A biologically inert photolabile precursor of carbamoylcholine has been synthesized; it is photolyzed to carbamoylcholine, a well-characterized acetylcholine analogue, with a half-time of 40 microseconds at pH 7.0 and a quantum yield of 0.8. The compound, N-(alpha-carboxy-2-nitrobenzyl)carbamoylcholine, was synthesized from (2-nitrophenyl)glycine. The photolysis rates (of five compounds) and the biological activity (of two compounds) were determined, and both properties were found to depend on the nature of the substituents on the photolabile protecting group. Laser pulse photolysis at wavelengths between 308 and 355 nm was used to investigate the wavelength dependence, quantum yield, and rate of the photolysis reaction. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis and by their ability to activate the nicotinic acetylcholine receptor. BC3H1 muscle cells containing those receptors and a cell-flow method were used in the biological assays. The approach described may be useful in the preparation and characterization of other photolabile precursors of neurotransmitters that contain amino groups. The importance of these rapidly photolyzed, inert precursors of neurotransmitters is in chemical kinetic investigations of the reactions involving diverse neuronal receptors; such studies have been hampered because the available techniques have an insufficient time resolution.

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.

We studied an analogue of bacteriorhodopsin whose chromophore is based on all-trans retinal. A five-membered ring was built around the 13-14 double bond so as to prohibit trans to 13-cis isomerization. No light-induced photochemical changes were seen, other than those due to a small amount (approximately 5%) of unbleached bacteriorhodopsin remaining in the apomembrane used for regeneration. The techniques used included flash photolysis at room and liquid nitrogen temperatures and Fourier-transform infrared difference spectroscopy. When the trans-fixed pigment was incorporated into phospholipid vesicles, no evidence of light-initiated proton pumping could be found. The results indicate that trans to 13-cis isomerization is essential for the photochemical transformation and function of bacteriorhodopsin.

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We studied the effects of hydrostatic pressure on the kinetics of the photocycle of purple membrane from Halobacterium halobium. The data were interpreted in terms of a unidirectional and unbranched model. We found that all of the distinct processes of the photocycle are retarded by pressure, with the earlier, fast processes showing less sensitivity to pressure than the later, slow processes. The qualitative similarity of these results with the effects of solvent viscosity on the photocycle kinetics suggests that the primary effects of pressure on the kinetics are via the intrinsic viscosity of the membrane and not via activation volumes. There is a strong quantitative correlation between the pressure effects and the solvent viscosity effects, further supporting this interpretation. We observed a monotonic decrease in the positive absorbance change signal at 640 nm near the end of the photocycle as the pressure is increased. This signal is usually ascribed to the O intermediate, and we interpreted our finding, along with evidence from other experiments, to mean that an ionizable group or groups, such as carboxylic acids, are undissociated and uncharged in O.