Multiple peptide composition of the large and small sub-units of Nicotiana tabacum fraction I protein ascertained by fingerprinting and electrofocusing

Abstract

14C-labeled Fraction I protein was synthesized by Nicotiana tabacum leaves during 20 h of photosynthesis in the presence of 14CO 2. The crystalline protein was carboxymethylated, dissociated by sodium dodecylsulfate and subjected to Sephadex G-100 chromatography which resolved the large and small subunits in a 3:1 ratio according to 14C content of the peptides. The large subunit migrated as 56 000 dalton monomers during sodium dodecylsulfate-polyacrylamide-gel electrophoresis; the small subunits as 12 500 dalton monomers. Fingerprinting resolved 54–56 tryptic peptides from the large subunit whereas 53–55 were expected from the number of arginine-lysine residues. Electrofocusing of carboxymethylated Fraction I protein showed the large subunit to be composed of three peptide bands which resolved in the pH 7 region and were nearly equal in density of staining. The number of tryptic peptides of the small subunit was twice the number of arginine-lysines; electrofocusing produced two approximately equal peptide bands in the pH 6 region. The same number of peptides as well as proportions of each was achieved by either staining of the bands or by autoradiography of 14C-labeled Fraction I protein. The three bands of different isoelectric points composing the large subunit have proved to be exceptionally useful as phenotypic markers for chloroplast DNA genes.