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J. Biochem, 2000, Vol. 127, No. 5 755-760
© 2000 Japanese Biochemical Society

other

Isolation and Characterization of Pepsinogen from Trimeresurus flavoviridis (Habu Snake)

Hiroo Yonezawa*,1, Tsuyoshi Nonaka*, Tetsuya UcMkoba*, Shousaku Hattori{dagger}, Motonori Ohno{ddagger} and Makoto Kaneda*

*Department of Chemistry, Faculty of Science, Kagoshima University 1-Konmoto, Kagoshima 890–;0065
{dagger}Amanu Laboratory of Injurious Animals, Institute of Medical Science, University of Tokyo Setouchi-cho, Oshima-gun, Kagoshima 894–h;1500
{ddagger}Department of Applied Microbial Technology, Kumamoto Institute of Technology 4-22-1, Ikeda, Kumamoto 860–0082

1To whom correspondence should be addressed Tel: +81–99–285–8113, Fax; +81–99–285–8117, E-mail: Yonezawa*sci.kagoshima-u.ac.JP

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5–;2.0, and it retained full activity at pH 6.2 and 30°C on incubation for 30 min. The optimum temperature for the snake pepsin was 50°C and it was stable at 40°C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oli-gopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors


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