Correction for amino acid loss during acid hydrolysis of a purified protein.
Hydrolyzing a protein in acid for a single hydrolysis interval, normally 24 h, will lead to inaccurate estimates of the amino acid composition of that protein due to an effect of the time of hydrolysis on peptide bond cleavage and amino acid degradation. The simultaneous yield and decay of amino acids during the hydrolysis of a protein can be described by a compartmental model with parameters for the hydrolysis and loss rates specific to each amino acid in a protein. The amino acid composition of the protein prior to hydrolysis can be determined by nonlinear 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 duplicate hydrolyses/interval system. Hydrolysis and loss rates were determined 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 which the hydrolysis and loss rates were estimated. Most of the amino acids 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. The determined amino acid composition of the protein, based on the nonlinear regression of the data from four different series of hydrolysis intervals, was compared with the known amino acid composition (sequencing). Using the routine duplicate sampling system, a nonlinear regression including 10 hydrolysis intervals (2, 6, 10, 14, 18, 22, 26, 30, 60, and 141 h) resulted in a mean amino acid recovery of 100% (range, 94-110%) and provided an acceptable compromise between accuracy and the cost of analysis.
Darragh
A J
AJ
Monogastric Research Centre, Massey University, Palmerston North, New Zealand.
Garrick
D J
DJ
Moughan
P J
PJ
Hendriks
W H
WH
D000596:Amino Acids / Q000032:analysis*; D000603:Amino Acids, Sulfur / Q000032:analysis*; D005561:Formates; D006851:Hydrochloric Acid; D006868:Hydrolysis; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D015203:Reproducibility of Results
D000596:Amino Acids; D000603:Amino Acids, Sulfur; D005561:Formates; C008817:peroxyformic acid; D009113:Muramidase; D006851:Hydrochloric Acid