ABSORPTION POLARIZATION PROPERTIES OF LIGHT-ADAPTED BACTERIORHODOPSIN IN THE 266–620nm REGION

Abstract— Using the method of polarized photochemistry, the polarization of the first daughter (K₆₁₀) in the photocycle of light-adapted bacteriorhodopsin (bR₅₇₀) is determined relative to the polarization of the photolysis pulse for different absorption regions of bR₅₇₀. The results are discussed in terms of recent spectroscopic assignments of bR₅₇₀, and in the 310–266 nm region, in terms of energy transfer processes between the apoprotein and the retinal.     

EFFECTS OF TRYPTOPHAN OXIDATION ON BACTERIORHODOPSIN STRUCTURE AND FUNCTION

Abstract— N -bromosuccinimide at low molar ratios specifically oxidizes tryptophan residues in purple membranes (bR). Loss of 1 mol tryptophan per mol bR (which corresponds to oxidation of Trp₁₀ or Trp₁₂ or part of both) produces slight changes in the absorption and CD spectra and in the photobleaching kinetics. The efficiency of reconstituting the retinylidene protein after bleaching and heptane extraction of these membranes was comparable to control values(–80%). Photocycling yield (M₄₁₂) with 265 nm or 530 nm excitation (15 ns pulse) was only slightly decreased. Another mol of tryptophan was reacted at higher NBS:bR molar ratio (10:1). Partial oxidation of several residues rather than titration of a specific residue occurred: HPLC indicated no reaction with the chromophore. A considerable loss of extinction at 570 nm including enhanced red and blue shifts of LD_max at low and high pH respectively, was observed. Also the photobleaching rate was faster and functional retinylidene membranes could not be reconstituted from heptane-extracted, apomembranes. Exogenous retinal could still locate the attachment site, based on formation of the fluorescent NaBH₄-reduced retinoyl adduct. Perturbation in the near UV and visible CD infer changes in helical conformation, trimer dissociation and decreased asymmetry of the chromophore. Photocycling efficiency was greatly decreased. The relative decrease was greater for 265 nm rather than 530 nm excitation. These results are consistent with co-operative destabilization of the protein conformation by oxidized tryptophan residues, which leads to a decrease in the hydrophobicity of chromophoric site.     

THE USE OF NEUTRAL RED TO MONITOR THE SURFACE POTENTIAL OF THE PURPLE MEMBRANE

Abstract— The binding of neutral red to purple membrane has been studied. The intrinsic p K _a and the apparent p K _a, of bound neutral red were determined by titration and by measuring the binding ratio of neutral red to purple membrane as a function of pH. The surface potential of purple membrane was inferred from the difference between these two p K _as. The H⁺/M412 ratio at different ionic strengths was also measured and compared with the surface potential. The results show that the H⁺/M412 decreased as the surface potential increased due to decreased salt concentrations. However, this correlation holds only for KCl concentrations higher than 30 m M . At lower salt concentrations, the change in surface potential is always less than the variation in the H⁺/M412 ratio.     

ON THE ROLE OF TYROSINE IN THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract -The rate of formation of the M intermediate ( k _M) in the photocycles of bacteriorhodopsin (bR₅₇₀) and of nitrated bacteriorhodopsin (bR₅₃₂ⁿ), is measured over the range between pH 6.5 and 11.5. In the case of bR₅₇₀, k _M is markedly pH dependent, exhibiting a titration-like curve with pK ∽ 10.3. The pH dependency is completely eliminated by nitration. On the basis of previous work by Lemke and Oesterhelt (1981), the effect is attributed to the specific modification of the Tyr 26 residue. The data are rationalized by a mechanism in which deprotonation of Tyr 26 at the stage of the L intermediate constitutes a prerequisite for deprotonation of the retinal-lysine SchifT base. Both reactions are intimately associated with the photo-induced proton pump mechanism.     

