ROTATIONAL DIFFUSION EFFECTS ON ABSORBANCE MEASUREMENTS: LIMITATIONS TO THE MAGIC‐ANGLE APPROACH*

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Abstract
Abstract–Optical absorbance changes are commonly used to characterize intermediates which appear in the bleaching sequence of rhodopsin and in the photocycle of bacteriorhodopsin. Absorbance changes can be caused by an intermediate's rotational diffusion, and when this occurs it can distort absorbance changes due to the structural evolution of intermediates. Linear polarization of an optical probe source at 54.7° (the magic angle) relative to the polarization direction of a linearly polarized actinic source has often been used to eliminate signals due to rotational diffusion. We used Jones calculus to investigate the validity of the magic‐angle strategy. Taylor expansion of the result in powers of the absorbances of the bleached ground state and of the intermediates leads to a relatively simple expression which can be used to determine whether rotational contributions are likely under various experimental conditions. This expression shows that in first order no dichroism‐dependent term appears in the absorbance measured at magic angle. In second order, however, linear dichroism contributes to signals. For the sequence of rhodopsin intermediates:rhodopsin → bathorhodopsin BSI → lumirhodopsin.where BSI is a recently discovered blue‐shifted intermediate, we determined the magnitude of the dichroism signals to be, on average, <2% of the true absorbance change due to the intermediates themselves (and hence undetectable). Freedom from dichroism artifacts in this case results from the fact that the transition dipoles of these intermediates are similar to that of rhodopsin. Larger and certainly detectable dichroism signals are predicted to occur, even at the magic angle, for later intermediates which have transition dipole moments which differ significantly from that of rhodopsin. Other strategies for eliminating rotational diffusion effects are compared to the magic‐angle approach.