PROTON-TRANSFER REACTIONS LINKED TO RHODOPSIN ACTIVATION

Purified bovine rhodopsin solubilized in dodecyl maltoside was photoly zed at 20 degrees C with 477 nm light, and difference spectra were col lected at time delays ranging from 10 mu s to 10 ms after photolysis. Bromocresol purple was added to the samples to detect pH changes in th e aqueous environment due to changes in the protonation state of rhodo psin. The data were analyzed using singular value decomposition and gl obal exponential fitting, which revealed three exponential processes i ndicating the presence of at least four intermediates, Spectral change s of the indicator dye were separated from those of rhodopsin, and pro ton release and uptake rates were analyzed within the framework of rho dopsin photoreaction kinetics. Proton release occurred during Lumi dec ay to Meta-I-380 followed by uptake upon Meta-I-380 decay and by a mor e significant proton uptake with the time course of Meta-I-480 decay. On the basis of the estimated number of protons released and taken up in each step of the rhodopsin photoreaction, we concluded that two for ms of Meta-II are present, The two forms of Meta-II, Meta-IIa' and Met a-IIb, differ in protonation state from one another as do both from th e earlier, 380 nm absorbing form, Meta-I-380.