Purification and characterization of anticoagulant protein from the tabanus, Tabanus bivittatus.
Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-Ile-Asp-Lys-Val-Arg. The protein was activated by Cu2+ and Zn2+, and the optimal conditions were found to be at pH 3-6 and 40-70 degrees C. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.
Ahn
Mi Young
MY
Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea. amy@rda.go.kr
Hahn
Bum Soo
BS
Lee
Pyeong Jae
PJ
Wu
Song Ji
SJ
Kim
Yeong Shik
YS
D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects*; D002413:Cations, Divalent; D002863:Chromogenic Compounds; D003300:Copper / Q000737:chemistry; D004175:Diptera / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D008970:Molecular Weight; D010314:Partial Thromboplastin Time; D011487:Protein Conformation; D013696:Temperature; D013917:Thrombin / Q000037:antagonists & inhibitors; D015032:Zinc / Q000737:chemistry
D000925:Anticoagulants; D002413:Cations, Divalent; D002863:Chromogenic Compounds; D019476:Insect Proteins; D003300:Copper; D013917:Thrombin; D015032:Zinc