A highly sensitive method for identification of amino termini of proteins: application to multiple forms of poly(C)-avid ribonuclease and 17β-hydroxysteroid dehydrogenase

Abstract

A radiochemical method for the determination of the amino terminus on very small amounts (0.5–5 nmol) of protein is described. The high sensitivity of the method is achieved by using undiluted 1-fluoro-2,4-dinitro-[3,5-3H]benzene ([3H]Dnp-F) as the labelling reagent under conditions in which a maximum amount of radioactive label is incorporated. Chemical homogeneity is achieved by reacting with excess unlabelled Dnp-F. High recovery is obtained by adding Dnp-albumin as carrier protein. A mixture of Dnp 14C-labelled amino acids is added prior to hydrolysis and identification of the amino terminus is made on the basis of the 3H/14C ratios of the separated Dnp-amino acids. The method was tested on insulin, pancreatic ribonuclease, and lysozyme which gave high 3H/14C ratios only in the expected amino-terminal amino acids. Application to multiple forms of poly(C)-avid ribonuclease gave only amino-terminal lysine. Two of four putative isozymes of 17β-hydroxysteroid dehydrogenase had serine as the amino terminus while the other two had aspartic acid or asparagine.