Purification and characterization of a chymosinlike protease from the gastric mucosa of harp seal (Pagophilus groenlandicus)

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Abstract
Four zymogens of acidic proteases A, B, C and D were isolated from the gastric mucosa of harp seals by ion-exchange chromatography on a DEAE-Sephadex A-50 column. The major zymogens were A and C, and the ratio of zymogen A to zymogen C was greater in extracts from 1-wk-old animals than in extracts from adult animals. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phenylalaninetriethylene tetramine Sepharose and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (EC 3.4.23.4) and porcine pepsin (EC 3.4.23.1). Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis [PAGE]. Zymogen A was homogeneous as judged by sodium dodecyl sulfate (SDS)-PAGE and high performance liquid chromatography, but was heterogenous by PAGE at pH 8.3. Zymogens A and C had MW of 33,800 and 44,000, respectively, as estimated by SDS-PAGE. Protease A had an isoelectric point of 4.90. Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk-clotting to proteolytic activity than those of seal protease C and porcine pepsin and had a pH optimum of 2.2-3.5 for Hg hydrolysis. It did not inactivate RNase, had very low activity on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine and lost activity in 6 M urea. Protease A is chymosinlike.