has determined by photocalorimetry that the enthalpy of bathorhodopsin was 35 kCal mol-' higher than rhodopsin. Such an endothermic reaction makes the visual pigment highly specific towards its stimulus but this very high efficiency to store light energy raises the question of the mechanism of energy uptake. It was first suggested that energy was stored by separation of charges between the protonated Schiff base and its counterion (Rosenfeld et al., 1977; Honig et af., 1979). Later, Birge and Hubbard (1980, 1981) suggested that the apoprotein could contribute to energy storage and proposed a model where 26 kCal mol-I were stored in bathorhodopsin. Finally, Warshel and Barboy (1982) predicted that bathorhodopsin could store as much as 42 kCal mol-I, a large fraction of this energy being stocked as strain energy in the protein cavity. Beside these theoretical considerations, it was recently shown that as few as 16 kCal mol-' were stored in the primary

*To whom correspondence should be addressed. tAbbreviations: PA, photoacoustic; ROS, rod outer

segments; Hepes, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic.

process of the related pigment bacteriorhodopsin which shares many features of primary photochemistry with rhodopsin (Birge and Cooper, 1983). In view of these different theoretical and experimental facts, we felt that energy uptake by visual pigment was worthy of re-evaluation by photoacoustic spectroscopy.

The photoacoustic spectroscopic technique consists in measurement of the fraction of absorbed light that is converted into heat. Among other applications to living organisms, it has been used to study energy transfer and energy uptake by a variety of photosynthetic systems (Cahen et al., 1978; Malkin and Cahen, 1979; Garty et al., 1982; Carpentier et al., 1983a and b; Boucher et al., 1983; O'Hara et a!., 1983). We already have shown that photoacoustic spectra of rhodopsin could be obtained at low temperature (Boucher and Leblanc, 1981a,b). In this report, we present the energy dissipation spectrum at 77 K for rhodopsin in the rod outer segment (ROS)$ membranes. The results confirm the storage of 145 kJ mol-' (35 kcal) in the primary photoreaction of the visual pigment obtained by Cooper (1979a). In addition, the monitoring of the process over a large wavelength range shows that the energy storage efficiency varies by less than 10% over the wavelength range where rhodopsin absorbs: at 350 nm, the quantum yield for the formation of bathorhodopsin is at least 0.6.

MATERIALS AND METHODS

All manipulations involving visual pigments were carried out in a dark room under dim red light (A > 650 nm), unless otherwise stated.

Preparation of rod outer segments. ROS were prepared from cattle eyes obtained from a local abattoir. Photo- receptor membranes were broken off the retinae by vortexing them for 10 s in 10 mM Hepes buffer (pH 7.5) containing 0.15 mM CaCI2, 0.1 mM EDTA and 1% (wtlvol) NaCl (0.5 mthetina). The slushy suspension was

459

460 FRAN~OIS BOUCHER and ROGER M. LEBLANC

diluted two times with 80% (wt/vol) sucrose solution, poured into 50 me centrifuge tubes, overlaid with approxi- mately 2 n i l of Hepes buffer and spun for 45 min at 12000 RPM in a Sorval HB-4 swinging bucket rotor. After centrifugation. the ROS membranes floating on the top of the 40% (wthol) sucrose solution were collected with a pipette, diluted 3 times with buffer and washed by centrifugation at 15000 RPM for 30 min in a Sorval SS-34 fixed angle rotor. Usually this flotation/sedimentation procedure was repeated twice, after which ROS were further purified by sedimentation through a density gradient. ROS suspended in 10 mM Hepes buffer were layered above four 26-36% (wtivol) continuous sucrose concentration gradients which were spun overnight at 22000 RPM in an IEC SB-110 swinging bucket rotor. This method yields principally one band of ROS in the gradient at a dcnsity of 1.14 g/mU. The band was collected and washed twice by centrifugation in buffer and finally in pure water. Rod outer segments prepared in that way had a 280 to 500 nm absorbance ratio of 1.9, when solubilized in 3% Amonyx LO (Onyx Chemical Co., NJ) and contained not more than 65 phospholipids per rhodopsin molecule, as assayed by phosphorus analysis.

