Mechanistic Studies of Retinal Schiff Base Formation and Hydrolysis in Relation to Visual Pigment Chemistry

Robb, Jenifer L (1987) Mechanistic Studies of Retinal Schiff Base Formation and Hydrolysis in Relation to Visual Pigment Chemistry. PhD thesis, University of Glasgow.

Abstract

The possible mechanism of chromophore hydrolysis during photolysis of visual rhodopsin has been investigated by a study of the formation and hydrolysis of the schiff base N-retinylidene n-butylamine in various aqueous anionic, cationic and neutral detergent micelle systems. The schiff base was used as a model of the visual pigment chromophore and the detergent micelles allowed some degree of control on the environment of the chromophoric group in an attempt to mimic the specific environmental effects within the binding site of rhodopsin. The classical schiff base reaction mechanism involves the formation of tetrahedral carbinolamine intermediates and transient species consistent with such intermediates have been observed during the hydrolysis reactions of N-retinylidene n-butylamine in the anionic, cationic and neutral detergents using stopped-flow spectroscopy. It was found that accumulation of the carbinolamine intermediate during hydrolysis is greatest when the pH is near the peak of the pH-rate profile and rapid transients were not observed during hydrolysis away from the pH maxima, nor under any accessible experimental conditions during the formation reactions. The non-steady-state kinetic data obtained from stopped-flow experiments have been analysed using a mainframe computer program called "Discrete", and these data can be described by a double exponential expression which is typical of biphasic reactions involving the formation and decay of an intermediate species. Analysis of the kinetic data, along with data obtained from steady-state kinetics, has allowed the determination of all the rate constants in the retinal schiff base reaction mechanism. The hydrogen ion titration behaviour of N-retinylidene n-butylamine has been studied and the results have shown that there are significant differences in the apparent pK of the schiff base in the various detergents. These differences can be explained by the fact that the micellar environment has an electrostatic effect on the protonation of the schiff base and it is likely that charged groups in the vicinity of the binding site of rhodopsin could affect protonation behaviour in a similar way. This would explain the anomalously low pK, value of the active lysine group in rhodopsin-electrostatic interaction of an adjacent positively charged group would make protonation of the lysine more difficult and so lower its pK. value. Similarly the apparent pK. of the retinal schiff base in rhodopsin is anomalously high because of the formation of a hydrogen bond with this adjacent group. The carbinolamine intermediate formed during schiff base hydrolysis has been observed at room temperature using continuous flow, pH - jump resonance Raman spectroscopy. The spectrum of the intermediate is very similar to the published resonance Raman spectrum of the metarhodopsin II intermediate formed during the bleaching of the visual pigment rhodopsin. This would suggest that the metarhodopsin transition represents the start of the hydrolysis of the schiff base linkage in rhodopsin. A possible mechanism for the hydrolysis step has been suggested and it seems likely that the change of configuration from trigonal to tetrahedral of the carbon atom of the schiff base linkage at the hydrolysis stage may provide the driving force for the activation of the enzyme cascade which results in the generation of the nerve impulse.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Biochemistry
• Date of Award: 1987
• Depositing User: Enlighten Team
• Unique ID: glathesis:1987-77557
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 11:53
• Last Modified: 14 Jan 2020 11:53
• URI: https://theses.gla.ac.uk/id/eprint/77557