Purification and Initial Characterization of 124-Kilodalton Phytochrome from Avena

Two procedures for the purification of 124-kdalton (kDa) phytochrome from etiolated Avena seedlings are described. These procedures involve combinations of existing protocols but with two key modifications aimed at precluding previously encountered proteolysis: phytochrome is maintained in its far-red absorbing form (Pfr), and the serine-protease inhibitor phenylmethanesulfonyl fluoride is included in all media until proteolysis is no longer a problem. The initial steps in both procedures are identical but are followed in one case by Affi-Gel Blue chromatography and in the other by immunoaffinity chromatography. The phytochrome preparations obtained by either procedure are >95% homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lack detectable levels of the proteolytically degraded 118- and 114-kDa species that constitute preparations obtained by previous protocols. The spectral properties of 124-kDa phytochrome purified by the Affi-Gel Blue procedure contrast with those of the extensively characterized 118/114-kDa products in several major respects. The purified 124-kDa molecule has a Pfr absorbance maximum at 730 nm, negligible dark reversion with or without sodium dithionite, and enhanced absorbance at 730 nm of the spectrum (predominantly Pfr) obtained after saturating red irradiation relative both to the 673 nm shoulder of this spectrum and to the absorbance maximum of the Pr spectrum at 666 nm. The difference spectrum, with a spectral change ratio (ΔA_r/ΔA_fr) of 1.07, is indistinguishable from that determined in vivo, indicating retention of the spectral properties of the native molecule through the purification procedure. Immunoaffinity-purified 124-kDa phytochrome on the other hand is spectrally denatured and has been used here only for compositional analyses. The amino acid composition of 124- and 118/114-kDa preparations does not differ significantly on a mole percent basis, but in contrast to published data for the 118/114-kDa species, 124-kDa phytochrome has a blocked NH₂ terminus. These data provide further evidence that the proteolytic conversion of 124- to 118/114-kDa phytochrome involves removal of polypeptide segments critical to the structural and spectral integrity of the native molecule and indicate that at least part of this proteolysis involves removal of the NH₂-terminal residue(s).