Spectroscopic rechniques. Absorption spectra were measured with a Cary 17D or a Unicam SP8-100 spectrophotometer, the latter being equipped with a diffuse sample holder which permits recording spectra of ROS without any distortion originating from scattered light. In the Cary spectrophotometer, ROS spectra were measured by using the opal glass technique.

Low temperature spectra were obtained by using a nitrogen cryostat (Oxford Cryomagnetic Systems, Oxford, England). The sample holder has been modified to accommodate glass microscope slides.

Photoacoustic spectra were recorded using a single beam photoacoustic spectrometer built in our laboratory. The spectrometer was equipped with a gas-microphone low temperature resonant cell described elsewhere (Boucher and Leblanc, 1981a) whereas the other components were standard equipment. The light source was a loo0 W Xe lamp (Schoeffel Instrum. Corp.. Westwood, New Jersey) focused on the entrance slit of a monochromator (500 nm blaze, Schoeffel Instrum. Corp.) driven by a variable speed DC motor. The monochromatic light was modulated by a mechanical chopper at a frequency that could be varied between 20 and 1000 Hz. The monochromator output was reflected and focused onto the cell. The output signal was amplified and fed into a lock-in amplifier (Ithaco Dynatrac model 393, Aptec, Montreal) receiving a reference signal from the chopper. The lock-in output vs wavelength was stored in a mini-computer (Challenger 11, Ohio Scientific. Aurora. OH) and normalized for variations in incident light intensity by dividing the PA signal of the sample at each wavelength by the previously stored PA signal of a totally absorbing material (carbon black). Since biological samples are always characterized by strong UV absorption and a weaker absorption in the visible. a computer controlled cut-off filter (50% T at 496 nm) was introduced in the light path at wavelengths longer than 500 nm in order to reduce background signal due to light of second harmonic frequencies. Between 350 and 650 nm, the average light intensity at the sample position was 30 W m-?.

Measurement of energy dissipation by photoacoustic spectroscopy. Measurement of energy dissipation in photochemically active systems by photoacoustic spectroscopy was first proposed by Malkin and Cahen (1979). According to these authors, in a nonfluorescent photochemically active sample, the photoacoustic signal (p) can be quantified by the following energy balance equation

[ Nhc p = k a 1 -

where k is an instrumental constant and a, the fraction of light absorbed by the molecules located within the thermally active layer. +i and AE, represent, respectively, the quantum yield and the internal energy change of a light driven process while N, h, c and A correspond to Avogadro's number, Planck's constant, speed of light and wavelength, respectively. For a given sample, if a can be measured, the dissipation spectrum will be given by the expression

r SbiAEiA 7

1 i ' Nhc - 1 p l a = k 1 -

For a single process having constant values for I$ and AE, p / a shows a linear dependence on A, and once extrapolated to p la = 0 equation (2) reduces to

4 AE = Nhch (3)

Application of this method to the primary photochemistry of rhodopsin not only requires the precise knowledge of a but also necessitates that the photoproducts of rhodopsin do not contribute to the generation of p. These two problems have been circumvented as follows:

(i) Determination of a. As already mentioned, a corresponds to the fraction of light absorbed by the molecules located within the thermally active layer. One can easily conceive the importance of this value by considering that the photoacoustic signal generated by molecules located deeply in the sample may be damped before reaching the surface and thus may not contribute to the generation of p. However this problem can be completely circumvented if the sample depth is smaller than the thermally active layer. This thermally active layer (p,) is defined by

p, = (a'/aw)' (4)

where a' is the thermal diffusivity of the sample and w, the modulation frequency. Among a variety of biological samples whose thermal diffusivity is known, the values range from 0.8 to 1.38 X cm' s-' (Touloukian et al., 1973). The thermal diffusivity of ROS should be within this range. According to equation (4) this is to say that depths ranging from 40 to 8 pm are probed between 20 and 500 Hz. For the purpose of our experiment, we have prepared very thin samples of ROS (see sample preparation) and experimentally checked that they were thinner than (see results section) so that a can simply be obtained by measuring the transmission spectrum of the sample.