SPECTRAL PROPERTIES OF THE RHODOPSIN-SYSTEM OF THE CRAYFISH Astacus leptodactylus

Abstract— The absorption spectra of the membrane-bound and of the digitonin-solubilized visual pigment of crayfish Astacus leptodactylus were investigated by conventional spectrophotometry. A method was developed to isolate purified rhabdoms almost entirely free from screening pigments from a single retina. The quantity of isolated and purified rhabdoms from a single retina was sufficient to measure the absorption spectra of the visual pigment.
The absorption spectra of the chromoprotein system (R and M) show that both the membrane-bound and the digitonin-solubilized visual pigment isomers are stable at 0°C and pH 7.0. Rhodopsin and metarhodopsin are photoreversible under these conditions without any light-induced denaturation. The difference spectra for the chromoprotein isomers and those of different photostationary states yield maximal values for ΔE at 570 and 485 nm.
At neutral pH, 0°C, Λ_max of rhodopsin is 530 nm. Irradiation with light of Λ= 630 to 640 nm isomerizes rhodopsin nearly quantitatively to metarhodopsin with Λ_max, of 500 nm. The molar extinction coefficient of metarhodopsin is greater than that of rhodopsin by a factor of ˜ 1.41. each measured at its respective Λ_max Metarhodopsin can be isomerized to rhodopsin by irradiating at Λ > 630 nm. As the absorption spectra of the two chromoprotein isomers overlap, only part of the metarhodopsin can be reversed to rhodopsin. The maximal photoreversion can be achieved by irradiating at 460 nm. The stability of the digitonin-solubilized chromoprotein is remarkably dependent on temperature. Warming the digitonin extract of rhabdoms from 0 to 20 or 30°C caused a shift of the rhodopsin spectrum to shorter wavelengths (Λ_max= 485 nm) accompanied by a decrease of E_Λmax by about 30%.     

A LASER FLASH PHOTOLYSIS STUDY OF PYRENE-1-ALDEHYDE. INTERSYSTEM CROSSING EFFICIENCY, PHOTOREACTIVITY AND TRIPLET STATE PROPERTIES IN VARIOUS SOLVENTS

Abstract— Triplet-and singlet-related photoprocesses of pyrene-1-aldehyde (PA) in various solvents have been investigated in detail using 337.1 and 355 nm laser flash photolysis in conjunction with time-correlated determination of fluorescence lifetimes (τ_F) and steady-state photochemical and absorption-emission spectral measurements. In benzene, the lowest triplet of PA (43 < E_T < 46 kcal/mol) has a lifetime of about 50 µs (τ_T) and displays the absorption maximum at 443 nm with a maximum extinction coefficient (ε_max) of 21000 M ^-1cm^-1; the corresponding ketyl radical has a sharp absorption maximum at 428 nm (ε_max≥ 25000 M ^-1cm^-1). The quantum yields (φ_T) of lowest triplet occupation are high in nonprotic solvents (0.6–0.8), decrease in protic solvents (alcohols) as the polarity of the latter is increased, and maintain a complementary relationship with the quantum yields (φ_F) of fluorescence. Quantum yields (φ_PC) of loss of PA due to photoreactions in some solvents have also been determined under conditions of steady irradiation at 366 nm; φ_PC is in the range 0.1–0.2 in electron-rich olefinic solvents such as cyclohexene and tetramethylethylene. These results concerning τ_F, τ_T, φ_F. φ_T and φ_PC as well as the effects of 1,2,4-trimethoxybenzene and 2,5-dimethyl-2,4-hexadiene as quenchers for fluorescence, triplet yield, and photochemistry are discussed in the light of possible state orders for PA in polar and nonpolar environments.     

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 ORIGIN OF THE RED-SHIFTED ABSORPTION MAXIMUM OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M₄₁₂ intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophore-protein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although s-trans ring-chain planarity prevails in the M₄₁₂ intermediate it does not contribute significantly (only ca 750 cm⁻¹) to the opsin shift observed in M₄₁₂. It is suggested that in M₄₁₂, the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.     

LIGHT-INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light-induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.
Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR₅₇₀) and M₄₁₂-intermediate, but decreased the decay rate of the M₄₁₂-intermediate. Light-induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light-induced proton transport. The role of amino group(s) in light-induced proton release and transport through the purple membrane is discussed.     

