CORRECTION FOR AMINO-ACID LOSS DURING ACID-HYDROLYSIS OF A PURIFIED PROTEIN

Hydrolyzing a protein in acid for a single hydrolysis interval, normal ly 24 h, will lead to inaccurate estimates of the amino acid compositi on of that protein due to an effect of the time of hydrolysis on pepti de bond cleavage and amino acid degradation. The simultaneous yield an d decay of amino acids during the hydrolysis of a protein can be descr ibed by a compartmental model with parameters for the hydrolysis and l oss rates specific to each amino acid in a protein. The amino acid com position of the protein prior to hydrolysis can be determined by nonli near regression of data derived from multiple hydrolysis intervals. In the present study egg-white lysozyme was hydrolyzed in 6 M HCl using 18 hydrolysis intervals (range, 2-141 h) using the conventional duplic ate hydrolyses/interval system. Hydrolysis and loss rates were determi ned for each amino acid. Increasing the number of hydrolysis intervals prior to the maximum point on the hydrolysis curve, and including an hydrolysis interval greater than 100 h increased the accuracy with whi ch the hydrolysis and loss rates were estimated. Most of the amino aci ds underwent some degree of loss during hydrolysis, Of particular note was the loss rate for cysteic acid, which was greater than that found for serine which is commonly regarded as an acid-labile amino acid. T he determined amino acid composition of the protein, based on the nonl inear regression of the data from four different series of hydrolysis intervals, was compared with the known amino acid composition (sequenc ing). Using the routine duplicate sampling system, a nonlinear regress ion including 10 hydrolysis intervals (2, 6, 10, 14, 18, 22, 26, 30, 6 0, and 141 h) resulted in a mean amino acid recovery of 100% (range, 9 4-110%) and provided an acceptable compromise between accuracy and the cost of analysis. (C) 1996 Academic Press, Inc.