Darragh A J, Garrick D J, Moughan P J, Hendriks W H
Monogastric Research Centre, Massey University, Palmerston North, New Zealand.
Anal Biochem. 1996 May 1;236(2):199-207. doi: 10.1006/abio.1996.0157.
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.
在酸性条件下对蛋白质进行单次水解（通常为24小时），由于水解时间对肽键断裂和氨基酸降解的影响，会导致对该蛋白质氨基酸组成的估计不准确。蛋白质水解过程中氨基酸的同时产生和衰减可以用一个房室模型来描述，该模型具有蛋白质中每种氨基酸特定的水解和损失率参数。水解前蛋白质的氨基酸组成可以通过对多个水解时间段得到的数据进行非线性回归来确定。在本研究中，使用传统的每个水解时间段重复水解系统，将蛋清溶菌酶在6 M盐酸中进行18个水解时间段（范围为2 - 141小时）的水解。测定了每种氨基酸的水解和损失率。在水解曲线的最高点之前增加水解时间段的数量，并包括一个大于100小时的水解时间段，提高了水解和损失率的估计准确性。大多数氨基酸在水解过程中都有一定程度的损失。特别值得注意的是半胱氨酸的损失率，它大于通常被认为是酸不稳定氨基酸的丝氨酸的损失率。基于四个不同系列水解时间段数据的非线性回归所确定的蛋白质氨基酸组成，与已知的氨基酸组成（测序）进行了比较。使用常规的重复采样系统，包括10个水解时间段（2、6、10、14、18、22、26、30、60和141小时）的非线性回归，平均氨基酸回收率为100%（范围为94 - 110%），并在准确性和分析成本之间提供了可接受的折衷方案。
