Formation of aggregates of tryptic fragments derived from the carboxyl-terminal half of pea phytochrome and localization of the site of contact between the fragments by amino-terminal amino acid sequence analysis.

Limited trypsinization of large pea (Pisum sativum cv. Alaska) phytochrome and subsequent size-exclusion chromatography (SEC) in 0.1 M Na phosphate, pH 7.8, yielded a high-molecular-mass aggregate of tryptic fragments of phytochrome. Further SEC in 0.1 M Tris-HCl, pH 7.5, plus various concentrations of NaSCN, indicated that the tryptic-fragment complex contained an aggregate of 7 fragments of molecular mass from 38 to 55 kDa. The amino-terminal sequence of each fragment was determined from the samples electroblotted from sodium dodecylsulfate polyacrylamide gels onto polyvinylidene difluoride membranes, in order to localize the various fragments on the phytochrome polypeptide chain. All of the 7 fragments in the aggregate were found to be derived from the carboxyl-terminal half of phytochrome. A portion of the polypeptide chain (from Ala-752 to Arg-1000) common to all the tryptic fragments has been assigned as the site(s) of contact of the fragments. The tryptic-fragment complex, as well as large phytochrome itself, has been shown by SEC to dissociate to monomers in 2 M NaSCN. The result indicates that the main force involved in maintaining the complex and in contacts between monomers of phytochrome is non-ionic in nature. Relationship between the contact site(s) of the tryptic-fragment complex and large phytochrome monomer is discussed.