Agkihpin, a Distinct SVTLE from the Venom of Gloydius halys Pallas: Purification, Characterization and Structure–Activity Determination

Abstract

Blood clots produced by snake-venom thrombin-like enzymes (SVTLEs) are cleared rapidly, which makes SVTLEs attractive as potential candidates for antithrombotic therapy. We isolated a SVTLE, agkihpin, from the venom of Gloydius halys Pallas. Agkihpin was confirmed to a single-chain TLE with molecular mass of 25.5 kD, pI of 7.43, optimal pH of 8.0 (hydrolyzing TAME), linked carbohydrate absent, and weak fibrinogen clotting activity. It was also found that (i) G. halys might be the latest species in SVTLEs phylogenetic tree; (ii) different level of conservation was shown among the SVTLEs from the Viperidae snakes. Some of those site may account for different activities exhibited by those SVTLEs, especially position 181, at which a fibrinogenolytic activity increase was found when a basic and larger amino acid substituted by a neutral and smaller one; (iii) an extra α-helix constructed with a 'Pro + acidic amino acid + aromatic amino acid' pattern was found in the SVTLEs from Gloydius and Agkistrodon snakes, although it does not necessarily imply an effect on the fibrinogenolytic activity of the SVTLEs. This study provided some new insight into the activity of SVTLE.

A Novel Fast and Efficient Approach to Purify the Thrombin-like Enzyme from Two <i>Bothrops</i>-genus Snake Venoms

“…One of the major consequences of using several steps during a protein purification is the loss of material due to freeze-drying intermediate steps. The purification of Leucuribin from B. leucurus, for instance, was described as 36.7% yield after three chromatographic steps [24]; Agkihpin from G. halys Pallas had a yield of 51% and 5.5 mg of protein were obtained from a gram of venom subjected to four stages of purification [39]; after three chromatographic steps the yield of VLCII from 500 mg of Vipera lebetina venom was about 4.35 mg [3]. The protocol presented in this work allowed us to process half a gram of each venom in a few days with fairly reproducible results.…”