(ii) Generation of photoacoustic signal by photoproducts. After several repeated scans of PA spectra of rhodopsin samples at low temperature, the maximum PA intensity gradually shifts towards higher wavelengths due to accumulation of bathorhodopsin (Boucher and Leblanc, 1981b). To prevent this accumulation, we took advantage of the photoreversibility of the primary reactions of rhodopsin to remove reaction products as they were formed. At 77 K, a wavelength and intensity dependent photoequilibrium exists between rhodopsin, bathorhodopsin and isorhodopsin. It is convenient for the purpose of this work to describe the photoequilibrium by the simpler expression proposed by Suzuki and Callender (1981):

t$ = 0.67

t$ = 0.5

.$I = 0.054

t$ = 0.1 Rhodopsin Bathorhodopsin lrorhodopsin

A,,, = 505 nm Am#, = 4XI)nm hmar = 543 nm

In order to remove batho- and isorhodopsin as they were formed, an actinic continuous light beam was superimposed on the modulated monochromatic beam. It consisted of light from a 250 W tungsten-halogen lamp (Oriel Corp.,

Photoacoustic study of rhodopsin 46 1

Stamford, Conn.) filtered through an aqueous solution of eosin. Eosin was chosen because it mimics absorption by rhodopsin. and thus permits photolysis of any reaction product having absorption characteristics different from rhodopsin. The transmittance of the eosin filter at 450,500, 550 and 570 nm are 12%T, 2%T, 33%T and 80%T, respectively. The light intensity at the sample position was around 150 W m-2. Since the actinic light is not modulated. it does not induce PA signals. I n the presence of that light, several PA spectra could be run over a period of >S h without changes in the spectra. Maximum PA intensity remained at 505 nm (the maximum absorption by rhodopsin at 77 K), indicating that little (if any) bathorhodopsin accumulated during the measurements.

Sample preparation. In order to get layers of ROS as uniform as possible and suitable for measurements of photoacoustic spectra, samples were prepared by centrifugation. The bottom of 50 me centrifuge tubes was filled with a Plexiglass adaptor that fitted exactly circular microscope glass slides (10 mm diameter x 1 mm thick). Tubes were filled with pure water, a carefully cleaned glass slide was introduced in each adaptor and a 0.2 to 2 rnt ROS sample was added. Tubes were spun for 45 min at 12000 RPM in a Sorval HB-4 swinging bucket rotor. After centrifugation, water was carefully removed and the glass slide supporting the pelleted ROS was placed in a dessicator at 3°C for 12 h. For each ROS slide, transmission spectra were measured at 298 and 77 K before measurement of PA spectra. The transmission spectra were not affected by the presence or the absence of a blank microscope slide in the reference beam. The glass slides used absorbed less than 3% of incident light at 350 nm. In the experimental determination of the thermal properties of ROS. the circular glass slides were coloured in green, prior to deposition of ROS. with a large felt tip commercial marker. Following the sedimentation of ROS, the slides were dried as previously described and then exposed to diffuse day light for 1 h. Absorbance measurement of these slides showed a maximum at 480 nm (metharhodopsin I) in addition to the absorbance of the green ink. Longer light exposure did not show any further advance in the photolysis of rhodopsin. indicating that samples were sufficiently dehydrated to block the photolytic sequence at the metarhodopsin I stage (Yoshizawa, 1972).

RESULTS

Determination of the thermal transfer properties of ROS

Measurement of energy dissipation in a biological sample by photoacoustic spectroscopy necessitates that an exact knowledge of the absorption characteristics of the molecules which efficiently contribute to the generation of the photoacoustic signal. For such achievement, we have tried to obtain photoacoustic spectra from samples sufficiently thin that all the depth of the sample contributes to PA signal generation. To determine if such thin layers could be prepared with ROS, they were deposited on microscope slides previously coated with water insoluble green ink. This arrangement is sketched in Fig. 1, and the transmission spectrum of such a sample is given in Fig. 2. The green undercoat has minimum transmittance at 680 nm, a wavelength at which ROS (in the metarhodopsin I state, A,,, = 480 nm) do not absorb light. Thus, it is possible to determine if the entire depth of the ROS layer is part of the

ROS LAYER

=EN INK LPVER

WROSWPE SLKE

t Figure 1. Schematics of the arrangement used for the determination of the thermally active layer in ROS samples. Glass slides are coloured with green ink, on which variable amounts of ROS are deposited by sedimentation. Straight arrows indicate light absorption at different depths and wavy arrows represent heat diffusion from different depths.

