QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

THE INVOLVEMENT OF WATER AT THE RETINAL BINDING SITE IN RHODOPSIN AND EARLY LIGHT-INDUCED INTRAMOLECULAR PROTON TRANSFER

Abstract Extensive dehydration of air-dried films of bovine rod outer segment membranes induces fully reversible changes in the absorption spectrum of rhodopsin, indicative of deprotonation of the retinylidene Schiff base in more than 50% of the rhodopsin molecules in the sample. This suggests that water is involved at the site of the Schiff base protonation in rhodopsin. In contrast, the spectrum of metarhodopsin I is resistant to similar dehydrating conditions, implying a significant difference in the mechanism for protonation in metarhodopsin I. The photochemistry of dehydrated membranes was also explored. Photoexcitation of deprotonated rhodopsin (λ_max 390 nm) induces a large bathochromic shift of the chromophore. The major photoproduct at room temperature was spectrally similar to metarhodopsin I (λ_max, 478 nm). These findings suggest that intramolecular proton transfer involving the Schiff base proton may occur in the earlier stages of the visual cycle, prior to or during the formation of metarhodopsin I.     

ABSORPTION SPECTRA OF TCA-DENATURED RHODOPSIN AND OF A SCHIFF BASE COMPOUND OF RETINAL

Abstract— When TCA-denatured rhodopsin was frozen in liquid nitrogen, Λ_max was markedly shifted to longer wavelengths as the concentration of TCA increased. After TCA denaturation, species specific absorption disappeared and the absorption maxima of the squid pigments became identical with those of corresponding pigments of octopus.
In solutions at 5° the bathochromic shift of Λ_max of TCA denatured rhodopsin was observed at higher concentrations of TCA than in the frozen state. Λ_max of N-retinylidene-butylamine (NRB) was also displaced towards longer wavelengths with increasing concentrations of TCA. This bathochromic shift was enhanced by freezing. The mode of the bathochromic shift of Λ_max provoked by TCA was very similar both in the cases of denatured rhodopsin and of NRB. The absorption spectrum of NRB was identical in shape with that of TCA-denatured rhodopsin, as the half-band widths of both materials were about 5500 cm^-1 in the liquid state and 5000 cm^-1 in the frozen state. Λ_max of retinal and NRB were red shifted in polar and polarizable solvents.
It was concluded that the strong acidity and the relatively large polarizability of TCA are responsible for the bathochromic shift of Λ_max of the Schiff base in TCA-denatured rhodopsin.     

FLUORESCENCE OF HOUSEFLY VISUAL PIGMENT

Abstract —The fluorescence of housefly photoreceptors was studied in vivo by using the deep pseudopupil technique. Whereas the rhodopsin R490 of the peripheral retinular cells fluoresces negligibly the metarhodopsin M580 fluoresces distinctly in the red. The newly discovered metarhodopsin M'is produced by intense blue light and can be reconverted into rhodopsin by intense long wavelength light. M'also fluoresces in the red; its excitation spectrum and emission spectrum peak at _max= 570 and 660 nm respectively.
Intense ultraviolet light irreversibly reduces the visual pigment fluorescence as well as the broad band autofluorescence (kmnx 470 nm) originating from non-visual pigments in the fly's eye.     

THE FORMATION OF TWO FORMS OF BATHORHODOPSIN AND THEIR OPTICAL PROPERTIES

Abstract— Using two kinds of rhodopsin preparations (digitonin extract and rod outer segments suspension), we measured changes in absorption spectra during the conversion of rhodopsin or isorhodopsin to a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (437 nm) at 77 K and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (> 650 nm) at 77 K. The reaction kinetics could be expressed with only one exponential in the former case and with two exponentials in the latter case. These data suggest that both rhodopsin and isorhodopsin are composed of a single molecular species, while bathorhodopsin is composed of two molecular species, designated as bathorhodopsin₁ and bathorhodopsin₂. The absorption spectra of these bathorhodopsin were calculated by two different methods (kinetic method and warming-cooling method). The former was based on the kinetics of the conversion of two forms of bathorhodopsin by irradiation with the red light. The spectra obtained by this method were consistent with those obtained by the warming-cooling method. Bathorhodopsin₁ and bathorhodopsin₂ have Λ_max at 555 and 538 nm, respectively. The two forms of bathorhodopsin are interconvertible in the light, but not in the dark. Thus, we suggest that a rhodopsin molecule in the excited state relaxes to either bathorhodopsin₁ or bathorhodopsin₂ through one of the two parallel pathways.     

