The photocycle of bacteriorhodopsin at high pH and ionic strength. II. Time-dependent anisotropy studied by partially saturating photoselection.

Photoselection measurements with moderate excitation intensity on bacteriorhodopsin (bR) immobilized in a polyacrylamide gel soaked in 3 M KCl in the pH range 8.0-9.5 resulted in an unusual time-dependent anisotropy. In the microsecond region, the anisotropy exhibits a constant level that is considerably less than 2/5 theoretically expected for the vanishing excitation intensity, indicating partial saturation. In the millisecond region, it becomes time-dependent. Theoretical models for such a time-dependent anisotropy are presented. These models include a consideration of: (i) reorientation of the retinal chromophore during or after excitation, (ii) parallel reactions of differently saturated photoselected species of a heterogenous bR population preexisting in the ground state or photochemically induced, (iii) branching in a photochemical step, and (iv) cooperativity of molecules within a trimer. All of these models describe the anisotropy as a ratio of sums of exponentials, where the rate constants correspond to the kinetics of the photocycle. An analysis of the fitted amplitudes of the exponentials favors the models involving parallel processes rather than those invoking chromophore reorientation.