title: Porcine muscle prolyl endopeptidase: limited proteolysis of tryptic peptides from hemoglobin beta-chains at prolyl and alanyl bonds.
abstract: Tryptic peptides from hemoglobin (Hb) beta-chains were used as model substrates for limited proteolysis by prolyl endopeptidase (EC 3.4.21.26) from porcine muscle. From the physicochemical and enzymatic properties of prolyl endopeptidase the conditions for routine digestion were established as follows: the molar ratio of enzyme to substrate was 1 to 100, and the reaction was carried out in sodium phosphate buffer (pH 6.4) at 37 degrees C for 4 h. Under these conditions the peptide bonds on the carboxyl terminal sides of proline and alanine residues in the tryptic peptides from Hb beta-chains (with Mr values of less than 2100) were hydrolyzed by the enzyme with the exception of the amino terminal alanyl bond and aminoacyl alanyl bond. In addition, one of five seryl bonds was cleaved by the enzyme. However, the Hb beta-chain itself, Mr 16,600, and its two CNBr-peptides with Mr 10,200 and Mr 6400, respectively, were not hydrolyzed. Under the same conditions a prolyl bond in oxidized B-chains of insulin, Mr 3400, was partially digested, and an alanyl bond was not hydrolyzed. The data indicate that the prolyl endopeptidase is useful for the limited proteolysis of peptides with relative masses of less than 3000 at both prolyl and alanyl bonds.
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D008251:Macaca; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011392:Proline / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases; D013552:Swine; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*
chemical_list: D006454:Hemoglobins; D011392:Proline; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D014357:Trypsin; D000409:Alanine