Proteins enzymatic cleavage, polypeptide chains

The sequence of amino acids in the polypeptide chain (i.e., the primary structure of a polypeptide or protein) can be established by selective chemical and enzymatic cleavage of the protein followed by separation, amino acid analysis, and sequence determination of all peptide fragments. The entire amino acid sequence is established by overlapping identical regions of the individual fragments. The following problem illustrates the procedure. 

The peptides obtained by specific chemical or enzymatic cleavage are separated by some type of chromatography. The sequence of each purified peptide is then determined by the Edman method. At this point, the amino acid sequences of segments of the protein are known, but the order of these segments is not yet defined. How can we order the peptides to obtain the primary structure of the original protein 7Te necessary additional infor mation is obtained from overlap peptides (Figure 3.22). A second enzyme is used to split the polypeptide chain at different linkages. For example, chy-motrypsin cleaves preferentially on the carboxyl side of aromatic and some other bulky nonpolar residues (p, 247). Because these chymotryptic peptides overlap two or more tryptic peptides, they can be used to establish the order of the peptides. The entire amino acid sequence of the polypeptide chain is then known. 

Many proteins contain hundreds of amino acids. To determine their sequences other reactions are required to provide sequences short enough to be determined by Edman degradation. Enzymatic cleavage by two enzymes, trypsin and chymotrypsin, is used to produce smaller peptides. Trypsin cleaves polypeptide chains on the C-terminal side of basic residues such as arginine and lysine. Chymotrypsin cleaves the polypeptide on the C-terminal side of aromatic residues. 

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