Rhodopsin bleaching: Relative effectiveness of high and low intensity flashes

Yislon Rrs.

Vol.

5. pp. 633-638.

RHODOPSIN

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Press 1965.

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BLEACHING:

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RELATIVE

LOW INTENSITY T. P.

EFFECTIVENESS FLASHES’

WILLIAMS

Division of Medical Science, Walter S. Hunter Laboratory R.I. 02912

of Psychology, Brown University, Providence,

(Received 1 July 1965)

INTRODUCTION

PHOTO-ISOMERIZATIONof the chromophore, 114s retinal, to the all-truns isomer triggers the bleaching of rhodopsin-a complicated process involving many transient species (cf., MATTHEWS et al., 1963). If certain of these species absorb a second quantum, the all-rruns chromophore is re-isomerized to the 1I-cis configuration thus regenerating rhodopsin (GRELLMANN et al., 1962; YOSHIZAWA and WALD, 1963). (The production of isorhodopsin is also possible but will not be considered since it was not detected in any of the solutions.) Bleaching thus can be halted by light-a process called photoreversal of bleaching. The rapid interconversion of rruns and cis isomers in this way is the basis for an upper limit on bleaching by short, intense flashes. HAGINS (1955) was the first to report that the upper limit on bleaching was about 50 per cent in his experiments on excised eyes. Later, statistical and kinetic formulations of bleaching were developed to explain this upper limit (WILLIAMS, 1964). These formulations are based on the photoreversal aspect of bleaching.

FIG. 1. Simplified bleaching scheme showing photoreversal. Wavy arrows are photochemical steps, straight arrows are thermal steps. Starting with rhodopsin. R, note that an odd number of quanta absorbed results in unstable P, I_.. or M species which decay to PRODUCTS. Even numbers of quanta absorbed result in regeneration of rhodopsin.

The statistical nature of photoreversal is, perhaps, the easiest to visualize and will be used in this report. Therefore, some space will be given to it now. First, consider the mechanism in Fig. 1. R is rhodopsin, P is pre-lumirhodopsin, L is lumirhodopsin and M is metarhodopsin. PRODUCTS refers to all species which result from the decay of M. Wavy lines indicate photochemical steps, i.e. steps which occur upon the absorption of a quantum. Straight lines are thermal processes. Absorption of a quantum by R starts the bleaching process but, if P, L or M absorb a second quantum during the flash, bleaching is reversed. 1 This work was supported by the U.S. Public Health Service Grant, NBO5256. 633

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On the other hand, if P, L or M do not absorb the second quantum before the flash is over, Hereafter, we define they are bound to bleach because they have the tram chromophore. “bleaching” as the formation of PRODUCTS. That is, a molecule must pass completely through the P, L and M stages and form PRODUCTS before it is considered bleached. Thus, if the flash duration is short enough (as in the experiments to be reported here), no PRODUCTS are formed during the flash; hence, no bleaching occurs during the flash. However, the number of molecules which do bleach afterwards is in fact those molecules which end up in the P, L or M stages when the flash is over. The number of such labile species can be formulated statistically. A brief description of this follows. (For a more detailed discussion compare WILLIAMS, 1964.) If a rhodopsin molecule absorbs one quantum it produces pre-lumirhodopsin and the bleaching process is started. However, if either P, L or M absorbs a second quantum, the system is stabilized and the molecule will not bleach. Once stabilized, however, the molecule is free to absorb a third quantum and

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FIG. 2. Statistical calculation of odd and even numbers of quanta absorbed. Notice that the number of odd equals the number of even absorptions at high intensities. This gives rise to the 50 per cent upper limit on bleaching. On the other hand, at low intensities, the odd curve predominates and all absorptions lead to bleaching.