PHOTOCHEMICAL BEHAVIOR AND EMISSION CHARACTERISTICS OF 9-VINYLANTHRACENE

9-Vinylanthracene (9-VA) is an efficient fluorescer of fluorescence quantum yield = 0.91 0.03 in methanol (_ex= 365 nm). This is consistent with its low photochemical quantum yields _c (_cx= 365 nm) of 0.005 in the absence of azobisisobutyronitrile (AIBN) radical initiator and _c= 0.02 in the presence of 10^-2 M dm^-3 AIBN. 9-Vinylanthracene was shown to behave as a 9-substituted anthracene undergoing photodimerization rather than the typical vinyl aryl monomer photopolymerization. The photodimer formation is supported by spectroscopic techniques. 9-Vinylanthracene undergoes efficient fluorescence quenching in the presence of AIBN. Stern-Volmer plots indicate a collisional mechanism. 9-Vinylanthracene crystals as well as polycrystalline films give excimeric emission (_max= 510 nm, _cx= 380 nm) which is considerably red shifted ( ca. 100 nm) compared with molecular emission.     

KYNURENIC ACID — I. SPECTROSCOPIC PROPERTIES

Abstract— The presence of different ground state species in ionic and/or keto s enol equilibrium has been demonstrated by studying NMR, IR and UV spectroscopic properties of kynurenic acid and related quinoline derivatives. The optical and emission properties of kynurenic acid and related compounds are thus very sensitive to pH conditions. Fluorescence and intersystem crossing (ISC) quantum yields have been measured in various solvents. The first excited singlet state is probably very short-lived in neutral aqueous solution. The triplet state is long-lived (10 μs) as shown by 353 nm laser flash spectroscopy. A high ISC quantum yield and a large molar extinction coefficient make it easy to detect the T₁→ T_n transient (Δ_max≃ 570 nm).     

BIOPHYSICAL AND BIOCHEMICAL ASPECTS OF PHENGODID (RAILROAD-WORM) BIOLUMINESCENCE

In studies of the bioluminescence of 11 species of phengodid collected in central and southeast Brazil, we have found that: (1) their lateral lanterns emit light in the yellow-green region (λ_max= 540–580 nm) and the head lantern color is shifted to the red region ( λ_max= 565–620 nm), (2) the luciferins of both types of lanterns are identical to that of lampyrids and elaterids and (3) the luciferase physicochemical properties are also similar to those of lampyrids and elaterids (optimum pH ca 8.1; K_m(ATP) = 260–370 μM , Kμ(luciferin) = 170–400 μM; molecular weight ca 60 kDa; apparent activation energy of in vitro bioluminescence ca 58 kJ/mol). Thus the bioluminescence system of phengodids appears to be essentially the same as that of lampyrids and elaterids. The different bioluminescence colors of the lanterns of Phrixothrix species (λ_head= 600–620 nm; λ_lateral= 535–565 nm) and other phengodid species are probably elicited by the presence of luciferase isoenzymes, as occurs in the case of elaterid prothoracic and abdominal lanterns.     

THE PRODUCTION OF SINGLET MOLECULAR OXYGEN BY ZINC(II) PHTHALOCYANINE IN ETHANOL AND IN UNILAMELLAR VESICLES. CHEMICAL QUENCHING AND PHOSPHORESCENCE STUDIES

Abstract— Zn(II)phthalocyanine (ZnPc) generates O₂(¹Δ_g) with a quantum yield of ca. 0.4 upon photocxcitation at 354 or 600 nm in ethanolic solution as determined by time-resolved phosphorescence studies at 1270 nm and photooxidation experiments using 1,3-diphenylisobenzofuran (DPBF) as substrate. The quantum yield of photooxidation slightly increases upon incorporation of ZnPc into unilamellar liposomes of dipalmitoylphosphatidylcholine. Under our irradiation conditions (600 nm, 18^°C, and short light exposure times), DPBF(5–50 μM) undergoes photooxidation by a pure Type II mechanism; the rate constant for the O₂(¹Δ_g) + DPBF reaction is (1.1 ±0.1) x 10⁹ M^-1 s^_1 in ethanol solution and determined to be about two orders of magnitude smaller when both ZnPc and DPBF are embedded into liposomes.     

