THE EFFECT OF VISCOSITY ON THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— We study the effect of solvent viscosity on the kinetics of the photocycle of bacteriorhodopsin (bR) from Halobacterium halobium. Solvent viscosity is altered by changing the glycerol concentration from 20 to 80% glycerol by volume. The kinetics of the photocycle are observed after flash photolysis at four wavelengths at several temperatures between 240 and 315 K. Assuming a sequential model, bR → K -→ L → M → O → bR, Arrhenius plots of the rate coefficients determine the activation enthalpies and frequency factors for each step. Kinetic data from all solvents are considered together and studied as a function of temperature for fixed solvent viscosities. The early steps of the cycle are insensitive to solvent viscosity, →; the later steps are retarded with increasing viscosity. Activation enthalpies are independent of viscosity; the frequency factors are proportional to η^−K, where the exponent k 0.25 for the transition K → L, 0.0 for L → M, 0.8 for M → O and 0.5 for O → bR.     

INTERACTION OF DIBUCAINEHCl LOCAL ANESTHETICS WITH BACTERIORHODOPSIN IN PURPLE MEMBRANE: A SPECTROSCOPIC STUDY

Several spectroscopic techniques (absorption, emission, transient absorption and differential scanning calorimetry--DSC) were used to investigate the deprotonation of dibucaine.HCl in a hydrophobic environment, and the interaction sites and mechanisms of the local anesthetic dibucaine.HCl on bacteriorhodopsin (bR) in purple membrane. The important results are summarized as follows: (1) the visible absorption features of native (lambda max = 568 nm) and deionized (lambda max = 608 nm) bR are sensitive to the amount of dibucaine.HCl added; (2) the emission spectrum of dibucaine.HCl embedded in the retinal-free mutant bR is similar to that of dibucaine free base in Triton X-100 micellar solutions; (3) the phosphorescence emission of dibucaine at 77 K is completely quenched by bR and the fluorescence quenching rate for the incorporated dibucaine.HCl in bR was determined as kq = 4.09 x 10(13) M-1 s-1; (4) the incorporation of dibucaine.HCl in bR inhibits the slow component rate of formation of M412 and decreases the amount of M412 formation in the photochemical cycle of bR; and (5) the thermal stability of native bR was measured by DSC in the presence and absence of dibucaine and yielded an endothermic transition at 95.9 +/- 1.0 degrees C with 13.6 J/g (3.25 +/- 0.12 cal/g) of enthalpy changes. All observations suggest that the action site of the local anesthetic, dibucaine.HCl, is near or at the chromophore, i.e. the retinal Schiff base of bR. The anesthetic action on bR purple membrane is probably via a specific site binding, but not a conformational mechanism.     

INTERACTIONS OF BOTH MELITTIN AND ITS SITE-SPECIFIC MUTANTS WITH BACTERIORHODOPSIN OF Halobacterium halobium: SITES OF ELECI'ROSTATIC INTERACI'ION ON MELITI'IN

Abstract Melittin and its site-specific mutants differentially delay the slow-decaying component of the photocycle intermediate M₄₁₂ of bacteriorhodopsin in the purple membrane and the acetylated purple membrane whose several lysine residues are modified. This effect is attributed to the interaction of the total positive charges of melittin or its mutants with the total negative charges of bacteriorhodopsin. The effects of melittin and its mutants on the Triton X-100–solubilized bacteriorhodopsin monomers are somewhat complicated but are associated with their charges. These results show that there is electrostatic interaction between bacteriorhodopsin and melittin and that both N-and C-termini of melittin function as sites of the interaction, with Arg 22 and Arg 24 making a prominent contribution to the effective surface charge of melittin. Melittin, at certain concentrations, partially restores the decreased photoactivity of the bacteriorhodopsin monomers trapped in the Triton-lipid-protein mixed micelles, which suggests that melittin may compete with Triton X-100 for the binding sites on the bacteriorhodopsin monomers. Other kinds of interactions between bacteriorhodopsin and melittin are also indicated. The possible states of melittin in membranes are discussed.     

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.     

