Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography

An enzymatic method for hydrolyzing bovine milk proteins was developed. Pur ified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein ) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, amin opeptidase IM, and prolidase at 37 degrees C for 20 h. Free glutamine and o ther amino acids were derivatized with phenylisothiocyanate and separated u sing a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glu tamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, an d beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical va lues, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to en zymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino aci ds. Free glutamine was found to be very stable (97% recovery) under the enz ymatic hydrolysis conditions. (C) 1999 Academic Press.