Light-induced linear dichroism in photoreversibly photochromic sensor pigments 3. Chromophore rotation estimated by polarized-light reversal of dichroism
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Phytochrome from oats (Avena sativa L. cv. Sol II), partially purified on brushite, was immobilized on Sepharose beads to which antiphytochrome immunoglobulin had been covalently linked. The immobilized phytochrome was first brought to the Pr form with unpolarized far-red light. The change in linear dichroism at 660 nm induced by plane polarized red light, and its reversal by plane polarized far-red light were then studied using a dual-wavelength spectrophotometer equipped with polarizing filters. The far-red light was most effective in reversing red-induced dichroism when the angle between the planes of polarization of red and far-red light was approximately 23.degree.. From this it was computed that the long-wavelength transition moment of phytochrome rotates about 29.degree. (or 180-29.degree.) with respect to the protein during conversion from Pr to Pfr. The reverse experiment, using unpolarized red light followed first by polarized far-red light and then polarized red light, with dichroism monitored at 730 nm, also gives most effective reversal for an angle of about 23.degree. between polarization planes, but this corresponds to a transition moment rotation of about 36.degree. (or 180.degree.-36.degree.). The present method is more straightforward but less accurate and confirms an earlier conclusion that the rotation angle is close to 32.degree. (or 180.degree.-32.degree.) in contrast to the "in vivo" value of 90.degree. found by several workers.