BIOSYNTHESIS AND ASSEMBLY OF PURPLE MEMBRANE OF HALOBACTERIUM HALOBIUM S9: LIGHT STIMULATION OF BACTERIORHODOPSIN FORMATION

Abstract— The effect of light on purple membrane biogenesis in Halobacterium halobium S9 strain was investigated. When bacteria were grown in the dark, the 570nm absorption due to bacteriorhodopsin increased more slowly than under illumination, but eventually after longer incubation, reached the same level as that seen in the illuminated culture.
Analysis of membrane fractions by sucrose density gradient centrifugation revealed that two different membrane fractions, containing purple and brown membrane could be detected in the exponential growth phase. Another fraction whose density was higher than that of purple membrane, disappeared concomitantly with the increase in purple membrane and brown membrane, indicating that it may be related to purple membrane formation.
HPLC analysis of membrane proteins showed that there was no significant difference in de novo synthesis of bacterio–opsin between dark and illuminated cultures. This led us to conclude that light stimulated retinal binding to bacterio–opsin and/or retinal biosynthesis rather than bacterio–opsin synthesis. Bacteriorhodopsin seemed to form the brown membrane fraction first, which then spontaneously reorganized into purple membrane.
When incorporated in liposomes, bacteriorhodopsin in brown membrane was found to have rather higher proton pump activity than that in purple membrane. The H⁺ pumping activity was quite heat labile. This and the CD spectra indicated that bacteriorhodopsin in brown membrane might exist without forming normal timer unit.     

THE EFFECT OF IONIC STRENGTH AND pH ON THE PROTONATED SCHIFF BASE AND TYROSINE DEPROTONATION KINETICS DURING THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— The deprotonation kinetics of tyrosine and the protonated Schiff base during the bacteriorho-dopsin photocycle were studied under different perturbations by transient absorption spectroscop Native purple membrane, as well as samples which were deionized (blue) then restored with Na⁺ or La³⁺ were used at pH's ranging from 7 to 10 at very low salt concentrations. The results were compared with previous studies at higher ionic strength. The important conclusions can be summarized as follows: (a) The rate constants of both the Schiff base and tyrosine deprotonation are not very sensitive to the changes of conditions. (b) An almost linear relationship is observed between the relative amplitudes of the tyrosine deprotonated during the cycle and the slow component of the Schiff base deprotonation under the different perturbations studied. This was taken to support the two site model for the protonated Schiff base, one near tyrosine and the other near its ionized form. (c) The pK_a value determined from the ratio of the amplitude of the fast to the slow component of the Schiff base deprotonation is found to decrease with increasing ionic strength of the medium. At extremely low ionic strength, it was found to equal that of the tyrosine phenolic group in solution.     

BRANCHING PATHWAYS IN THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— The pulsed laser photolysis of light-adapted bacteriorhodopsin (BR₅₇₀) is carried out over the temperature range between 25°C and—92°C in neutral and alkaline water-glycerol solutions. The results arc indicative of considerable complexity, introduced by two temperature dependent branching reactions associated with the intermediates K₆₁₀, L₅₅₀ and M₄₁₂, of the BR₅₇₀ photocycle. (a) At relatively low temperatures the primary photoproduct K-₆₁₀ equilibrates with a blue-shifted species, K_p. Both K₆₁₀ and the new intermediate subsequently decay into another species, K'_r, in a process which competes with the formation of L₅₅₀. Finally, K'_p converts very slowly to L₅₅₀. This branched pathway delays the formation of L₅₅₀ and thus of M₄₁₂, without affecting the final yield of either species, (b) A thermal back-reaction regenerating BR₅₇₀ takes place at the stage of L₅₅₀, inhibiting the formation of M₄₁₂. The reaction which also predominates at low temperatures, is relatively inefficient at high pH when the forward L₅₅₀→ M₄₁₂ step is highly catalyzed. It is the superposition of both branching mechanisms, (a) and (b), which accounts for the complex effects of temperature and pH on the photo-cycle of BR₅₇₀. Mechanism (b) is accounted for by a molecular scheme in which deprotonation of a tyrosine moiety at the stage of L₅₅₀ constitutes a prerequisite for deprotonation of the retinal-lysine schiff-base as required for forming M₄₁₂. This scheme appears to be directly related to the proton pump. Mechanism (a) introduces additional complexity in the photocycle at low temperatures but its molecular aspects are still unclear.     

TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY OF THE BACTERIORHODOPSIN PHOTOCYCLE ON THE PICOSECOND AND NANOSECOND TIME SCALES

Abstract— Resonance Raman spectra of the picosecond bacteriorhodopsin intermediate(s) have been obtained by microbeam, flow and subtraction techniques using a synchronously pumped, cavity-dumped dye laser. Nanosecond spectra also were measured with this laser by cavity dumping without mode-locking. The picosecond spectra in the fingerprint region, which is sensitive to the configuration of the retinal chromophore, differ from spectra of the parent bR₅₇₀ but could be correlated to the spectrum of bR^DA₅₅₀ , a “13-cis” species which has been determined from spectra of bR₅₇₀ and bR^DA₅₆₀. The picosecond transient and bR^DA₅₅₀ also are similar in the 950–1050 cm^-1“deuteration fingerprint” region when the medium is changed from H₂O to D₂O. These results suggest that trans—cis isomerization occurs during the 40-ps pulse duration. The shift relative to the parent bR₅₇₀ in the ethylenic stretch region suggests that the picosecond and nanosecond transients absorb at wavelengths longer than 570 nm. The C band at 1646 cm^-1 is found to shift or to broaden upon photolysis in the picosecond time scale. This might suggest a change in the electronic structure of the group and its environment on the picosecond time domain. The nanosecond spectra obtained in this work (with 15-ns pulses) are similar to the spectra previously observed on the 100-ns time scale but are slightly different from the picosecond spectrum. These data suggest that more than one transient species appears on the picosecond-to-nanosecond time scale. The temporal evolution of Raman bands in the fingerprint as well as the low energy (950–1050 cm^-1) region and its implications are discussed.     

PHOTOREACTION OF THE ACIDIFIED FORM OF BACTERIORHODOPSIN AND ITS 9-CIS DERIVATIVE IN PURPLE MEMBRANE AT LOW TEMPERATURES

Abstract— The photoreaction of the acidified form of bacteriorhodopsin and its 9-cis derivative was studied by low temperature spectroscopy.
A short exposure of the acidified form of bacteriorhodopsin, which was prepared by adding 2 m M HC1 to purple membrane suspension in 67% glycerol at 0°C, to red light at – 72°C resulted in the blue-shift of the spectrum. The feature of the shift was very similar to that accompanied by the formation of stable 9- cis acidified form of bacteriorhodopsin at 0°C, but only 13- cis - and all- trans -retinals were found in the extract from this product. No blue-shifted product was found on irradiation at – 190°C.
Irradiation of the 9- cis form of acidified bacteriorhodopsin at -72°C with blue light caused the isomerization of its 9- cis -retinylidene chromophore to 13- cis and all- trans forms without a significant spectral change. It became greater only after the sample was warmed above – 24°C. These results indicate the presence of the light-induced product which has trans configuration on the 9-10 double bond and exhibits the 9- cis type spectrum.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

Ni－Cu和MgO－SiO_2间的相互作用及其对催化性能的影响

本文用ＴＰＲ，ＩＲ，ＴＰＤ和ＴＰＳＲ等技术研究了以表面改性法制备的ＭｇＯ－ＳｉＯ２（ＭＳＯ）表面复合物担载的Ｎｉ－ＣＵ合金间的相互作用及其对ＣＯ加氢反应催化性能的影响．结果表明，ＮｉＯ－ＣｕＯ与ＭＳＯ间的相互作用导致部分ＭＯ与ＭＳＯ形成表面物种从而使金属组分氧化物还原温度明显升高；还原后的Ｎｉ－Ｃｕ／ＭＳＯ表面上存在着两类活性中心，即合金相中的Ｎｉ与载体相中的Ｍｇ２＋（或Ｍｇ２＋－Ｏ）；在两类活性中心的协同作用下，ＣＯ吸附除有孪生、线式和桥式吸附态物种外，还有一种新的卧式吸附态（Ｎｉ．．．Ｃ＝Ｏ→Ｍｇ２＋）．这种吸附态的活化程度较高，易在表面上发生裂解形成Ｎｉ－Ｃ和Ｍｇ２＋－Ｏ，其中Ｎｉ－Ｃ是加氢反应的碳源；Ｈ２在催化剂表面上发生解离吸附形成Ｎｉ－Ｈ和Ｍｇ２＋－ＯＨ，前者比较活泼，是加氢反应的氢原．ＣＯ加氢生成烃类的反应在Ｎｉ中心上按＂表面碳＂机理进行，其生成ＣＺＨ４的选择性高于８０％；Ｈ２Ｏ的生成反应按２（Ｍｇ２＋－ＯＨ）－→Ｍｇ２＋＋（Ｍｇ２＋－Ｏ）＋Ｈ２Ｏ方式进行．     