430 bso

WNELENGTH M I 1

1

Figure 2. Transmission spectrum of glass slides coloured with green ink, before (-) and after (-----) deposition of ROS. The ROS spectrum (.-.) is obtained by subtraction of (-) from (-----). Once deposited on the slides, ROS are dried and kept at room temperature under diffuse day light so that the visual pigment is kept in the metarhodopsin I

stage (A,,, = 480 nm).

“thermally active layer” by analysis of the PA signals of ROS undercoated with green ink at 480 and 680 nm. In samples having a sufficient depth, PA signals emerging from molecules superficially and deeply located should be out of phase. As an example, Fig. 3 shows the phase pattern of the PA signals recorded at 480 and 680 nm for ROS undercoated with green ink. In that case, ROS absorbed 68% of the incident light at 480 nm and the modulation frequency was 400 Hz. A phase lag of 29 degrees exists between the ROS signal (480 nm) and the green ink signal (680 nm) indicating that if the phase is adjusted for maximum PA intensity at 480 nm, the green undercoat will be only partially detected. In Table 1, the phase lag between ROS and the green undercoat is given for samples of different transmissions (depth). It appears that for ROS samples absorbing less than

462 FRAN~OIS BOUCHER and ROGER M. LEBLANC

0-1 I

150 200 250 300

PHPSE DEQRIESI

Figure 3. Acoustic signal intensity against phase at 480 nm (4,) and 680 nm (+?) corresponding respectively to the ROS layer and the green ink layer. Modulation frequency: 400 Hz. Temperature: 295 K. Absorption by ROS layer:

68% of incident light at 480 nm.

Fable 1. Phase lag between the top pigment layer and the green undercoat layer

1981a) but with samples absorbing less than 25% of the incident light. All those samples were thus thinner than their thermally active layer and their transmission spectra could then be taken as a.

Measurement of energy dissipation by rhodopsin at 77 K

When rhodopsin is cooled down to 77 K, its absorption characteristics are slightly modified. The changes in the absorption spectrum of ROS deposited on microscope slide is shown in Fig. 4. Upon cooling down to 77 K, absorption increases in the 350-550 nm region, the absorbance maximum shifts from 498 nm to 505 nm and the edge of the main absorption band is sharpened. This effect is essentially the same than that described for solubilized rhodopsin. However, unlike solubilized rhodopsin, the final form of the low temperature

Fraction of light absorbed by the pigment layer at

Phase lag (degrees)

480 nrn* (%) 50Hz 100Hz 400Hz

13 0 0 0 17 0 0 0 33 8 11 19 68 15 19 29 76 50 66 t

*Green undercoat absorbs 4676% of the incident light at 680 nm. Signal of undercoat is not measurable at this frequency.

one third of the incident 480 nm light, the phase lag between the ROS layer and the green ink layer is negligible, but as absorption by ROS (depth of ROS layer) increases, the phase lag becomes larger. In extreme cases (low transmission by ROS and high modulation frequency), the PA signal from the undercoat is not only out of phase but sometimes completely attenuated, indicating that the green ink layer is completely out of the thermally active layer (pJ. In such a case, the deeper part of the ROS layer should be considered also to be out of the thermally active layer.

From Fig. 3, it can be seen that the most deeply located molecules (the green ink layer) do generate a PA signal with a phase lag of 30 degrees, which will be attenuated by about 10% if phase is adjusted to measure the superficially located molecules (ROS). It can also be seen that a two times smaller phase lag would induce a negligible attenuation. We thus choose to measure energy dissipation by using samples absorbing less than 50% of the incident energy and at a frequency of usually 100 Hz. In some experiments, to improve the signal to noise ratio, PA spectra were measured at 325 Hz, the resonance frequency of the PA cell (Boucher and Leblanc,

WAVELENQTH I P m )

Figure 4. Transmission spectra of ROS deposited by centrifugation on glass slides. Spectra are recorded against an air blank at room temperature (-) and 77 K (--).

