DEPENDENCY OF APPARENT RELATIVE QUANTUM YIELD OF ISORHODOPSIN TO RHODOPSIN ON THE PHOTON DENSITY OF PICOSECOND LASER PULSE

Abstract— The quantum yield of bleaching of isorhodopsin relative to that of rhodopsin was measured by irradiation of both pigments with a steady light source or a picosecond laser pulse. The each pigment was prepared by incubation of 11- cis or 9 -cis retinal with opsin, respectively. The ratio of isorhodopsin to rhodopsin in the quantum yield was estimated to be 0.37 using irradiation with a steady light, while with a weak picosecond laser pulse (excitation photon density: below 20 μ.J/1.8 mmφ), it was estimated to be 0.39. Both values are in good agreement with each other. On the other hand, excitation with a strong picosecond laser pulse (above 20 μ.J/1.8 mmφ) produced a larger ratio than 0.39, indicating that saturation effects can be easily observed by irradiation with strong picosecond laser pulses.     

EXISTENCE OF HYPSORHODOPSIN AS THE FIRST INTERMEDIATE IN THE PRIMARY PHOTOCHEMICAL PROCESS OF CATTLE RHODOPSIN

It has long been believed that bathorhodopsin is the first intermediate of visual process for cattle rhodopsin. In the present paper hypsorhodopsin is shown to be the first intermediate by the use of picosecond spectropic technique. The main first intermediate, hypsorhodopsin, is formed with the time constant of 15 ± 5 ps. The time constant of the formation of bathorhodopsin from hypsorhodopsin is 50 ± 20 ps. Bathorhodopsin intermediates formed directly from excited state rhodopsin and those formed indirectly through hypsorhodopsin are 71/2^#% and 93%, respectively, of total bathorhodopsin intermediates in octylglucoside buffered solution. Batho intermediates formed directly and indirectly are 0% and 100%. respectively, of total batho intermediates in LDAO buffered solution.     

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.     

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.     

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.     

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.     

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.     

INFLUENCE OF PLASMA DENSITY ON NUCLEATED DENSITY OF DIAMOND

The predeposition method for mereasing CH_4 concentration ininitial stage of diamond synthesis by plasma chemical vapor deposition.isused to enhance nucleated density of diamond films.The plasma parametersare diagnosed in situ using the Langmuir double probe.The relation betweenplasma ion density and nucleated densitv of diamond is revealed Increasingplasma ion density results in enhancement of nucleated density of diamondobviously     

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.     

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.     

EFFECT OF THE PHYSICAL STATE OF PHOSPHOLIPID ON RHODOPSIN REGENERATION FROM RETINYLIDENE PHOSPHOLIPID

Abstract— Rhodopsin regeneration in rod membranes involves reactions of all -trans retinal (released from bleached pigment) with phosphatidylethanolamine, photic isomerization of retinal, and binding of 11-cis retinal to opsin. This investigation demonstrated that formation of retinylidene phospholipid and retinal binding to opsin were both affected by the physical state of phospholipid. A fluid membraneous environment provided by the acyl chains of phospholipid was essential for these reactions to proceed efficiently. The retinal moiety of retinylidene phospholipid appeared to be directly transferred to opsin by transimination.     

RETINAL-OPSIN LINKAGE OF CEPHALOPOD RHODOPSIN

Abstract— The pK_a of the Schiff base of N-retinylidene butylamine was determined in anionic (sodium dodecylsulfate), cationic (cetyltrimethylammonium bromide) and nonionic (polyoxyethylene (9) lauryl ether) detergent solutions. The pK_a of the Schiff base was raised from 6.4 to 9.9 by the effect of the neighboring anion. The rise of the pK_a was affected by the ion strength. Squid metarhodopsin behaved in a manner similar to the model Schiff base in the anionic detergent solution. The cationic group showed the opposite effect on the pK_a of the Schiff base. The retinal Schiff base in rhodopsin might be heavily influenced by adjacent anionic groups. The nature of the interaction is discussed.     

CIRCULAR DICHROISM OF BOVINE RHODOPSIN

Abstract— When rhodopsin is associated with membrane particles, phospholipid may be involved in maintaining a preferred conformation of the opsin and the asymmetric structure of the chromophore of rhodopsin.     

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.     

NANOSECOND LASER PHOTOLYSIS OF RHODOPSIN AND ISORHODOPSIN

Kinetic and spectral measurements have been carried out on the primary intermediate in the photolysis of rhodopsin and isorhodopsin, initiated by a 457 nm, 6 ns (FWHM) laser pulse. In rhodopsin the kinetic decay of bathorhodopsin was found to be 140 ± 15 ns at 20°C. The decay of bathorhodopsin to lumirhodopsin has an activation energy of 51 ± 4 kJ/mol (12.2 ± 1 kcal/mol). The decay kinetics of bathorhodopsin were found to be the same for rhodopsin in membrane and detergent solubilized suspensions. The kinetic decay of the batho product in the photolysis of isorhodopsin was found to be the same as rhodopsin.
The corrected transient spectrum 50 ns following excitation in rhodopsin has two peaks near 560 and 440 nm. A peak was also observed in isorhodopsin near 550 nm at 50 ns following excitation but no transient was observed in the blue. The 550 nm peak in isorhodopsin has an intensity similar to that in rhodopsin indicating that the quantum yields for the formation of batho products of rhodopsin and isorhodopsin are similar under the irradiation conditions used here. Transient spectra for rhodopsin and isorhodopsin 1 μs following excitation are also different. In isorhodopsin the corrected transient spectrum has a peak at 500 nm, similar to low temperature steady state irradiation spectra. The 1 μs transient spectrum in rhodopsin is more intense than in isorhodopsin and shows a peak at 475 nm.     

加强实验室管理工作初探

实验室管理是科研工作的重要保障,对实验室实行科学系统的管理是规范实验秩序、保证实验室正常运行和提高实验效果的必要手段.阐述了实验室管理的基本任务.提出了加强实验室管理的建议.     

QUANTUM EFFICIENCIES OF THE REVERSIBLE PHOTOREACTION OF OCTOPUS RHODOPSIN

The classic method of photometric curves for photosensitivity determination has been extended to the case of photoreversible reactions and applied to the octopus rhodopsin --> acid metarhodopsin photoreaction. In such cases, measurements at one irradiation wavelength yield the sum of the photosensitivities of the forward and reverse processes. However, by using different irradiation wavelengths, together with appropriate molar extinction coefficients, the quantum efficiencies for both reactions may be resolved. For detergent-solubilized octopus rhodopsin at room temperature, pH 7, the quantum yields are found to be 0.69 (+/- 0.03) for rhodopsin --> metarhodopsin, in line with values observed in a range of vertebrate visual pigments, and 0.43 (+/- 0.02) for the reverse photoregeneration process. The similarities in overall photosensitivities of the forward and reverse reactions in the visible region are consistent with a significant physiological role for photoreversal in the cephalopod visual cycle.