EFFECTS OF METAL CATIONS, RETINAL, AND THE PHOTOCYCLE ON THE TRYPTOPHAN EMISSION IN BACTERIORHODOPSIN

Abstract— The picosecond fluorescence kinetics of tryptophan residues in bacteriorhodopsin and some perturbed analogs are measured to study the different tryptophan environments and their changes upon metal cation removal, retinal removal, and M₄₁₂ trapping. In bacteriorhodopsin, the emission shows four decay components designated Or, C2r, C3r, and C4r in order of increasing lifetimes. The emission wavelength of C3r and C4r is near that found in aqueous solution, while that of C1r is the shortest. The removal of retinal triples the total emission intensity and reduces the number of components to two, suggesting that the observed variation of the lifetimes in bacteriorhodopsin results from the variation of the energy transfer efficiency between different tryptophans and retinal. We conclude that the Or and C2r emission is from the closest tryptophans to the retinal. The quenching of the C3r emission by all metal cations, including those that cannot act as energy acceptors, e.g. Ca²⁺, is attributed to protein conformation changes caused by metal cation binding which leads to a stronger energy transfer coupling between tryptophans and retinal. The additional quenching of the C2r emission in Eu³⁺bound bacterioopsin is proposed to result from direct energy transfer between tryptophans and Eu³⁺.     

KINETIC MODEL OF BACTERIORHODOPSIN PHOTOCYCLE: PATHWAY FROM M STATE TO bR

A model of the last parts of the bacteriorhodopsin (bR) photocycle is proposed on the basis of experimental data for the kinetic behavior of the 'O' intermediate during a temperature pulse in distilled water suspension. The model includes the previously proposed (but not well characterized) intermediate 'N' between the 'M' and 'O' states of bR. This intermediate exists in fast temperature-dependent quasi-stationary equilibrium with the red-shifted intermediate 'O' and has a maximum of absorption close to the bR spectrum.     

EFFECT OF INTERACTION BETWEEN SURFACTANTS,HLB AND ZETAPOTENTIAL IN EMULSIFICATION

Emulsification of xylene was carried out using mixed surfactant system. HLB concept was used to select the right surfactant mixture. Variation in the stability of emulsion with respect to HLB was investigated by studying interaction between mixed surfactant. The interaction was quantified interms of interaction parameter β. Exact location and nature of the interaction was (hydrophilic / hydrophobic) studied by 1H NMR spectroscopy. Charge present on emulsion droplet was calculated by zetapotential measurement.     

Ni－Cu和TiO_2－SiO_2间的相互作用及其对催化性能的影响

用表面反应改性法制备了ＴｉＯ２－ＳｉＯ２（ＴＳＯ）表面复合物载体．采用ＴＰＲ，ＩＲ，ＴＰＤＭＳ和ＴＰＳＲ－ＭＳ等技术研究了ＮＩ－Ｃｕ／ＴＳＯ间的相互作用及其对ＣＯ加氢反应的催化性能．结果表明，ＮｉＯ－ＣｕＯ与ＴＳＯ间的相互作用导致ＣｕＯ的还原温度降低和ＮｉＯ的还原温度升高，并有少量表面物种生成；还原后的Ｎｉ－Ｃｕ／ＴＳＯ催化剂表面上存在着两类活性中心，即合金相中的Ｎｉ及载体相中的Ｔｉｎ＋（或Ｔｉｎ＋－Ｏ）；ＣＯ在催化剂表面存在孪生、线式、桥式和卧式等４种吸附态；Ｈ２在催化剂表面上发生解离吸附形成Ｎｉ－Ｈ和Ｔｉｎ＋－Ｈ，前者比较活泼，是加氢反应的主要Ｈ源；卧式吸附态极易在催化剂表面裂解形成Ｎｉ－Ｃ和Ｔｉｎ＋－Ｏ，前者是加氢反应的Ｃ源，使ＣＯ加氢生成烃类的反应在Ｎｉ中心上按＂表面碳＂机理进行，其生成乙烯的选择性大于６０％．Ｈ２Ｏ的生成反应在Ｔｉｎ＋中心上按Ｔｉｎ＋－Ｏ与Ｔｉｎ＋－Ｈ或Ｎｉ－Ｈ反应的途径进行．     