start the process all over again. Thus, a molecule which absorbs an odd number of quanta during a flash must bleach while one which absorbs an even number does not. The fraction of molecules absorbing even or odd numbers of quanta is assumed to be given by the Poisson distribution, Figure 2 shows curves for the number of odd and number of even absorptions as a function of the logarithm of the flash intensity. One set of measurements of bleaching is tied to each curve and the odd-curve fits the data nicety but the even curve does not. Thus, it seems that the only molecules which bleach are those which absorb an odd number of quanta during a short fiash. The important aspects of these curves for the present work are their limits. Notice, for example, that when the intensity is high enough, the number of odd equals the number of even absorptions and the 50 per cent limit on bleaching obtains. The important point is that when the intensity is very high the energy of the flash is used very inefficiently for bleachinghalf of the absorbed quanta lead not to bleached rhodopsin but to photoreversed rhodopsin. Indeed, the quantum efficiency, y, must approach zero as the intensity of the flash approaches infinity. On the other hand, when the intensity of the flash is low, say it bleaches less than about 15 per cent of the rhodopsin, the odd-curve is dominant-almost no even absorptions occur. This is because, at low intensities, the probability is very low that any one molecule will

Rhodopsin

Bleaching: Relative Effectiveness of High and Low Intensity Flashes

635

absorb more than one quantum. One is an odd number and hence virtually all the molecules, which absorb, bleach. This is an efficient (for bleaching) use of the absorbed quanta. In fact, the quantum efficiency approaches unity as the intensity (or fraction bleached) approaches zero. (The relationship between y and fraction bleached will be discussed later.) The purpose of this article is to present data on the relative bleaching effectiveness of high and low intensity flashes. (The definition of bleaching efictiveness will become clear later, but note at this time that it is not synonymous with quantum eficiency.) It will be shown, for example, that a single, very high intensity flash bleaches less rhodopsin than two flashes at one-half that intensity, i.e. the fraction bleached does not respond simply to the total energy input when the intensity is very high. However, when the intensity is low enough so that double (in general, multiple) absorptions are precluded, the extent of bleaching does indeed respond only to the total energy input. METHODS

AND

MATERIALS

Rhodopsin solutions were digitonin extracts of frog (Rana pipiens) rod particles. The solutions were maintained atpH 65 with M/15 phosphate buffer. Small volumes of NHeOH (1 M) were added to the extracts to make its final concentration O-01 M. NHeOH removes photoproducts and permits meaningful measurements of the extent of bleaching. The apparatus is described in detail elsewhere (WILLIAMS, 1964). Briefly, it involves a weak monitor source of monochromatic radiation (500 rnp) whose intensity is modulated by the rhodopsin concentration of a sample. The change of intensity reaching a photomultiplier tube upon bleaching (partial or complete) is related to the extent of bleaching by the Beer-Lambert law. The bleaching source is a strobe light whose duration is short enough (90 per cent of the light is dissipated in 800 psec) so that no bleaching occurs during the flash. The experiments were done as follows: A sample, 0.2 ml, was put into a cuvette and thence into a constant temperature (25°C) aluminum holder. The optical density was measured against digitonin. The appropriate intensity flash was given, say a maximum intensity one. The density change due to bleaching was determined. Additional maximum flashes were given 5 min apart, and the density change measured after each. Finally, the sample was bleached completely. In order to compare the fraction bleached by one such maximum intensity flash with, in general, n flashes at l/n the intensity, the appropriate filter was imposed between strobe and sample, and n flashes were given and counted by a solidstate metronome device. No attempt was made to monitor the output of the strobe in this multiple flash situation. Previous experience with this instrument has shown that, so long as the flashes are not given too close together (within 100 msec), the flash intensity varies only slightly and, apparently, randomly with a spread of about 10 per cent. RESULTS

Figure 3 shows the fraction of rhodopsin bleached as a function of the total energy input. Energy input is defined as the intensity per flash, If, times the number of flashes, n. The highest intensity is arbitrarily set at 1.0. Then, for example, nlf=l+O means one flash at intensity l-0, two flashes at intensity 0.5, etc. Notice that a single flash at If= 1-Obleaches 42 per cent of the rhodopsin but that two flashes at 0.5 bleach 53 per cent. Furthermore, four flashes at O-25 bleach 61 per cent. In general, there is substantially more bleaching at any given energy input as one decreases 11 from 1*Oto O-25. This is as predicted in the

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WILLIAMS

introduction. It is due to the fact that much more photoreversal of bleaching occurs with the higher intensity flashes. In fact when 42 per cent bleaching occurs it means that 42 per cent of the molecules have absorbed an odd number of quanta. Figure 2 shows that, at this time, 18 per cent of the molecules have absorbed an even number of quanta and have been photoreversed. This is a very inefficient (for bleaching) use of the input. 1.0

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BI~chjng with short flashes of various intensities. Energy input is defined as the number of flashes times the intensity per flash. The highest flash intensity is assigned unit value. Note that adecrease in flash intensity from 1.0 to @25 results in more rhodopsin bleached at any given value of energy input. For fl&h intensities of 0.25 and lower, the fraciion bleached depends only on the total energy input. FIG. 3.