A MICROSECOND KINETIC STUDY OF THE PHOTOGENERATED MEMBRANE POTENTIAL OF BACTERIORHODOPSIN WITH A FAST RESPONDING DYE

Abstract— Bacteriorhodopsin is a light activated proton pump which generates proton and electric gradients across the cytoplasmic membrane of Halobacterium halobium. In this study, a dye whose fluorescence intensity responds rapidly to membrane potential was used to follow the evolution of the potential on liposomes reconstituted with bacteriorhodopsin, in the microseconds time domain. By comparing the formation kinetics of the potential to those of the long-lived intermediate species in the bacteriorhodopsin photocycle, M₄₁₂, both in H₂O and ₂H₂O suspensions, we can draw the following conclusion: the electric potential onset time is 20 μs after initiation of the illumination. The triggering of the potential is not the formation of the M₄₁₂ intermediate, which was hitherto considered to be the first species in the bacteriorhodopsin cycle which has an unprotonated Schiff base linkage at the retinal chromophore. Rather, the potential forms at the transition of the L₅₅₀ intermediate to the species X which precedes M₄₁₂ or even at the preceding conversion of K₅₉₀ to L₅₅₀.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

AN INVESTIGATION OF RETINAL AS A SOURCE OF SINGLET OXYGEN

Abstract— All -trans retinal is dissolved in alcohols and illuminated at 365 nm in the presence of a singlet oxygen acceptor, 2,5-dimethylfuran. Illumination produces the photosensitized oxidation of the acceptor which is measured by the disappearance of its 215 nm absorption band. A kinetic study is carried out and β_DMF is 1.6 × 10^--⁴ M . The quantum yield of ¹O₂ production from the light-excited retinal is estimated to 0.096. The retinal sensitized photooxidation of dimethylfuran is inhibited by a ¹O₂ quencher, 1,4-diazabicyclo(2,2,2)-octane, and enhanced by deuteration of the solvent. Deuterated solvents are known to increase ¹O₂ lifetime.
The production of ¹O₂ from retinal is briefly discussed in relation to the damage which may be induced by light in the visual cells.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.     

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.     

TIME-RESOLVED RESONANCE RAMAN STUDIES ON THE PHOTOCHEMICAL CYCLE OF BACTERIORHODOPSIN

Abstract— Kinetic resonance Raman (RR) experiments were designed to study the time-behaviour of the retinal-binding protein bacteriorhodopsin (BR) in its photochemical cycle. The unphotolyzed chro-mophore B-570 and the two intermediates L -550 and M-412 were probed by the characteristic C=C stretching vibrations of the retinal moiety. Time resolution was achieved with a spinning cell as flow system in combination with two CW lasers in a pump-probe configuration. RR spectra were probed at 475 nm at various delay times between pump and probe event. The deconvolution of the spectra into the various components B-570, L-550 and M-412 was carried out by curve fitting procedures. It was found that at pH7.4 L-550 decays — with a time-constant of 62 μs — not completely but to a residual level of 35% of its initial value. This intermediate L -amplitude finally disappears in the ms-range (4.5 ms) synchroneously with the intermediate M -412. An analogeous time-behaviour was found at pH 4.6. In the basic range also an " L " -intermediate could be identified which is coupled to the long-lived M-component. To explain the peculiar time-dependence it is proposed that during the fast decay of L a dynamic equilibrium between L and M is established. Then during the reconstitution of B -570 the two intermediates disappear synchroneously. A molecular model is presented in which the dynamic equilibrium between L and M is explained by an oscillatory motion of a proton from the Schiff base group of the chromophore to its counterion.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY—II. PHOTOPHYSICAL PROPERTIES OF [26] PORPHYRIN

Abstract— –Photophysical properties of [26] porphyrin (26 P) were investigated in chloroform. The quantum yields of fluorescence, of S₁→ T₁ intersystem crossing and singlet oxygen formation were measured. The purity, stability, the strong absorption in the red (δ_max= 783 nm; ε_max= 28 000 M ¹ cm^-1) and the ability of singlet oxygen formation recommend 26 P as potential photosensitizer for tumor therapy.