Absorption Spectra of Planarian Visual Pigments and Two States of the Metarhodopsin Intermediates

Abstract— Absorption spectra measurements of isolated planarian ocelli by a microspectrophotometer (MSP) and intra-ocellar recordings of the early receptor potential (ERP) were carried out in order to characterize in situ planarian rhodopsin (pRh) and its photoproducts. The MSP spectra of the isolated ocelli revealed Λ_max at about 500 nm. The ERP evoked by a test stimulus was a positive monophasic waveform in the dark but became negative during exposure to violet light. During subsequent darkness, the ERP rapidly reverted to a positive waveform but with a smaller amplitude than before exposure. The ERP amplitude recovered to its initial level upon exposure to red light. The ERP experiments suggest that pRh produces two metarhodopsin intermediates, with Λ_max longer than that of pRh: the metastate responsible for the negative ERP converts to another metastate that results in a smaller ERP in the dark-adapted ocellus.     

PHOTOCHEMISTRY OF RETINOCHROME

Abstract— Retinochrome is a photopigment found in the visual cells of cephalopods. It has been considered to act as a supplier of the 11- cis -retinal required for synthesis of rhodopsin, because its all-trans chromophore is isomerized to 11- cis form in the light. Light and thermal reactions of squid retinochrome were investigated by low-temperature spectrophotometry.
On irradiation with green light at liquid-nitrogen temperature, retinochrome (λ_max 496 nm, – 190°C) is converted mainly to an intermediate lumiretinochrome (λ_max 475 nm, – 190°C), its chromophore being changed to 11- cis -retinal. On irradiation with blue light at - 190°C, retinochrome is changed to a photosteady–state mixture (λ_max 487 nm, – 190°C) composed mainly of retinochrome and lumiretinochrome, since lumiretinochrome is partially regenerated back to retinochrome. Similarly, irradiation of lumiretinochrome with blue light also results in the same photosteady-state mixture, which can be completely reverted to lumiretinochrome on re-irradiation with green light.
Lumiretinochrome is stable at a wide range of temperatures from – 190°C to about – 20°C. Above – 20°C, it is further converted, thermally, into metaretinochrome (λ_max 470 nm), which is the same bleached product as has been observed on irradiation of retinochrome at room temperatures. Thus, the light-bleaching process of retinochrome is rather simple compared with that of rhodopsin.     

FLUORESCENCE SPECTRA OF BLOWFLY METAXANTHOPSINS

Abstract— The main visual pigment of blowflies (xanthopsin, Vogt, 1983 Z. Naturforsch. 38c ,329–333) photoconverts into two thermostable metaxanthopsin states M and M'(e.g. Stavenga et al. , 1984 Photochem. Photobiol. 40 ,653–659). The fluorescence spectra of the two photoproducts were studied by microspectrofluorometry in vivo. The emission spectra of M and M'are very similar and peak at 660 nm. The excitation spectra of M and M'have peak wavelengths at Λ_max ' 584 nm and = 568 nm respectively.     

TIME RESOLUTION OF A BACK PHOTOREACTION IN BACTERIORHODOPSIN

Abstract— The back photoreaction from the M(412nm) intermediate in the photocycle of light-adapted bacteriorhodopsin, BR_LA(570 nm), is studied using pulsed laser excitation. The decay of a primarily produced species, M_P, regenerates BR_LA(570nm) in a process characterized by a half life of 200 ns at 25°C. The absorption maximum of M_P is blue shifted (Λ_max≃ 395 nm) relative to that of M(412nm). The primary photochemical step, M(412nm) → M_P, is attributed to a conformational change in the polyene residue. The energy and entropy of activation of the subsequent M_P→ BR_LA (570 nm) relaxation are reported and discussed.     

REFLECTANCE SPECTROSCOPY FOR THE CHARACTERIZATION OF PHOTOCHROMISM IN THE CRYSTALLINE STATE

Abstract— The Kubelka-Munk theory for diffuse reflectance has been applied to a quantitative study of photochromism in the crystalline state. For three systems investigated it was found possible to assign first order rate constants to the thermal relaxation process and estimate the pre-exponential factor A and the activation energy E_a in Arrhenius equation. For the fading of the red photocolored form, Λ_max=490 mμ, of benzaldehyde phenylhydrazone A = 1.4×10⁸ min^-1 and E_a= 15.7 kcal mole^-1. For the fading of the blue photocolored form, Λ_max=590 mμ, of 2–(2,4-dinitrobenzyl)pyridine A= 5×10¹⁴ min^-1 E_a =23.3 kcal mole^-1, Cinnamaldehyde semicarbazone showing 'reversed phototropy' has a photoactivated state, Λ_max=400 mμ, which in dark is transformed into a strongly absorbing yellow species, Λ_max= 430 mμ with A = 14 × 10¹⁰ min^-1 and E_a= 18.7 kcal mole-¹.     

