DEPENDENCY OF PHOTON DENSITY ON PRIMARY PROCESS OF CATTLE RHODOPSIN

The primary photochemical reactions of cattle rhodopsin suspended in H2O or D2O were compared between excitation with both a weak and an intense picosecond laser pulse (wavelength, 532 nm; duration, 25 ps) at room temperature. The time-dependent change of absorbance at about 575 nm demonstrated that photohodopsin, a precursor of bathorhodopsin, was produced immediately after the excitation with a weak picosecond laser pulse. It decayed to bathorhodopsin with a time constant of 45 ps which is close to the value reported previously [Shichida et al., (1984) Photobiochem. Photobiophys., 7, 221-228]. No deuterium effect was observed in this process. Excitation with an intense laser pulse induced instantaneous increase of the absorbance at about 575 nm and remained at almost constant level on the picosecond time scale, which was in good agreement with the pioneering work [Busch et al., (1972) Proc. Natl. Acad. Sci., USA, 69, 2802-2806]. No deuterium effect was observed in this photochemical process.     

LIGHT-INDUCED CONFORMATIONAL CHANGES IN CATTLE RHODOPSIN AS PROBED BY MEASUREMENTS OF THE INTEFFACE POTENTIAL

Abstract— A novel technique of capacitative coupling of oriented rhodopsin at a polar/apolar interface allows the time resolved investigation of conformational changes following a flash. Electric signals arise as a consequence of changes of the interface potential. A signal occurring within milliseconds behaves like the R₂-phase of the "early receptor potential" (= ERP). This response is interpreted as a conformational change of rhodopsin. No correlation of this signal is found to the spectroscopically defined metarhodopsin I-metarhodopsin II transition.
The temperature dependence of the conformational change coincides with the temperature dependence of the latency of the 'a-wave' of the electroretinogram, reported by Arden and Ikeda (1968). It is suggested that the command step of visual excitation is the conformational change and not one of the spectroscopically defined photolysis steps of rhodopsin.
Analysis of slower electrical signals following the fast response is in accordance with the model of a light-induced pore formation.     

FEMTOSECOND STUDIES OF PRIMARY PHOTOPROCESSES IN OCTOPUS RHODOPSIN

Abstract— Femtosecond spectroscopy of octopus rhodopsin in H₂O and D₂O was performed over a very wide spectral region of 400–1000 nm. Transient gain and absorption from the excited state were observed for the first time around 650 and 700 nm, respectively, just after 300 fs pulse excitation. Bathorhodopsin was formed within 400 fs from the excited state; therefore, the cis-trans isomerization completes within 400 fs. The first intermediate "primerhodopsin" found in our previous paper is most likely "quasi-thermal" bathorhodopsin, in which the local thermalization of the chromophore is achieved. Then cooling down of the chromophore to the surrounding protein temperature takes place with 20 ± 10 ps along with blue-shifting of a spectrum of 10 ± 5 nm. In addition to these observations, a prominent gain in the region of > 850 nm was observed and decayed with 2–3 ps in H₂O. A similar time constant was estimated for a partial decay of an induced absorption around 600 nm. This process may be related with two forms of bathorhodopsin reported previously. In this scheme, two forms of bathorhodopsin are formed with time constants of about 400 fs and 2 ps. In the sample in D₂O, time constant of 3–4 ps was obtained for the slower process.     

ANALYSIS OF PRIMARY PHOTOCHEMICAL PROCESSES IN BACTERIORHODOPSIN

Abstract— The sequence of primary events following light absorption by light adapted bacteriorhodopsin (bR₅₇₀) is considered by analyzing recent picosecond absorption and emission data. The analysis is facilitated by theoretical calculations which allow us to characterize the properties of the first excited singlet state. It is concluded that excitation leads to the eventual population of a photochemically important nonfluorescent excited state (I) which decays into a photoproduct (J₆₂₅)- In J₆₂₅, which is most probably a ground state molecule, the chromophore has undergone a structural change, presumably trans → 13- cis isomerization. It is suggested that the subsequent process

reflects a relaxation of the protein environment involving proton transfer.     

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

Abstract— Photoacoustic spectroscopy is used to investigate the uptake of energy in the primary photoreaction of bovine visual pigment (Rhodopsin → 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^-1 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 10%, even in the spectral region of the β-band of rhodopsin.     

