The gecko visual pigment: the anion hypsochromic effect.

The 521-pigment in the retina of the Tokay gecko (Gekko gekko) readily responds to particular physical and chemical changes in its environment. When solubilized in chloride deficient state the addition of Class I anions (Cl-, Br-) induces a bathochromic shift of the absorption spectrum. Class II anions (NO3-, IO3-, N3-, OCN-, SCN-, SeCN-, N(CN)2-), which exhibit ambidental properties, cause an hypsochromic shift. Class III anions (F-, I-, NO2-, CN-, AsO3-, SO2(4-), S2O2(3-) have no spectral effect on the 521-pigment. Cations appear to have no influence on the pigment absorption and Class I anions prevent or reverse the hypsochromic shift caused by Class II anions. It is suggested that the spectral displacements reflect specific changes in the opsin conformation, which alter the immediate (dipolar) environment of the retinal chromophore. The protein conformation seems to promote excited-state processes most in the native 521-pigment state and least in the presence of Class II anions. This in turn suggests that the photosensitivity of the 521-pigment is controlled by the excited rather than by the ground-state properties of the pigment.

The visual pigment cyanide effect.

The visual pigment of the Tokay gecko (Gekko gekko) with its in situ absorption maximum at 521 nm has its spectral position at 500 to 505 nm when chloride-deficient digitonin is used for the extraction. In this case the addition of chloride or bromide to the extract restores the maximum to 521 nm. This property, characteristic of gecko pigments in general, does not occur with any of the rhodopsins that have been tested. Simple salts of cyanide, a pseudohalogenoid with an ionic radius close to those of chloride and bromide and/or its hydrolysis product attacks both this gecko pigment and rhodopsins in the dark. This is seen as a slow thermal loss of photopigment if (sodium) cyanide is present at concentrations above 40 mM for the gecko pigment and 150 mM for the rhodopsins of the midshipman (Porichthys notatus) and of the frog (Rana pipiens). In all cases the loss of the photopigment is accompanied by the appearance of a spectral product with maximum absorption at about 340 nm. Cyanide addition has no effect on the photosensitivity of the native pigments and neither does it alter, as do chloride, bromide and other anions, the spectral absorbance curve. The spectral product at 340 nm also appears when the visual pigments are photolyzed in the presence of cyanide salts below the threshold concentrations given above. Incubation of digitonin-solubilized all-trans-retinal with (sodium) cyanide leads to a reaction product with absorption spectrum similar to that obtained with visual pigments under comparable conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

The Gecko visual pigment: the dark exchange reaction and the effects of anions.

A dark reaction is known to occur in retinal extracts of the gecko (Gekko gekko), in which the natural 11-cis-chromophore of the 521-pigment is apparently replaced by adding 9-cis-retinal to form the 9-cis-photopigment. With chloride-deficient extracts the reaction involves some 70% of the 521-pigment. Anions like nitrate, azide, thiocyanate and cyanate that shift the spectrum toward the blue do not affect this 70% exchange. Anions like fluoride, iodide and sulfate likewise do not alter this magnitude of reaction. In contrast, chloride and bromide that induce a bathochromic spectral shift lead to a decrease in this dark replacement of the 11-cis chromophore. This protection is similar to the action of these two anions in antagonizing the pigment loss by NH2OH and by temperature, both occurring in the dark. Apparently, chloride and bromide alter the opsin conformation so as to stabilize and/or protect the Schiff's base linkage but nitrate, azide, thiocyanate and cyanate act at a different opsin site or by a different mechanism.

Discrete charge carrier transfer sites on proteins? A D.C. conductivity study on ultrathin protein films.

D.C. electrical conduction in thin protein films (200 mg/cm2; approximately 2 mu in thickness) of bovine serum albumin and its dinitrophenylated derivatives with different stoichiometric composition was investigated at 17, 20 and 23% relative humidities and at room temperature. Statistically significant decrease in conductivity due to derivatization was observed even at protein-2,4-dinitrophenol stoichiometry as low as 1:2. A charge injection mechanism based upon discrete charge carrier transfer sites could account for the observations. Analogous events may operate in cellular signaling and coding.

The gecko visual pigment: its photosensitivity and the effects of chloride and nitrate ions.

By use of the method of photometric curves, the photosensitivity of the major and ion-sensitive pigment of Gekko gekko has been determined and compared with that of rhodopsins of the frog (Rana pipiens) and of the fish (Porichthys notatus). In the presence of Cl- (or Br-), the gecko pigment has the same photosensitivity as the other A1 rod pigments, but unlike these, the addition of NH2OH does not lead to a Dartnall effect, i.e. an enhancement in the measured rate of photic bleaching. This is because the gecko pigment has no meta-III intermediate. In the Cl- -deficient state the gecko pigment has a photosensitivity 0.8 times that of the Cl- -provided system. The increase in photosensitivity brought on by Cl- is quantitatively accounted for by the Cl- -induced hyperchromic effect. The addition of NH2OH to the system without added Cl- leads to a small increase in measured rate of photic bleaching with an apparent 13% increment in photosensitivity. This is not a classical Dartnall effect for here again no meta-III is involved. The possibility is raised of an additional, yet undiscovered, action of NH2OH on the opsin moiety. Nitrate ions (NO3-) are known to produce an increase in extinction coefficient similar to that of Cl- and a hypochromic shift in the spectral absorbance. Despite the hyperchromic action, NO3- produces a reduction in the measured rate of photic bleaching, an effect explained by the appearance of a meta-III type intermediate absorbing at about 470 nm. While Cl- is able to antagonize the NO3- -induced hypochromic shift, it is unable to reverse the NO3- -induction of meta-III. This, along with other differences in responses of the gecko pigment to these two ions, suggests that Cl- and NO3- act at two different sites and produce unique conformational changes in the protein molecule.

