Rhodopsin photoenergetics: lumirhodopsin and the complete energy profile

Abstract

1. Introduction Various intermediate stages in the photolysis of visual pigments have been recognised, but neither their stereochemical form nor their relation to the actual process of visual excitation have been resolved (review [I]). The relative energies of many of the intermedi- ates in the bovine rhodopsin bleaching sequence have been determined in recent years by direct photocalo- rimetric methods [2-I]. Here I report the measure- ment of the enthalpy change associated with the light- induced formation of lumirhodopsin in bovine photo- receptor membranes at -75°C. This completes the sequence of calorimetrically accessible intermediates, and allows construction of an energy profile for the entire process which establishes firm thermodynamic constraints for proposed molecular mechanisms. 2. Materials and methods Bovine rod outer segment membranes (ROS) were prepared by a standard sucrose density gradient tech- nique [5] from the retinas of fresh, dark-adapted cattle eyes, obtained locally. Samples for photocalo- rimetry were prepared by suspension of ROS in a mixture of glycerol (2 vol.) and 0.1 M sodium phos- phate buffer (1 vol.), pH 7.2 at room temperature. Rhodopsin concentrations and the extent of bleaching during calorimetry were determined from room tem- perature absorbance spectra of aliquots of the ROS suspension dispersed in 2% (w/v) CTAB (cetyltrimeth- ylammonium bromide, Sigma Chem. Co.), 0.1 M phosphate buffer, pH 7 .?I, containing 10 mM hydrox- ylamine hydrochloride, assuming a molar extinction coefficient of 41 000 at 500 nm. Unless otherwise stated, all operations were performed in the dark or under dim red light. 324 Instrumental details of the low-temperature photo- calorimeter have appeared in [3,6]. For these experi- ments the entire calorimetric unit was surrounded by a copper circulation coil and suspended in a Dewar vessel filled with methanol. The system was further insulated with expanded polystyrene foam and mounted in a temperature-controlled ( 20 f 1°C) darkroom. The calorimeter was cooled to -75°C by circulation of methanol from a low-temperature bath, modified for proportional temperature control with a Pt resistance probe and precision electronic controller. Sample illumination was via glass fibre optics from a grating monochromator and a stabilised 200 W xenon arc lamp. Electrical calibration gave an instrumental sensitivity of 0.071 pV/pW under these conditions. The amplified signal, integrated over 60 s intervals, was recorded digitally as a function of time through- out an experiment. In a typical experiment the photocalorimeter was loaded with 2 .O ml ROS suspension (Esoo = 1) together with a reference sample of the same material totally bleached at room temperature prior to loading. After thermal equilibration at -75°C for several hours (usually overnight) the sample was irradiated briefly at 480 or 520 nm and the differential thermal response recorded. The sample was then removed, warmed to room temperature, and an aliquot taken for spectral determination of the amount of photoreaction in comparison with starting material. Control experi- ments included trials with bleached material in both calorimeter cells. 3. Results and discussion Lumirhodopsin is the kinetically stable photo- product of bovine rhodopsin at -75°C and the reac- tion only proceeds to subsequent stages on warming