WIVELCNBTM In- 1

Figure 5. Photoacoustic (p--) and dissipation (p/a b) spectra of ROS at 77 K. Modulation frequency: 325 Hz. Absorption by ROS: 22% of incident light at 505 nm. Maximum PA intensity: 505 nm. Error bars indicate the uncertainty in the calculation of p/a. PA spectrum was measured in the presence of the superimposed non-

modulated actinic light.

Photoacoustic study of rhodopsin 463

spectrum of the dry ROS never appeared to depend on the speed at which samples were cooled (Yoshizawa, 1972). These spectra also show that rhodopsin is not dehydrated during drying of ROS on microscope slides. Indeed, extensive dehydration shifts rhodopsin absorption to 390 nm (Rafferty and Shichi, 1981). Since absorption characteristics of dry ROS slides are not different from those of solubilized ROS, we assume that their photochemical behavior also do not differ from that in solution.

Figure 5 shows the photoacoustic and dissipation spectra of dry ROS. Measurements on ROS discal membranes, prepared according to Smith and Litman (1982), give the same results. The PA spectrum has maximum intensity at 505 nm in the presence of the superimposed red actinic light. It corresponds to the same maximum as found in the transmission spectrum. If significant amounts of bathorhodopsin were present, the maximum PA intensity should be red shifted compared to the transmission spectrum, an effect that will be seen later. We thus assume that the actual PA spectrum corresponds to the PA spectrum of pure rhodopsin. In the wavelength range of maximal absorption by rhodopsin (i.e. 450-550 nm), the relative dissipation spectrum ( p / a ) behaves like predicted by Eq. (2). The spectrum linearly depends on wavelength, suggesting a constant value of b, AE (see Materials and Methods) for this wavelength range. This part of the dissipation spectrum can be used to calculate the amount of energy absorbed in the a band of rhodopsin that is not converted into heat. From the slope of the 450-550 nm region of the dissipation spectrum of Fig. 5, the X-axis intercept was found equal to 1207 nm. Once this value of A introduced into Eq. 3 together with a quantum yield of 0.67 for the formation of bathorhodopsin from rhodopsin (Dartndll, 1972; Hurley el a!. ,1977), a value of 148 kJ mol-' is found for AE. Since this result strongly depends on the precision of the slope of the dissipation spectrum, we performed several deter- minations on five different batches of ROS and an average value of 1229 nm k 62 (k 5%) was found for A, corresponding to a AE of 145 kJ mol-' (34.7 kcal) for the reaction. This is to say that as much as 60% of a 500 nm photon energy is stored in the formation of bathorhodopsin from rhodopsin. This value is precisely the same than that obtained by Cooper (1979a) from photocalorimetric measurements.

Figure 5 also shows that at wavelengths shorter than 450 nm, p / a progressively deviates from a typical single process energy dissipation spectrum. The deviation from linearity goes in the sense of a lower value of 4 AE at shorter wavelengths. The phenomenon was observed in all rhodopsin samples studied and it will be discussed later.

During the scan of PA spectra at low temperature, if care is not taken to avoid accumulation of bathorhodopsin (and/or possibly other inter-

