Studies on the Energetics and Mechanism of Visual Pigment Rhodopsins

Dixon, Sheila F (1986) Studies on the Energetics and Mechanism of Visual Pigment Rhodopsins. PhD thesis, University of Glasgow.

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Three aspects of vision are examined in this work. Firstly, the enthalpy of formation of each of the intermediates in the octopus rhodopsin photocycle were measured photocalorimetrically. The formation of the first intermediate (bathorhodopsin) proceeded endothermically, with storage of ~50% of the exciting photon energy. Formation of the later intermediates, lumirhodopsin, mesorhodopsin, acid metarhodopsin and alkaline metarhodopsin involved release of the stored energy. Experiments were performed on both octopus microvilli membranes and L1690 detergent extracted octopus rhodopsin and there was no significant difference in the results obtained. Variation of the buffer system used in the experiments showed that the enthalpy of the rhodopsin to acid metarhodopsin reaction involved no contributions from protonation changes. In contrast to this, the rhodopsin to alkaline metarhodopsin reaction was found to proceed with the release of ~1 proton/rhodopsin molecule. The energy difference between acid and alkaline metarhodopsin was estimated calorimetrically to be ~ 50 KJ/mole, a value which was confirmed by Van't Hoff analysis on the temperature dependence of the reversible acid ? alkaline metarhodopsin equilibrium. The protonation changes in the acid ? alkaline metarhodopsin equilibrium were studied in two ways: 1. Directly, by formation of both acid and alkaline metarhodopsin in the absence of buffer. This gave a value of 1.08 protons per molecule of rhodopsin, which reinforced the calorimetrically determined value. 2. By acid-base titration of the metarhodopsin equilibrium. This showed a non-ideal titration curve with a value of 0.8 protons per molecule of rhodopsin. The energy profile obtained for octopus rhodopsin was compared with that which had been measured for bovine rhodopsin and a general mechanism for the primary photoreaction step is proposed. Differences which occur between vertebrates and invertebrates at the later intermediates are considered in relation to their environment and pigment regeneration requirements. In the second part of the work, the quantum yield for the forward and backward octopus rhodopsin photoreaction was measured. Potassium ferrioxalate and cattle rhodopsin were used as actinometers. Both systems gave the same result within experimental error. The rhodopsin to acid metarhodopsin reaction was found to have a value of 0.689 +/- 0.016. The regeneration reaction acid metarhodopsin to rhodopsin had a quantum yield of 0.430 +/- 0.017. Two different methods of analysis were used to interpret the results. One was based on theory developed by H. Dartnall, the other on a method devised by A. Kropf. Both methods had to be modified to allow for photoreversal of the acid metarhodopsin to rhodopsin. The results for both methods of analysis were essentially identical. In the third part of the work, the question of hydrolysis of the chromophore-protein bond during the metarhodopsin I to metarhodopsin II transition in cattle rhodopsin was investigated using 0 18 labelled water. The uptake of label was monitored by mass spectroscopy and infrared spectroscopy. Both methods seem to suggest that Schiff base hydrolysis does indeed occur during the metarhodopsin I to metarhodopsin II transition in bovine rhodopsin.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-77367
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 09:10
Last Modified: 14 Jan 2020 09:10

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