Vapor-Phase Hydrazinolysis for Microdetermination of Carboxyl-Terminal Amino Acids of Proteins

Abstract

The “vapor-phase” hydrazinolysis method was devised for the microdetermination of the carboxyl-terminal residue of a protein. With this method, a polypeptide sample is degraded with vaporized hydrazine. The optimum conditions for hen egg-white lysozyme were established to be 2 to 4 h at 90 or 100°C, the recovery of the carboxyl-terminal leueine being about 70%. With this vapor-phase method, side reactions are reduced and the time of hydrazinolysis is shortened. The limit of quantitation for the earboxyl-terminus of a protein is about 50pmol, as judged so far with hen egg-white lysozyme. The carboxyl-termini of several proteins were determined using this novel procedure.