THE AMINO ACID COMPOSITION OF RIBONUCLEASE

Abstract

Among the properties of ribonuclease which make the protein particularly suitable for structural studies are its low molecular weight and its availability in chromatographically homogeneous form (2). Studies on the chemical structure of the enzyme have been inaugurated by Anfinsen, Redfield, Choate, Page, and Carroll (3) and are also being pursued in this laboratory (1). As complete information as possible on the amino acid composition of the molecule is fundamental to such investigations. The first amino acid analyses of the protein were carried out by Brand (4). The present investigation concerns the application of more recent analytical methods to a chromatographically purified preparation of ribonuclease A. In the following experiments, 22 and 70 hour acid hydrolysates have been analyzed by chromatography on columns of Dowex 50-X4 (5). As in the studies of Harfenist (6) on insulin, Smith and Stockell (7) on carboxypeptidase, and Smith, Stockell, and Kimmel (8) on papain, the use of at least two periods of hydrolysis has proved desirable. The procedure permits corrections to be made for the decomposition of amino acids that appear to be unstable under the hydrolytic conditions and, furthermore, the use of the longer time of heating also insures nearly complete liberation of isoleucine and valine from peptide linkages that may be relatively resistant to hydrolysis. The data thus obtained from the hydrolysates have made it possible to estimate the integral number of residues of each amino acid present in the intact molecule of ribonuclease.