LUMINESCENCE AND PHOTOCHEMISTRY OF 4-THIOURIDINE IN AQUEOUS SOLUTION

Abstract— In bidistilled water, 4-thiouridine (4TU) exhibits a weak unusual luminescence, the quantum yield of which is 3 × 10^-4 at 25°C. The excitation spectrum corresponds well to the 4TU absorption spectrum. The emission lies at longer wavelengths (Λ_max 550 nm) than the 4TU phosphorescence observed at 77 K (Λ_max, 470–480 nm). From the emission signal obtained after an excitation flash of 3 ns half-width, an "apparent" rate constant for the radiative deactivation process, shorter than 5 × 10⁶ s, can be inferred. The 300 K emission is efficiently quenched by halides and by oxygen: quenching involves a long-lived intermediate (⋍ 200 ns).
Clearly the emissive state X is populated through the S₀-S₁ electronic transition π→π* of 4TU. The nature of X cannot be unambiguously determined: it cannot be an excimer but can be either the 4TU triplet state or another chemical state distinct from the 4TU excited singlet or triplet states.
An interesting finding is that the 300 K emission and the ability of 4TU to photoreact are related: they are quenched with the same efficiency by halide anions. This indicates that quenching occurs at the same long-lived intermediate species , which is either a precursor of the emitter or the emitter itself.     

PHOTOCHEMISTRY OF FLAVINS—I. CONVENTIONAL AND LASER FLASH PHOTOLYSIS STUDY OF ALLOXAZINE

Abstract— The photobleaching of alloxazine in buffered aqueous solution has been studied by means of flash photolysis using conventional and laser excitation sources. Several transient species have been characterized. The alloxazine triplet state (Λ_max 420 nm and 550 nm, times; = 9 μs) was identified with the aid of low-temperature comparison experiments in ethanol. Transient absorption with Λ_max 440 nm, which appears after decay of the triplet state, and whose second-order decay is pH-dependent, is postulated to be due to the semiquinone radical (AH₂*) and a radical derived from alloxazine by addition of water and loss of a hydrogen atom (HAOH*), which are in equilibrium with their conjugate cation radicals. The results of experiments in the presence of oxygen indicate that these species are not primarily formed from the triplet state. The enhanced second-order decay of the flavin radicals in oxygen-containing solutions is interpreted in terms of their reaction with the peroxy radicals. The proposed mechanisms account for the production of hydroxylated alloxazines.     

FLUORESCENCE DETECTION OF AN 8-METHOXYPSORALEN METABOLITE IN HUMAN INTERSTITIAL FLUID

Abstract— A fluorescent method has been used to study the suction blister fluid of human volunteers collected after 8-methoxypsoralen (8-MOP) oral intake. A fluorescent chromophore with spectral characteristics (Λ_max= 390 nm, Λ_max=470nm) distinct from 8-MOP has been detected. Our results suggest the existence of a metabolite form of 8-MOP within the patients's skin prior to any UV irradiation. This form might result in the opening of the4–5' double bond of the 8-MOP molecule.     

A NEW FACET IN RHODOPSIN PHOTOCHEMISTRY

Abstract— A structure is proposed for the prosthetic group of the visual pigments rhodopsin, prelumirhodopsin (bathorhodopsin) and lumirhodopsin. The intrinsic photochemical step in this model is tautomerization of the prosthetic group of rhodopsin to a hexaeneamine retrotautomer with an exomethylene group for prelumirhodopsin. Based on the proposed structures, molecular orbital calculations were carried out; the absorption maxima calculated snowed the same trends as the Λ_max values observed. An exact fit was not obtained because many interactions had to be neglected. Essential information of the laser Raman resonance spectrum of prelumirhodopsin can be interpreted based on the structures proposed by our model.
The model elucidates why some retinal derivates can and others cannot form visual pigments with opsin and visual pigments having vastly differing absorption maxima yet employ the same mechanism for their photoreaction.     

LASER PHOTOLYSIS OF THE PURPLE MEMBRANE OF Halobacterium halobium IN THE PHOTOSTATIONARY STATE: THE PHOTOBRANCHING PROCESS FROM THE O640 INTERMEDIATE

Abstract The photobranching process from the O₆₄₀ intermediate (O) in the photocycle of bacteriorhodopsin was studied. The O form accumulated with continuous wave visible light (390–800 nm) irradiation of the acidic (pH 3.9–6.0) purple membrane of Halobacterium halobium at 22°C. The photocycle of O via an L-like (or N-like) intermediate was driven by 630 nm pulsed light. The newly found intermediate has an absorption band in the 450–560 nm region. The "green-light"-absorbing pigment, tentatively called G₅₂₀, was converted to O with a time constant of (1.2 ± 0.2) ms. No M-like species was found in the cycle. The quantum yield of the cycle was estimated to be 0.30 ± 0.15.     