mediates), the maximum PA intensity gradually shifts towards higher wavelengths (Boucher and Leblanc, 1981b). Figure 6 shows a PA spectrum scanned in the absence of the superimposed actinic beam. In that case, maximum intensity is at 510 nm, indicating that significant amounts of bathorhodopsin has accumulated. Due to the photoreversibility of the reaction, pure bathorhodopsin cannot be obtained in that way. After a few scans, maximum intensity stabilized at 515 nm. It was not possible to observe more red shifted maxima with our experimental set-up. The dissipation spectrum obtained from those PA measurements is also shown in Fig. 6. Its major features are a large peak in the 550 nm region and the absence of slope changes below 450 nm. We explain both as follows: We must first point out that this dissipation spectrum is obtained by dividing the PA spectrum (p) of a rhodopsin- bathorhodopsin mixture (maximum intensity at 510 nm) by the transmission spectrum (a) of a sample containing only rhodopsin. Thus, absorption differences between rhodopsin and bathorhodopsin must be partly responsible for the appearance of the 550 nm peak. In addition, instead of storing 145 kJ m o l - I , t h e format ion of rhodops in f rom bathorhodopsin should give off an equivalent amount of energy. It is thus likely that the PA signal should be higher in the spectral region of maximum absorption by bathorhodopsin. Bathorhodopsin must also have significant absorption in the 350-450 nm region. The dissipation spectrum should thus correspond to both processes (i.e. energy taken up by rhodopsin and energy given off by bathorhodopsin) in that spectral region too and result in the attenuation of the slope which is effectively observed by comparison with Fig. 5. This effect together with the fact that there are differences between the values of Q

used to calculate p / a and the effective a make difficult to assess a quantitative significance to the dissipation spectrum of Fig. 6. It is noteworthy that

1

WAVELENOTH ( nm 1

Figure 6. Photoacoustic ( F ) and dissipation (p/a &) spectra of ROS containing a mixture of rhodopsin and bathorhodopsin at 77 K. Maximum PA intensity: 510 nm. Modulation frequency: 110 Hz. Absorption by ROS: 27%

of incident light at 505 nm.

464 FRANCOIS BOIJCHER and ROGER M. LEBLANC

we never have obtained experimental results indicative of the formation of isorhodopsin from bathorhodopsin probably because of the lower quantum yield of the reaction.

DISCUSSION

The amount of energy dissipated by excited rhodopsin has been calculated using the photoacoustic and absorption data. Much attention has been paid to the light scattering effect on absorption measurements. This effect is not so important in PA spectroscopy. The ROS slides prepared as previously described are much less opaque than an aqueous suspension of ROS. The method used (diffuse sample holder and opal glass) to measure absorption spectra appears to be adequate since essentially the same result has been obtained for ROS discal membranes which diffuse still less light. Moreover, significant distortion in the absorption spectra should gradually increase the apparent value of a and result in a downward curvature of the dissipation spectra at shorter wavelengths. Such an effect has not been observed.

From the dissipation spectrum of rhodopsin at 77 K (Fig. 5), we have calculated by the slope-intercept method used by Garty et al. (1982) that 145 f 6 kJ mol-' (34.7 kcal) were stored in the primary process of the bleaching sequence of rhodopsin. An identical value has been obtained by Cooper (1979a) from photocalorimetric measurements during irradiation of detergent solubilized and suspended ROS at 450 and 485 nm.

We found that the dissipation spectrum of rhodopsin revealed a constant value of cf~ AE at wavelengths higher than 450 nm. As the quantum yield for the formation of bathorhodopsin from rhodopsin is known to be temperature independent (Hurley et al., 1977), there is no reason why it could be dependent on the physical state of the sample. Thus, the fact that our measurements lead to the same value of AE as that obtained from photocalorimetric measurements, not only confirms the validity of our method but offers the possibility to extend the study of the photochemical process at shorter wavelengths. As a matter of fact, if only one single endothermic process occurs, whatever the irradiation wavelength, we should observe only one straight line in the dissipation spectrum. Below 450 nm, the dissipation spectrum of rhodopsin shows a leveling off which may be explained in the following way. First of all, we must exclude the possibility that this effect could be due to an enhancement of light scattering at low temperature. Despite the fact that the spectra of ROS slides (Fig. 4) show a rather large decrease in transmission in the 350-450 nm region upon cooling, the PA data indicate that this effect is not an artefact caused by the increase in scattered light. If that were the case, the PA spectra, which are not so much distorted by scattering, would not have