Primary step in the bacteriorhodopsin photocycle: photochemistry or excitation transfer?

The absorption polarization of the first intermediate (K610) formed at room temperature in the proton-pumping photochemical cycle of bacteriorhodopsin (bR) shows a strong correlation with the polarization direction of the photolyzed parent molecule. The results suggest that, unlike other photosynthetic systems, excitation transfer does not take place prior to the primary photochemical change in bR. These observations together with the previously observed circular dichroism and the polarization temperature dependence are discussed in terms of the exciton structure and the nature of the absorption bandwidths (i.e., homogeneous vs. inhomogeneous) of the bR monomers within the trimer structure.

A model system for bacteriorhodopsin chromophore.

The absorption characteristics of bacteriorhodopsin chromophore cannot be understand on the basis of a simple protonated Schiff-base linkage. A possible hypothetical explanation may be an interaction of the aromatic amino acid residues also with retinal. Mixtures of retinal and tryptophan analogues were reacted in organic solvents. Many similarities were found in the absorption spectra of the different products of these reactions and in those of the main forms of bacteriorhodopsin photocycle. Such products are suggested to model the purple complex of bacteriorhodopsin as well as the chromophores of the photointermediates.

Time-resolved protein fluorescence studies of intermediates in the photochemical cycle of bacteriorhodopsin.

The photolysis-induced changes in the protein fluorescence intensity (at 320 nm) during the proton-pumping cycle of bacteriorhodopsin were examined by a delayed two-pulse technique in the time range 1 microsecond-20 msec at room temperature. No detectable change in the protein fluorescence intensity was observed on the earliest time scale within the lifetime of the intermediate K590, when retinal apparently undergoes the largest structural changes. The time dependence of the relative changes in fluorescence intensity did, however, display a close correlation with the population of the L550 and M412 intermediates. From a computer numerical fit of the data, with available published kinetic parameters, the protein fluorescence quantum yields of the K590, L550, and M412 intermediates are found to be 1.0, 0.92, and 0.80 of that for native bR570, respectively. The probable mechanisms of the observed fluorescence quenching during the photochemical cycle are qualitatively discussed.

Laser Raman studies of molecular interactions with phosphatidylcholine multilayers. II. Effects of mono- and divalent ions on bilayer structure.

Laser Raman spectroscopy was applied to characterize structural behavior of dipalmitoyl phosphatidylcholine multibilayer systems in the presence of several cations (K+, Na+, Cs+, Rb+, Ca2+, Mg2+, Cd2+, Ba2+) and anions Cl-, Br-, I-, NO-3, SO2-3, SO2-4, S2O2-3, S2O2-8). To evaluate the Raman-spectroscopical data quantitatively, characteristic intensity ratios, lateral and trans order parameters were used and compared. It was shown that the different trans order parameters are rather sensitive to ion-polar head group interactions and thus, they cannot give unequivocal information on the trans-gauche isomerization of hydrocarbon chains of phospholipids. The observed effects of ions on Raman spectra of phospholipid multilayers could not be explained simply on the basis of electrostatic interactions. The possible involvement of other factors (changes in polarizability, hydrogen bonds, etc.) is also discussed. It was demonstrated that the order parameters defined in different ways may result in different effectiveness sequences of ions. Of monopositive ions Na+ was found to be the most effective to influence the bilayer structure. For dipositive ions, of which Ca2+ proved to be the most effective, concentration-dependent effectiveness sequences were obtained. A plausible interpretation and some consequences of the concentration-dependent two-step binding of divalent cations were also outlined. Bilayered phospholipid structures turned out to be more responsive to anions than to most cations investigated. Interdependent actions of cations and anions, as well as the possible relevance of the charge distribution on anions are postulated.

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane.

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M412) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.

Comments on the quantitative interpretation of biomembrane structure by Raman spectroscopy.

The possibility of quantitation of information obtained from laser Raman spectra of aqueous lipid dispersions is discussed. It is shown that the all-trans chain order parameter ST introduced by Gaber and Peticolas ((1977) Biochim. Biophys. Acta 465, 260) for the characterization of biomembrane structure is of restricted applicability. This order parameter may give adequate information if polar head groups are not affected at all by the interaction resulting in trans-gauche isomerization. To demonstrate this, data on the effects of mono- and divalent ions on the all-trans chain order parameter are given. The lateral order parameter proved to be suitable for quantitative studies even in the case of ion-head group interaction.

Laser Raman studies of molecular interactions with phosphatidylcholine multilayers. I. Effect of iodine-iodide system.

The interactions of iodine, iodide and triiodide with phosphatidylcholine multilayers have been investigated in order to find supporting data for conduction mechanisms of iodine-doped bimolecular lipid membranes. On the basis of order-disorder parameters it can be concluded that phospholipid multilayers interact with iodine rather than with its ionic forms. No evidence for the existence of triiodide chains in iodine-doped multilayers has been found. Consequently, the resistance-lowering effect of iodine must be based upon direct iodine-lipid interaction and iodine penetration rather than upon ion transport processes.