THE PHOTOCHEMICAL INTERACTION BETWEEN THE TRIPLET STATE OF 8-METHOXYPSORALEN AND THE MELANIN PRECURSORL–3, 4 DIHYDROXYPHENYLALANINE

Abstract— The photochemical interaction between 8-methoxypsoralen (8-MOP) and the melanin precursorL–3,4-dihydroxyphenylalanine(dopaH₂) has been studied using laser flash photolysis. Triplet excited 8-MOP was thus found to abstract electrons from dopaH₂ ( k ∼ 2 × 10⁹ dm³ mol^-1 s^-1) to form semireduced 8-MOP and semioxidised dopaH₂.The technique of pulse radiolysis was used to establish separately the spectra of (a) the semi-reduced form of 8-MOP at pH 6.5 and (b) the semioxidised forms of dopaH₂ at pH 6.5, 5.8, 4.6 and 3.3. The corresponding λ_max and extinction coefficients found were: for 8-MOP at pH 6.5, λ_max= 350 nm (= 9050 dm³ mol^-1 cm^-1); for dopa at pH 6.5, λ_max= 305 nm (ε= 12000 dm³ mol^-1 cm^-1) and for dopaH at pH 3.3, λ= 305 nm (ε= 5900 dm³ mol^-1 cm^-1).     

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis , strainM–2, in the presence and absence of photosynthetic CO₂ fixation. The action spectrum for inorganic carbon accumulation in the presence of CO₂ fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO₂ fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO₂ fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.     

PHYTOCHROME INTERMEDIATES IN VIVO-I. EFFECTS OF TEMPERATURE, LIGHT INTENSITY, WAVELENGTH AND OXYGEN ON INTERMEDIATE ACCUMULATION

Abstract— Accumulation of weakly absorbing phytochrome intermediates has been demonstrated in Pisum epicotyl tissue under conditions of pigment cycling using a quasi-continuous measuring spectrophotometer. An action spectrum shows 690–700 nm to be the most efficient wavelength range in this process. Difference spectra for the decay of intermediates maintained by 690 nm light show that, if the experiment is done at 0°C, only P_fr is formed. At – 11°C, intermediates decaying to P_r can also be observed. At – 20°C, P_r is produced as well as a pigment with peak absorption at 710nm. Kinetic analysis of intermediate decay at – 11°C reveals that at least two intermediates are maintained by 690 nm light. The level of intermediate maintained by incandescent light at 0°C was 25% higher in air than in nitrogen.     

ABSORBANCE and CIRCULAR DICHROISM SPECTRA OF 1-C1S PHOTOPRODUCT FORMED BY IRRADIATING FROG RHODOPSIN

Abstract— We showed by spectrophotometry and HPLC that a photoproduct having 7-cis retinal (1-cis photoproduct) can be derived from the photoisomerization of frog lumirhodopsin (L) and metarhodopsin I (M I). The efficiency of the isomerization was higher in M I than in L. The absorption maximum of the 1-cis photoproduct at -20°C is at 455 nm, and its maximum absorbance 1.1 times as large as that of rhodopsin. The photoproduct exhibited two positive CD bands at 450 nm α-band) and 320 nm (β-band); the molecular ellipticity at a-band ([θ] = 73000) being larger than that of rhodopsin ([θ] = 61000). Re-examination of the absorption spectra of rhodopsin intermediates gave the absorption maxima of L. M 1 and M 111 to be 522, 482 and 475 nm, respectively.     

SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN-FLUORESCENT PROTEINS

Abstract— Spectral properties of guanidine-denaturated and pronase-digested green-fluorescent proteins (GFP) from two species of bioluminescent coelenterates have been investigated. Spectrophotometric titrations of Renilla and Aequorea GFP, following denaturation in 6 M guanidine HCl at elevated temperature, revealed identical absorption peaks in acid (383–384 nm) and in alkali (447–448 nm) and a single isosbestic point in the visible region at 405 nm. Both proteins exhibited a spectrophotometric pK. of 8.1 in guanidine -HCl. Pronase digestion of the heat-denaturated GFP's generated a methanol-soluble blue-fluorescent peptide with identical fluorescence emission spectra (λ_max= 430 nm, uncorrected; φ_f1= 0.003) for both coelenterate species. These data suggest that the large absorption differences between native Renilla and Aequorea GFP molecules result from unique protein environments imported to a common chromophore.