PROTON TRANSFER IN THE PRIMARY PROCESS OF VISION

Abstract— Proton transfer was theoretically examined as a possible primary process of vision. The motion of protons in the adiabatic potential of the Schiff base hydrogen bond was investigated in terms of quantum mechanics. The probability of proton transfer from the Schiff base nitrogen (i.e. the unprotonation of Schiff base) was found to increase as the retinal rotated around 11–12. double bond by 90°. The results also suggested that the proton transfer can take place before or during the transition from the excited to ground state (excited state proton transfer). We proposed that such excited state proton transfer is one of the elementary processes in primary visual photochemistry, and this process leads to the unprotonated visual pigment, hyposorhodopsin, which has been experimentally verified as one of the primary photoproducts of rhodopsin. The probability of this process could be comparable to the conventional process leading to the protonated intermediate, bathorhodopsin. The relation of these results with the recent experimental data is discussed.     

FUNCTIONAL PROPERTIES OF CATTLE RHODOPSIN IN SOLUBLE COMPLEX WITH PHOSPHOLIPIDS and DEOXYCHOLATE

Abstract— The necessary conditions of bleached rhodopsin to activate GTPase and to regenerate α-band were studied by changing the number of bound phospholipids to rhodopsin using gel filtration procedure. The number of bound phospholipids per mole of rhodopsin (bPL/rho) in the eluants was reproducibly controlled by the concentration of sodium deoxycholate (DOC) in the elution buffer. The eluants were soluble complexes composed of rhodopsin with original a-band, disk phospholipids and DOC. The regenerability of α-band depended on bPL/rho but neither on the concentration of DOC nor on state of aggregation of rhodopsin. The lowest number of bPL/rho for this activity under our experimental conditions was estimated to be30–50 in bPL/rho. GTPase was activated only by such complexes that had a nearly original quantity of bPL/rho in disk membranes. Other complexes with less bPL/rho showed aggregation upon bleaching and did not activate GTPase. The amount of phospholipids present in the disk membranes is sufficient to prevent aggregation of rhodopsin upon bleaching.     

ON THE STATE OF CHROMOPHORE PROTONATION IN RHODOPSIN: IMPLICATION FOR PRIMARY PHOTOCHEMISTRY IN VISUAL PIGMENTS

Resonance Raman multicomponent spectra of bovine rhodopsin, isorhodopsin, and bathorhodopsin are obtained at low temperature. Application of the double beam, 'pump-probe' technique allows an extraction of the rhodopsin and bathorhodopsin spectra in both protonated and deuterated media. Our results show that the Schiff bases of both rhodopsin and bathorhodopsin are fully protonated and the degree of protonation is unaffected by the rhodopsin-bathorhodopsin transformation. Further, the data support the concept or cis-trans isomerization as occurring in this transition. The effect of these results on various models for the primary photochemical event in vision is discussed.     

PRIMARY PHOTOCHEMICAL PRODUCTS OF DITHIOGLYCOLIC ACID IN AQUEOUS SOLUTION

Abstract— The photochemistry of dithioglycolic acid at 254 nm was investigated in deaerated aqueous solutions in the pH range 1.4–7.3. Initial yields of the primary photochemical products H₂S,–SH and aldehyde-(probably glyoxylic acid) were determined. The complex pH dependence of these simultaneously formed first stable products is interpreted in terms of the ground-state ionic equilibria, and in addition pH-dependent processes occurring in the excited state and labile intermediate sequence.
It is suggested that the photochemical mechanism involves two parallel pathways: one a hydrolytic splitting of S - S leading to H₂S formation (unaffected by the presence of O₂ or isopropanol), the other a free radical mechanism via C–S breakage, which is affected by the presence of O₂ or isopropanol.     