EVIDENCE FOR BRANCHING IN THE PHOTOCYCLE OF BACTERIORHODOPSIN AND CONCENTRATION CHANGES OF LATE INTERMEDIATE FORMS

Abstract— Flash photolysis transients of bacteriorhodopsin were recorded with a spectrograph -multielement photodiode array combination and the recordings were analyzed to determine the concentrations of bacteriorhodopsin intermediates "M" and "O" relative to the amount of "bR" cycling (pH 7.1,10–40°C). Estimated concentration time courses were simulated with solutions to two kinetic decay models which could account for photocycle temperature dependence. A unidirectional unbranched decay model overpredicts our estimated levels of [O(r)], whereas a model branched at the "M" intermediate describes each of the later intermediate levels well (with no evidence for an independent "N" form). Our results are consistent with "M" decay regulating the level and rates of change of [bR (t)] and (bR(f)]- and also suggest that two temperature-dependent pathways form "bR" from "M", one directly, and the other indirectly through "O".     

THE EFFECT OF CROSS-LINKING ON PHOTOCYCLING ACTIVITY OF BACTERIORHODOPSIN

Abstract— Purple membrane preparations from Halobacterium halobium were chemically modified with imidoesters. Dimethyl adipimidate (8.3 Å chain length) amidinates about five of the six free lysine residues whereas dimethyl suberimidate (11.3 Å) under the same conditions reacts with only 2–3 residues. Gel electrophoresis showed that the shorter chain length imidoesters were less effective than dimethyl suberimidate in oligomer formation. However, dimethyl adipimidate resulted in a more marked inhibition of the photoreaction activity. Monofunctional imidates, methyl acetimidate and methyl butyrimi-date, at comparable degrees of amidination, did not appreciably affect activity indicating that the presence of bulky groups on the exposed lysine residues does not cause the effects observed. Hence, the introduction of molecular mobility constraints by intramolecular cross-linking slows photocycling, and, therefore, inhibits proton pumping activity of bacteriorhodopsin. This indicates that conforma-tional changes of the protein moiety of bacteriorhodopsin occur during photocycling activity.     

TIME-RESOLVED ULTRAVIOLET ABSORPTION CHANGES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN*

Abstract– The kinetics of the absorption changes associated with the perturbation of aromatic acids during the photocycle of bacteriorhodopsin (bR) were studied at room temperature with microsecond time-resolution. Flash experiments with nanosecond excitation at 532 nm were performed on the purple membrane suspension at a number of measuring wavelengths in the spectral range250–630 nm (to monitor both non-chromophore changes and the photocycle kinetics). The kinetic data collected at different wavelengths were simultaneously fitted with a sum of exponentials to obtain time-resolved UV-VIS difference spectra of photocycle intermediates. This approach allowed us to separate kinetically distinct contributions coupled with tryptophan(s) and tyrosine(s) perturbations. Contributions associated with a reversible perturbation of tryptophans appeared with complex (multistep) kinetics during the bRM transitions and relaxed in a single step during the M0 transition. A contribution associated with perturbation of the local environment of tyrosine appeared before the L and relaxed during the Ob̊ transition.     

BLEACHING OF BACTERIORHODOPSIN IN PURPLE MEMBRANE, BROWN HOLO-MEMBRANE ANDL–1690-SOLUBILIZED MONOMERS WITH HYDROXYLAMINE IN LIGHT AND THE DARK

Abstract— The reactions of hydroxylamine with bacteriorhodopsin in the states of the purple membrane, the brown holo-membrane andL–1690-solubilized monomers were studied in light and the dark. The bleaching rate was strongly dependent on the state of the bacteriorhodopsin and largely enhanced by light. Analysis of the isomeric composition of the resultant retinaloximes by high performance liquid chromatography (HPLC) showed that more all- trans retinaloximes were present in the dark than 13- cis retinaloximes. However, under illumination, the relative amounts of 13- cis retinaloximes increased greatly, indicating the occurrence of trans to cis isomerization during the photochemical cycle of bacteriorhodopsin. Molar extinction coefficients of 13- cis retinaloximes were determined to estimate the isomeric composition of retinaloximes by HPLC analysis.