New procedures for isolating polypeptides and proteins from tissues. Metabolic incorporation of l-[14C]valine into fractions of intermediate molecular weight in broad-bean (Vicia faba L.) leaves

Abstract

Phenol-acetic acid-water (11:1, w/v/v) extracted as much as 95% of the N of leaves. Dialysis and chromatog-raphy on Sephadex G-75 of such extracts in the same solvent mixture were used for systematic isolation of polypeptides having molecular weights of a few thousand. After the removal of nonprotein fractions from broad-bean leaves by conventional procedures, a few per cent of the leaf N could be obtained from the protein-rich residues as poly-peptides in this molecular-weight range. These polypeptides are unlikely to be products of degradation of protein formed during the extraction; the leaf proteins do not yield products of low molecular weight during prolonged storage in the solvent mixture. L-[C14]Valine was administered to broad-bean leaves in the transpiration stream under conditions in which substantial incorporation into leaf protein resulted. The polypeptide valine attained specific radioactivities lower than those for the bulk protein. The impurity said to be present in the L-[C14]val-ine by Synge and Youngson (1961) arose during chromatography on paper in the presence of phenol and not by radiochemical decomposition during storage. Extraction with aqueous trichloroacetic add was more effective than the Chibnall ether-water procedure for removing water-soluble radioactive nonprotein substances of low molecular weight from the leaves. The results are discussed in relation to current theories of protein biosynthesis, and some possible uses are suggested for the new solvent mixture in attempting isolation of 0-polypeptidylribonucleic acids. Some of the procedures now described may prove generally useful for fractionation of biological material, particularly when hydrolysis of proteins or polysaccharides must be avoided. Attention is directed to a largely aromatic and glycosidic group of leaf components, which tend to fall into several different fractions both in the present and in traditional schemes for analysis of plant material.