On the other hand, Fig 3 shows that decreasing 1, from 0.25 to 0.062 has little or no effect on extent of bleaching for any given value of ~1,. Only one line has been drawn through these data since they are all so similar. This result is in accord with the idea that, when the intensity of a flash is low enough, few even absorptions occur during a flash and virtually all the molecules, which absorb at all, do in fact bleach. (Naturally, this is because they absorb one quantum each.) This is an efficient use of the input for bleaching. Note that one flash at 0.25 bleaches 21 per cent of the rhodopsin. Again, referring to Fig. 2, we see that 3 per cent of the molecules in the solution have absorbed an even number of quanta and are photoreversed. This small amount is apparently not detectable here and hence decreasing the intensity per flash further shows no further progression of the fraction bleached to higher values for a given nlz. DISCUSSION

The previous results show that more bleaching occurs if a large amount of light energy is administered in the form of many weak flashes rather than one (or a small number) of very intense ones. In a sense, this has been an experiment on the degree to which the rhodopsin system obeys the Bunsen-Roscoe law. Usually, one varies intensity and time of some steady

Rhodopsin

Bleaching: Relative Effectiveness of High and Low Intensity Flashes

637

source in such experiments. Here, however, an electronic strobe source was used whose duration was constant throughout the experiment. The only way to vary exposure time was to give different numbers of flashes. In this way the exposure time is a product of the flash duration and number of flashes. This is an unconventional yet, ostensibly, reasonable way to define the time parameter of the Bunsen-Roscoe constant. If the results are viewed in this way, then, the conclusion is that the law is obeyed only at low flash intensities. In the present results, a low intensity flash is, operationally speaking, one which bleaches about 20 per cent or less of the available rhodopsin. If a flash is able to bleach much more than this, the number of even absorptions rises rapidly and the Bunsen-Roscoe law is severely disobeyed. Strictly speaking, the 20 per cent figure depends on the accuracy of the measurements of bleaching. If 3 per cent photoreversal could be detected reliably in these experiments, the 20 per cent figure should be lowered. In fact, there is always a finite probability for a double absorption-even at the low intensity which corresponds to an input of only 2 quanta. Hence, theoretically, the rhodopsin system obeys the Bunsen-Roscoe law only in the limit as the intensity approaches zero. While the data of this paper have been concerned with relative effectiveness of bleaching, it seems appropriate to discuss, in a theoretical way, the closely related subject, quunturn number of molecules bleached eficiency, y. We shall define y in the usual way as, y= number of quanta absorbed ’ The results presented here have been explicable in terms of the statistical formulation of odd-even absorptions. It has been clear that the higher the intensity per flash, the higher is the number of even, non-bleaching, absorptions. Nevertheless, these even numbers of quanta are in fact absorbed and must be counted when computing y. In fact, all multiple absorptions, both odd and even, must be summed to get the total number of quanta absorbed. This is conveniently done on a fraction basis using tables of individual Poisson terms. For example, when the average number of quanta absorbed per molecule is 0.5, the Poisson function shows that 30.33 per cent have absorbed 1 quantum, 7.58 per cent absorbed 2 quanta, 1.26 per cent absorbed 3,0*16 per cent absorbed 4 and 0.02 per cent absorbed 5. Summing all the odd per cents shows that 31.61 out of every 100 molecules do bleach. However, summing all the quanta absorbed (1 x30.33+2 x7*58+etc.) shows that it takes 49.88 quanta to bleach these 31.61 molecules. Hence, y will be less than unity. In fact 31.61 1’=m=O*632. When y was calculated in this way and plotted against the fraction

FIG. 4. Calculated values of the quantum efficiency, y. for different values of the fraction bleached with short flashes. Note that 17approaches zero and unity as the fraction bleached approaches 0.5 and zero. respectively. RR

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bleached, the curve in Fig. 4 obtained. The quantum efficiency is essentially linear with fraction bleached for low values of the latter. This function reflects statements made earlier: y approaches unity and zero as the intensity of a short flash approaches zero and infinity, respectively. REFERENCES GRELLMANN, K. H., LIVINGSTON, R. and PRATT, D. (1962). A flash-photolytic investigation of rhodopsin at low temperatures. Nature, Lund. 193, 1258. HAGINS, W. A. (1955). The quantum efficiency of bleaching in situ. J. Physiol., Lond. 129, 22. MATTHEWS, R., HUBBARD, R., BROWN, P. K. and WALD. G. (1963). Tautomeric forms of metarhodopsin.

