Effect of NADPH on formation and decay of human metarhodopsin III at physiological temperatures

Difference absorption spectra were recorded during the formation and decay of metarhodopsin III after sonicated membrane suspensions of rhodopsin were bleached at 37 degrees C. The data were analyzed using SVD, spectral decomposition and global exponential fitting. By comparison of the results in the presence or absence of 70 microM NADPH and those for bovine or human rhodopsin, a single comprehensive scheme was fit to all the data, including reduction of retinal to retinol by the intrinsic retinol dehydrogenase. On the time scale studied the mechanism involves two 382 nm absorbing species and two 468 nm, absorbing species, supporting the notion that human metarhodopsin III is not a homogeneous species. The results confirm that metarhodopsin III forms and persists sufficiently long in the human retina under physiological conditions that it could undergo secondary photoisomerization.