muramidase and peroxyformic-acid

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.

Topics: Amino Acids; Amino Acids, Sulfur; Formates; Hydrochloric Acid; Hydrolysis; Muramidase; Oxidation-Reduction; Reproducibility of Results

A method for the hydrolysis of peptides and proteins in a hermetically sealed microcapillary tube has been developed. The method is based on the concept that oxidative degradation of labile amino acids during acid hydrolysis of proteins and peptides at high temperature can be reduced to a minimum by limiting the ratio of air to liquid (v/v, less than 1:10) in a microcapillary tube. Furthermore, the physical constraints imposed by the capillary tube will restrict the exposure of the protein solution to air at a very limited area at the meniscus of the liquid. This method eliminates the necessity of time-consuming sealing under vacuum and/or flushing with nitrogen to remove oxygen in the hydrolysis tube. High recovery of labile amino acids can be obtained in a reproducible manner. Because of the simplicity and high reproducibility of the method described, it could be the method of choice for the hydrolysis of protein and peptide intended for quantitative amino acid analysis. Performic acid oxidation is performed at 50 degrees C for 10 min instead of 4 to 20 h at 0 degrees C to achieve an equally good yield of cysteic acid and methionine sulfone from peptides and proteins.

Topics: Amino Acids; Animals; Cattle; Formates; Hydrolysis; Microchemistry; Muramidase; Oxidation-Reduction; Proteins; Reproducibility of Results; Ribonuclease, Pancreatic; Trypsinogen