Spectral and kinetic evidence for the existence of two forms of bathorhodopsin

Transient-absorption difference spectra from 320 nm to 700 nm were obtained at times ranging from 30 ns to 1200 ns after 532-nm photolysis of rhodopsin at room temperature. Kinetics on this time scale at various wavelengths are also presented. The isosbestic points between spectra acquired at successive times after photolysis shift from 510 nm to 530 nm. This shift is inconsistent with a simple process of one bathorhodopsin (BathoR) intermediate being transformed into one lumirhodopsin (LumiR) intermediate on this time scale. The kinetics at 425 nm, 515 nm, and 575 nm could not be fit well to a single-exponential expression. The data are consistent with the existence of two forms of BathoR (BathoR1 and BathoR2) that exhibit different spectra and decay kinetics. The BathoR1 absorption maximum lies near 565 nm, and the BathoR1/LumiR1 isosbestic point is near 430 nm. The BathoR2 absorption maximum lies near 535 nm, and the BathoR2/LumiR2 isosbestic point is near 480 nm. The kinetics at the isosbestic wavelengths were fit to single-exponential expressions corresponding to BathoR1 and BathoR2 lifetimes of 170 +/- 20 ns and 36 +/- 15 ns, respectively.