Simplified protein hydrolysis with methanesulphonic acid at elevated temperature for the complete amino acid analysis of proteins.

The importance of an accurate and convenient method of amino acid analysis for proteins and peptides is becoming increasingly obvious with the recent developments in biotechnology and protein engineering. The amino acid analyzer based on ion-exchange chromatography developed by Moore and Stein’,’ has remained the method of choice. The most commonly used conventional protocol of Hirs et aL3 utilizes 6 M hydrochloric acid and 11&120’%/24 h for hydrolysis of most proteins. A time-course study of hydrolysis extended to 72 h is always needed to ensure a complete hydrolysis of peptide bonds next to some hydrophobic amino acids such as valine or isoleucine, in addition to correction for losses of some labile amino acids such as cystine, tyrosine, serine and threonine residues in the protein samples. The rate-determining step in a successful determination of amino acid compositions is thus dependent on the careful anaerobic preparation of protein hydrolysates and timeconsuming calibration and extrapolation of the data from different time courses of hydrolysis for these amino acids. The determination of tryptophan by amino acid analysis is non-trivial. More than a decade ago, Simpson et aL4 introduced the non-volatile solvent, 4 M methanesulphonic acid containing 3-(2-aminoethyl)indole in place of hydrochloric acid for complete amino acid analysis from a single protein hydrolysate. However, it has not become a standard method compared to the 6 M hydrochloric acid/l 10°C protocol. Recently we have introduced rapid heating for protein hydrolysis using microwave irradiation’. During the refinement and application of this new technique, we have determined the temperature inside the microwave oven to be higher than 150°C. This high temperature offers the advantage of shortening the hydrolysis time from 24 h to several minutes. With this finding in mind, we feel that it is important to study the effect of temperatures higher than the conventional 110°C for hydrolysis upon the accuracy of amino acid analyses for some standard proteins. Previous reports on the application of higher temperatures (> 1 SO’C) in peptide and protein hydrolysis have emphasized the use of mixed-acid solvents such as hydrochloric acid-trifluoroacetic acid or -propionic acid ‘3’ . In this report the use of a high temperature and shorter time has been applied to the amino acid analysis of proteins with a single non-volatile acid, 4 Mmethanesulphonic acid, which yields amino acid composition data including tryptophan and half-cystine. Some simplification and increase in speed relative to the conventional protocol of employing vacuum-sealed tubes in the 6 M hydrochloric