Characterization of a Novel Thrombin-like Enzyme, Globlase from the Venom of Gloydius blomhoffii (Japanese Mamushi)
Yumiko Komori *
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan.
Yusuke Takashima
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan.
Shoko Ono
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan.
Toshiaki Nikai
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan.
*Author to whom correspondence should be addressed.
Abstract
Aims: To elucidate the coagulation mechanisms of a novel clotting factor isolated from Gloydius blomhoffii venom, its hydrolytic activity on various substrates were examined. Furthermore the primary structure was determined and compared with the other snake venom components.
Methodology: A thrombin-like enzyme was isolated from the crude venom of G. blomhoffii by DE52 Cellulose and CM52 Cellulose column chromatography. Enzyme activity was measured by using synthetic substrates (arginine esters, MCA-substrates and 3-(Acyloxy)-4-nitrobenzoic acid). Effect on fibrinogen was detected with bovine and human fibrinogen. Isoelectric point and molecular mass were measured by polyacrylamide gel electrophoresis and MALDI-TOF-MS. Amino acid sequence was decided with a protein sequencer by analyzing enzymatically cleaved peptides.
Results: A clotting factor was found to be homologous as indicated by a single band on SDS-PAGE, and the final preparation was named as globlase. Molecular mass of this enzyme was determined to be 13,876.36 Da and the isoelectric point was 8.8. Globlase showed arginine ester hydrolytic activity, and specificity for substrates of thrombin. Proteolytic activity and phospholipase A2 (PLA2) activity were not detected. Complete amino acid sequence analysis indicated that the primary structure of globlase is similar to PLA2. However the aspartic acid which exists in the active site of PLA2 was found to be substituted by glutamine.
Conclusion: It was shown in our current investigation that globlase is a novel thrombin-like enzyme isolated from G. blomhoffii venom. It was revealed that this enzyme had structure unlike the serine-protease such as the other thrombin-like enzymes.
Keywords: Gloydius blomhoffii, thrombin-like enzyme, primary structure, phospholipase A2.