Characterization of Peptides and Proteins with Enzymes

Abstract

Publisher Summary It is useful to degrade the proteins to be analyzed with proteolytic enzymes because the specificity of enzymes permits the isolation of uniform fragments. The spectrum of the peptides obtained in this way is sometimes the characteristic of a certain protein. The hydrolysis of proteins to peptides can be done with trypsin, chymotrypsin, pepsin, and other proteinases. The high specificity of trypsin permits the isolation of defined fragments. Trypsin splits peptide bonds wherever the basic amino acids lysine and arginine occur. In this way, peptides with a basic amino acid at the carboxyl end are obtained. The proteins are usually hydrolyzed at room temperature in a buffer solution at the pH optimum of trypsin or if it is desired to work in salt-free conditions in an autotitrator. Chymotrypsin hydrolyzes proteins at the carboxyl group of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan. However, the enzyme is also capable of hydrolyzing amino acid residues that have a space similar to that of the aromatic amino acids. Pepsin hydrolyzes synthetic substrates in aromatic amino acids so that the amino groups of these acids are liberated. Most of the proteins are not completely hydrolyzed by pepsin; often only peptides from the amino or carboxyl end are split off. Peptides obtained by the tryptic hydrolysis of a protein as well as proteins can be degraded stepwise from either the carboxyl or the amino end by enzymes. As these two enzymes are typical exopeptidases, they split off successive amino acids from the carboxyl and the amino end respectively of the protein.