J. gen. Physiol. 47, 215. WILLIAMS, T. P. (1964). Photoreversal of rhodopsin bleaching. J. gem Physiol. 47, 679-689. YOSHIZAWA, T. and WALD, G. (1963). Prelumirhodopsin and the bleaching of visual pigments. Land. 197, 1279.

Natrr~e,

Abstract-The extent of rhodopsin bleaching by high and low intensity flashes is investigated. A statistical view of bleaching by short flashes leads to the prediction that large amounts of light energy are more effective at bleaching if administered in the form of many weak flashes rather than one intense one. This prediction, based on the photoreversal of bleaching, is verified. A brief discussion of Bunsen-Roscoe law considerations is presented and finally, a theoretical calculation of the dependence of quantum efficiency on the fraction bleached is given. R&sum&On etudie le degre de d&coloration de la rhodopsine par des eclairs lumineux de forte et faible intensit6. La statistique permet de ptivoir qu’une forte energie lumineuse est plus efficace pour la d&coloration si on l’applique sous forme de nombreux &lairs faibles qu’un seul intense. Cette prkdiction, fondee sur la reversibilite photochimique de la d&coloration, est verifide par l’exp&rience. On presente une breve discussion de la loi de Bunsen-Roscoe et pour terminer on calcule theoriquement la variation de l’efficacite quantique avec la fraction d&coloree. Zusnmmenfaasung--Der Grad der Ausbleichung von Rhodopsin durch Blitze hoher und niedriger Intensitat wird untersucht. Eine statistiche Betrachtung der Ausbleichung durch kurze Blitze fiihrt zu der Vorhersage, dass grosse Lichtenergiemengen bei der Ausbleichung wirkungsvoller sind, wenn sie in der Form vieler schwacher Blitze gegeben werden als in einem einzigen starken Blitz. Diese Vorhersage, die auf der Photoumkehr der Ausbleichung basiert, wird bestitigt. Oberlegungen zum Bunsen-Roescoe’schen Gesetz werden diskutiert und endlich eine theoretische Rerechnung der Abhangigkeit der Quantenausbeute vom Grad der Ausbleichung angegeben. Pa3mMe-&‘iCCJIeAOBanaCb CTeneHb BbIUBeTaHUa pOAOnCAHa IlpH BbICOKOH H HU3KOfi WHTCHCWBHOCTA CBCTOBblX BCnblLtICK. CTaTUCTH’IeCKUi? aHaJIH3 BblUBeTaHUR IIpH KOpOTKWX BCnbIIIIKaX AaJI OCHOBaHHe AAII IIpeACKa3aHIUI 0 TOM, YTO 6onbmw KOAHYCCTBO CHCTOBO~~sHeprIm ~@$~KTHBH~~ AAR npouecca BbIUneTaHuX, ecm 0~0 noAaeTcfl B $opMe Muornx cna6bIx BcnbuueK, 9eM ri TOM cnyyae, ecmi Aaerca B cneAe 0AH0H HHTeHCUBHOfiBCIIbIIUKH. 3TO IlpeACKa3aHUe IlOATBep~AeHO Ha OCHOBaHHHWSyYeHHR @OTOO6paTUMOCTH BbIUBeTaHEIl. B 3aKnIOYeHUe IIprZAnaraeTCSI KpaTKOe 06CyXAeHUe 3aKOHa RyH3eHa-POCK0 Ii, HaKOHeU, AaeTCR TeOfJeTHYCCKHZipaCYeT 3aBUCHMOCTU KBaHTOB0i-i 3&#ieKTHBHOCTM OT TO# ~paKUUU, KOTOpaR 06ecUBeYeHa.