Spectral properties and chromophore rotation of phytochrome bound to substituted Sepharose

Abstract

Brushite purified phytochrome from Avena sativa L. cv. Sol II was bound to phenyl Sepharose, octyl Sepharose, CNBr‐activated Sepharose and to anti‐phytochrome immunoglobulins immobilized on Sepharose. The spectral properties of phytochrome bound to anti‐phytochrome immunoglobulins and to phenyl Sepharose were similar to phytochrome in solution. Phytochrome bound to CNBr‐activated Sepharose or to octyl Sepharose showed reduced Pfr formation after red irradiation. The reversal to Pr with far‐red light was only partial but a further increase at 667 nm took place slowly in the dark. A peak at 657 nm was seen in the difference spectrum between CNBr‐activated Sepharose‐bound phytochrome kept in darkness and the identical sample immediately after a far‐red irradiation.The change in linear dichroism at 660 nm and 730 nm, induced by plane polarized red or far‐red light, was measured. It was computed that the long‐wavelength transition moment of phytochrome had an average rotation angle of 31.5° or 180°–31.5°. The substrate used for immobilization had a limited effect on the rotation angle. Phytochrome immobilized on CNBr‐activated Sepharose gave an angle of 27.8° and phytochrome immobilized on phenyl Sepharose gave an angle of 32.6°.