LINEAR DICHROISM MEASUREMENTS ON ORIENTED PURPLE MEMBRANES BETWEEN PARALLEL POLARIZERS - CONTRIBUTION OF LINEAR BIREFRINGENCE AND APPLICATIONS TO CHROMOPHORE ISOMERIZATION

Absorption measurements with linearly polarized light were performed w ith oriented purple membranes in the wavelength range from 350 to 750 nm. The membranes were oriented in a magnetic field and subsequently i mmobilized in a gel. Alternatively orientation was achieved by anisotr opic swelling of a dehydrated gel. This new method of orientation, whi ch is similar to the gel-squeezing technique, yields a negative second -order parameter S-2 (e.g., the membranes orient preferentially with t heir planes parallel to the direction of expansion). In contrast to st andard linear dichroism measurements, we used a setup where the orient ed sample is inbetween two parallel polarizers. The dependence of the transmitted intensity on the angle between the orientation axis and th e polarizer direction shows that the phase difference due to linear bi refringence contributes substantially to the signal. Experimental data of high accuracy were obtained by taking measurements at 14 angles fr om -20 degrees to 110 degrees in steps of 10 degrees. Fitting of the p olarization data at each wavelength yields the absorption spectrum, th e anisotropy and the linear birefringence. Analogous measurements were carried out on chromophore-free samples to correct for light scatteri ng and to extract-the chromophore contribution to the linear birefring ence. The chromophore part of the linear birefringence is in good agre ement with numerical calculations from absorption and anisotropy via t he Kramers-Kronig transform. The method was applied to investigate pos sible changes in transition dipole moment orientation associated with the isomerization of the chromophore in light-dark adaptation (all-tra ns to 13-cis) and the blue-pink transition (all-trans to 9-cis). Analy sis of the anisotropy indicates that there is no change in the orienta tion of the transition dipole moment with respect to the orientation a xis between the light and dark adapted states. Only a small increase ( approximate to 2.5 degrees) of the chromophore angle occurs between th e blue state and the pink state. Comparison of the measured birefringe nce changes and the calculations from absorption and anisotropy change s confirm the validity of the approach.