Abstract
A thrombin-like enzyme named TLBbar was isolated fromBothrops barnettisnake venom and its biochemical and pharmacological characteristics were determined. TLBbar was purified using size exclusion chromatography and reverse phase HPLC, showing molecular mass of 28750.7 Da determined by mass spectrometry. TLBbar serine protease is basic (pI 7.4) and its structure shows similarity with other serine proteases of snake venom. Optimal proteolytic activity was at 37°C and pH 8; this activity was strongly inhibited by PMSF and Leupeptin, however; heparin, and soybean trypsin inhibitor (SBT-I) were ineffective. Kinetic studies on BApNA chromogenic substrate have revealed that TLBbar presents a Michaelis-Menten kinetics, with values ofKmandVmaxof 0.433 mM and 0.42 nmol/min, respectively. TLBbar showed high clotting activity upon bovine and human plasma, presenting IC of 125 and minimum dose coagulant (MDC) of 2.23 μg/μL. TLBbar cleavages the Aαchain of bovine fibrinogen, with maximal efficiency at 30–40°C in the presence of calcium after two hours incubation; this fibronogenolityc activity was inhibited by PMSF and Leupeptin, confirming its classification in the group of serine proteases. In addition, TLBbar is capable of aggregating platelets in the same way that thrombin in concentrations of 2.5 μg/μL.
Highlights
The snake venom contains a variety of proteins that are studied in the world for biological and pharmacological importance; within their complex composition has proteolytic enzymes which belong to two groups: serine proteases and metalloproteases
Serine proteases from snake venom (SVSPs) generally have 12 cysteine residues, 10 of which form five disulfide bonds based on homology to the trypsin, with the other two forming a single disulfide bridge conserved among SVSPs found in length C-terminus [11, 12]
5 mg of fraction was dissolved in 250 μL of solution A (0.1% trifluoroacetic acid—TFA) and centrifuged at 4500 ×g and the supernatant was applied on the analytical reverse phase HPLC Supelco C8, previously equilibrated with solution A for 15 min
Summary
The snake venom contains a variety of proteins that are studied in the world for biological and pharmacological importance; within their complex composition has proteolytic enzymes which belong to two groups: serine proteases and metalloproteases. A large number of studies, including molecular cloning, have led to the isolation and identification of TLEs mainly from the venom of subfamily Viperinaeand Crotalinae [3,4,5,6,7,8,9] These enzymes have a common catalytic mechanism, which includes high reactivity of the serine residue that has an important role in the formation of the transient acyl-enzyme complex, which is stabilized by the presence of histidine residues and aspartic acid within the active site. Serine proteases from snake venom (SVSPs) generally have 12 cysteine residues, 10 of which form five disulfide bonds based on homology to the trypsin, with the other two forming a single disulfide bridge conserved among SVSPs found in length C-terminus [11, 12]
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