SIMULATION ANALYSIS OF THE PHOTOCONVERSION PROCESS OF SQUID RHODOPSIN AT LIQUID HELIUM TEMPERATURE

The photoconversion process among squid rhodopsin, bathorhodopsin isorhodopsin and hypsorhodopsin was studied at liquid helium temperature. We evaluated the relative quantum yields of the photoconversion among four pigments by analysing the time-dependent change of absorption spectra. The result suggests that hypsorhodopsin is the common intermediate of rhodopsin and isorhodopsin, and there is no direct conversion between rhodopsin and isorhodopsin. Furthermore, rhodopsin converts to hypsorhodopsin or bathorhodopsin much more efficiently than does isorhodopsin, and bathorhodopsin does not convert directly to hypsorhodopsin. It was also found that rhodopsin and isorhodopsin convert to bathorhodopsin more efficiently than to hypsorhodopsin. In particular, the quantum yield of conversion from rhodopsin to bathorhodopsin was found to be about twice as large as that from rhodopsin to hypsorhodopsin. This result is somewhat in disagreement with the result obtained from the laser flash experiments at room and liquid nitrogen temperatures. The reason for the difference between the two experimental results is discussed.     

RAPID AND SENSITIVE OPTICAL ROTATION MEASUREMENT: KINETICS OF CONFORMATIONAL CHANGE OF RHODOPSIN INTERMEDIATE

A rapid and sensitive optical rotation (OR) measurement system using laser light has been developed to meet the demands of flash-photolysis experiments. The optimal OR resolution of the apparatus is 0.3mdeg with 10 μs time constant with single shot. The OR resolution could be easily improved less than 0.1 mdeg with accumulation of data by a kinetic processor. Applications are briefly described for the study of transient conformational change of photochemical intermediates of octopus rhodopsin following a blue light flash.     

激光诱导荧光衰减探测光化学反应

激光荧光光谱为重要光谱分析手段之一,广泛用于物理、化学、生物学等研究领域。 但对于一些含有发色基团的大有机分子,由于分子振动自由度很大,振动能级连续分布, 其荧光谱成为宽展的光谱带,振动带结构不复存在,使荧光光谱难于用作样品的成份鉴定。对于溶液样品由于分子间的碰撞及长程力的作用,也可能造成荧光猝灭和分子间电 子能量转移,特别是对那些吸收光子后易发生光分解的物质,所产生的新分子或碎片往 往也有荧光特征,叠加在原有分子的荧光带上,使得所要检测的任何一种成份的荧光强 度对所有其它荧光猝灭物和敏化物的浓度及其荧光特征有很复杂的依赖关系,这就增加 了对实验所得到的荧光光谱解释的难度。     

LOW TEMPERATURE SPECTROPHOTOMETRIC STUDY OF CATTLE BATHORHODOPSINS PRODUCED FROM RHODOPSIN AND ISORHODOPSIN IN TRANSPARENT MEDIUM WITHOUT CRACKS

Abstract— Cattle rhodopsin (11-cis-Rh) and isorhodopsin (9-cis-Rh) were prepared from the same extract of cattle opsin by incubation in the dark with 114– and 9-cis-retinals, respectively. The preparations obtained were mixed with glycerol (67% in the final concentration) and then cooled to 77 K by a rapid cooling method for freezing the samples as a clear glass, without any cracks. The spectral changes during their photochemical reactions were measured. A computer analysis of the spectra obtained verified that bathorhodopsin from 11-cis-Rh was identical in spectrum with that from 9-cⁱs-Rh not only in photosteady state but also under a short time irradiation.     

ORIENTATION OF RETINYLIDENE CHROMOPHORE OF HYPSORHODOPSIN IN FROG RETINA

Abstract— Hypsorhodopsin and bathorhodopsin were formed in the frog retina by irradiating rhodopsin at liquid He temperature (9 K) with orange light (> 520 nm) and blue light (437 nm), respectively. Hypsorhodopsin was converted to bathorhodopsin in the retina by warming above 32 K in the dark. Similar phenomena were observed in the rod outer segment suspension. A difference spectrum between hypsorhodopsin and bathorhodopsin in the retina produced by warming was almost identical with that in the rod outer segment suspension. This suggests that the transition dipole moment of hypsorhodopsin is parallel to the disk membrane plane which is also parallel to that of bathorhodopsin.     

PRIMARY PHOTOCHEMICAL PROCESSES OF CATIONIC ACRIDINE ORANGE IN AQUEOUS SOLUTION STUDIED BY FLASH PHOTOLYSIS

Abstract— A transformation system in Escherichia coli was employed to verify the extent of the lesions caused by ultraviolet (UV) and ionizing radiations. DNA inactivated at 280 nm could be reactivated to some extent by exposing the transforming DNA solutions at 240 nm. This reactivation has been tested using more than one strain of E. coli as recipient. Transforming DNA inactivated by ionizing radiations (⁹⁰Sr beta rays and ⁶⁰Co gamma rays) was not reactivable. Low doses of beta rays, however, reactivated the DNA inactivated by 280 nm UV to a slight but significant extent.     

