Komori Y, Nikai T, Ohara A, Yagihashi S, Sugihara H
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan.
Toxicon. 1993 Mar;31(3):257-70. doi: 10.1016/0041-0101(93)90144-8.
A thrombin-like proteinase, named bilineobin, was isolated from Agkistrodon bilineatus venom by Sephadex G-75, DEAE-Sephacel and Heparin-Sepharose CL-6B column chromatography. The purified enzyme has a mol. wt of 57,000 and catalysed the hydrolysis of arginine esters and thrombin substrates Boc-Val-Pro-Arg-MCA and Boc-Asp(OBz)-Pro-Arg-MCA. Although bilineobin converted fibrinogen into fibrin resulting in the production of fibrinopeptides, the activity was relatively low (0.65 NIH units/mg). Fibrinopeptides released upon hydrolysis by this proteinase were identified as fibrinopeptide A (FpA) and fibrinopeptide B (FpB) by measuring fast atom bombardment (FAB) mass spectra and amino acid sequence. This indicates that bilineobin hydrolyses the Arg(19)-Gly(20) bond in the A alpha chain and the Arg(21)-Gly(22) bond in the B beta chain of the bovine fibrinogen molecule. Kinetic study of FpA and FpB release reveals that bilineobin has a preference for cleaving the B beta chain. In addition, bilineobin is resistant to thrombin inhibitors such as hirudin. These suggest that the mechanism of action of bilineobin is similar but not identical to that of thrombin. It was demonstrated that the NH2-terminal region of bilineobin has significant similarities in sequence with thrombin-like proteinases from other snake venoms; however, only three residues were common with thrombin up to residue number 24.
从双斑锦蛇毒液中通过葡聚糖凝胶G - 75、二乙氨基乙基葡聚糖凝胶（DEAE - Sephacel）和肝素 - 琼脂糖CL - 6B柱层析分离出一种类凝血酶蛋白酶，命名为双斑蛇毒素。纯化后的酶分子量为57000，可催化精氨酸酯以及凝血酶底物Boc - Val - Pro - Arg - MCA和Boc - Asp(OBz) - Pro - Arg - MCA的水解。尽管双斑蛇毒素能将纤维蛋白原转化为纤维蛋白并产生纤维蛋白肽，但其活性相对较低（0.65 NIH单位/毫克）。通过测量快原子轰击（FAB）质谱和氨基酸序列，鉴定出该蛋白酶水解后释放的纤维蛋白肽为纤维蛋白肽A（FpA）和纤维蛋白肽B（FpB）。这表明双斑蛇毒素水解牛纤维蛋白原分子Aα链中的Arg(19) - Gly(20)键和Bβ链中的Arg(21) - Gly(22)键。对FpA和FpB释放的动力学研究表明，双斑蛇毒素更倾向于切割Bβ链。此外，双斑蛇毒素对水蛭素等凝血酶抑制剂具有抗性。这些表明双斑蛇毒素的作用机制与凝血酶相似但不完全相同。已证明双斑蛇毒素的NH2末端区域在序列上与其他蛇毒中的类凝血酶蛋白酶有显著相似性；然而，在第24位残基之前，与凝血酶只有三个残基相同。