comparably high signals in this spectral region and the dissipation spectra would show a downward curvature contrary to what is observed. The possibility that the 350-450 nm leveling could be due to accumulation of isorhodopsin (Amax = 480 nm) can also be ruled out. Formation of bathorhodopsin from isorhodopsin should store about 15% less energy than the rhodopsin bathorhodopsin reaction (Cooper, 1979b) but its quantum yield is only 0.1 (Suzuki and Callender, 1981). Higher values of plct are then expected for that reaction and if isorhodopsin was present in the sample, a leveling effect should effectively be seen in the 450-500 nm region. We never observed such an effect. On the other hand, the leveling effect producing increased value of plct in the 350-450 nm region might be due to contamination of the sample by late bleaching intermediates (metarhodopsin I1 or retinal). Appearance of these intermediates during the time of measurements or sample manipulation is unlikely since ROS are dried and metarhodopsin I (A,,, = 480 nm) is the last bleaching photoproduct. What about the possibility of having these products present before the preparation of the samples? We must mention that in these experiments, ROS have never been regenerated precisely to avoid eventual contamination by residual free retinal. These ROS typically contain 20% regenerable opsin. However, absorption spectra of the solubilized membrane preparations do not show any indication of the presence of equivalent amount of free retinal since even in the presence of hydroxylamine only the $ band of rhodopsin can be detected in the 300-400 nm region. In order to evaluate the concentration of an eventua l non-rhodopsin absorbing species responsible for the observed effect, we have traced the absolute energy dissipation spectrum observed for ROS. This spectrum is shown in Fig. 7. Since rhodopsin has a 67% efficiency for storing 145 kJ mol-' upon excitation, it is possible to trace the absolute dissipation spectrum from the relative dissipation spectrum of Fig. 5. Assuming that an eventual contaminant is photochemically inactive (100% of the absorbed energy is dissipated), we estimate that it should contribute about 10% of the absorption in the 350-450 nm region to produce the observed effect. Reported on the transmission scale of most of the ROS slide samples, this absorption should contribute less than a 1% decrease in transmission between 350 and 450 nm, which is difficult to observe in the transmission or PA spectra. The fact that the 350-450 nm leveling off is absent from the dissipation spectrum of samples containing bathorhodopsin in addition to rhodopsin (Fig. 6) suggests consideration of another explanation, even if we cannot completely rule out the possibility that some low concentration contaminants might be responsible for the leveling observed.

The second way to explain the irregularity in the dissipation spectrum is to argue that the quantum

Photoacoustic study of rhodopsin 465

yield for the formation of bathorhodopsin is smaller at 400 nm than at 500 nm. To our knowledge, measurement of quantum yield for this reaction at 77 K has never been done at wavelengths lower than 458 nm. From the value of A E (145 kJ mol-') and the dissipation spectrum of Fig. 7, it is possible to estimate the drop in quantum yield that could cause the observed increase in p h . At each wavelength, we have calculated 4 by the relation:

Energy dissipated = Energy absorbed (1 - +AE}.

The result is also shown in Fig. 7 (dotted line). A value of + = 0.67 is found over 450 nm. At shorter wavelengths, the apparent quantum yield gradually decreases to stabilize at a value of 0.60 between 350 and 420 nm. The high sensitivity of rhodopsin in that spectral region was reported as early as 1942 by Goodeve and coworkers who found that the spectral sensitivity of frog retina could be matched with the absorption spectrum of extracted visual pigment. Since photochemistry occurs from the first excited singlet state, we think that excitation to the second excited state of the pigment (p band) could easily lead to slightly lower efficiency of photochemistry due to direct deactivation to ground state andlor energy channeling into a triplet state.

I I 110 450 ow O W

WAVELENGTH lnm I

Figure 7. Solid line: Absolute dissipation spectrum of ROS calculated on the basis that 67% of the excited rhodopsin molecules store 115 kJ mol-I. Dotted line: Apparent quantum yield for the storage of 145 kJ mol-' in the

formation of bathorhodopsin (see text for details).

In conclusion, photoacoustic spectroscopy can be used to investigate the energy storage in primary photochemistry of rhodopsin. Our results confirm that the visual pigment stores a very large fraction of the energy absorbed. In addition, energy storage is not limited to excitation in the main absorption band. The efficiency of the process is maintained down to 350 nm. Thus, the poorly defined absorption of ROS

in the violet spectral region is not the result of a wavelength dependent scattering but reflects the real absorption characteristic of the membranes.

Acknowledgements-This work was supported by the Natural Sciences and Engineering Research Council of Canada.

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