PHOTOEXCITATION OF RHODOPSIN: CONFORMATION CHANGES IN THE CHROMOPHORE, PROTEIN AND ASSOCIATED LIPIDS AS DETERMINED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— The visual pigment rhodopsin is the major membrane protein in the rod photoreceptor membrane. Rhodopsin's function is to transduce the light induced isomerization (ll-cis to all-trans) of its internally located retinylidene chromophore into transient expression of signal sites at the surface of the protein. Fourier transform infrared (FTIR) difference spectroscopy has been used to study all of the steps in the photobleaching sequence of rhodopsin. Early protein alterations involving the peptide backbone and aspartic and/or glutamic carboxyl groups were detected which increase upon lumirhodopsin formation and spread to water exposed carboxyl groups by metarhodopsin II. The intensified and frequency shifted hydrogen-out-of-plane vibrations of the chromophore that are present in bathorhodopsin are absent in lumirhodopsin. This indicates that by lumirhodopsin, the chromophore has relaxed relative to its more strained all-frans form in bathorhodopsin. Finally, the transition to metarhodopsin II is found to involve perturbation of the acyl tail region of unsaturated phospholipid molecules possibly in response to small changes in the shape of the rhodopsin.     

UPON THE OPTIMAL GRAPHICAL REPRESENTATION OF FLASH DATA FROM PHOTOCHEMICAL SYSTEMS OBEYING FIRST ORDER KINETICS

Abstract— It is recommended that plots of absorbance change vs. log time be considered as a way to represent many decades of flash spectroscopy data when several separate decays are present. The values of the rate constants k , for the decays are easily revealed by nearly linear portions of the plots centered near t _j= k ⁻¹_j, which are inflection points in the graphs. The amplitudes of the decays are easily seen because they are proportional to the slopes of the linear portions of the plots. Such plots introduce no spurious singularities and they are easy to construct using semi-log paper. Examples, including one from bacteriorhodopsin, are presented. A simple mathematical proof of these features is provided.     

A POSSIBLE MECHANISM FOR THE ROLE OF N-RETINYLIDENE PHOSPHATIDYL ETHANOLAMINE IN THE REGENERATION OF RHODOPSIN

Abstract— A series of molecular orbital calculations on a model Schiff base comparable to protonated N -retinylidene phosphatidyl ethanolamine isomers has been made. The effect of the charged oxygen atoms of the phosphate moiety on the distribution of positive charge along the polyene chain of these isomers has been calculated. The stabilizing coulombic energy of interaction of these opposite charges and the possibility of free rotation around carbonxarbon double bonds in the electronically excited state has led to the conclusion that an 11- cis Schiff base isomer is the most probable product of the photoisomerization of an all-trans Schiff base.
The formation of a stable unprotonated all-trans Schiff base in aqueous detergent dispersion and its subsequent conversion to the protonated form, both with absorption spectra in conformity with the literature, is demonstrated.     

RECONSTITUTION OF SQUID RHODOPSIN IN RHABDOMAL MEMBRANES

Abstract— Squid opsin which is capable of combining with 11- cis or 9- cis retinal to reconstitute photo-pigment has been prepared by irradiation of rhabdomal membranes with orange light (> 530 nm) in the presence of 0.2 M hydroxylamine. When the irradiation is carried out either at concentrations of hydroxylamine higher than 0.2 M or with light of wavelength shorter than 530 nm, rhodopsin in the membranes is bleached quickly, but the ability of the resultant opsin to form rhodopsin is greatly reduced.
The optimum pH for rhodopsin regeneration in rhabdomal membranes was found to be between 6.5 and 8.5. The rate of regeneration of rhodopsin increases with raising temperature, and at about 20°C it is almost the same as that of isorhodopsin. Even after solubilization in digitonin solution, opsin still preserves the ability to reform rhodopsin.
All- trans retinal can be incorporated into retinochrome-bearing membranes, in which it is isomerized into 11- cis isomer by the photoisomerase activity of retinochrome. Rhabdomal membranes retaining active opsin can take up 11- cis retinal from retinochrome membranes so as to synthesize rhodopsin.