Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: Molecular sequence analysis of its cDNA and biochemical properties

Abstract

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.

Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease fromBothrops pirajaiSnake Venom

“…The BpirSP-39 showed a high thermostability similar to BpirSP27 and BpirSP41 [20], BjussuSP-I [48], and Barnettobin, a coagulant thrombin-like enzyme isolated and characterized from Bothrops barnetti venom [10]. This data confirms the expected results of an enzyme belonging to this class, which possesses considerable thermal stability, differing from metalloproteases which are quickly inactivated when exposed to extreme variations in temperature and pH [43].…”

“…Some of these enzymes are able to cleave only the α or β chains or both chains of the fibrinogen and are therefore known as svTLE-A, svTLE-B, or svTLE-AB, respectively [9]. New serine proteases are constantly being described and/or characterized [10–13]. …”

Structure Insights and Fibrinogen Peptides Molecular Recognition by Bothrombin, A Snake Venom Serine Protease from Bothropus jararaca

“…The treatment usually falls into inhibitor such as diisopropyl fluorophosphates but limited therapies due to serious and life threatening side effects. At present, modern branches such as biochemistry, molecular biology, genetics and pharmacology have grown significantly in the ability to identify specific biological targets [6]. In this continuation, computational tools may add some fast and low-cost input to make data more important to explore such targets in designing new drugs with the aim to decrease fatality.…”

Biochemical and Pharmacological Characterization of TLBbar, a New Serine Protease with Coagulant Activity fromBothrops barnettiSnake Venom

“…The vast majority of snakebites in Peru are inflicted by species of the genus Bothrops [19]. The biology and the biochemistry of a lot of toxins from bothropic venoms have been documented; however, there is scantiness of information about the B. barnetti snake venom, recently been reported the proteomic characterization of the venom and some biochemical characteristics of barnettobin, a thrombinlike enzyme from B. barnetti [20,21]. In this paper we report the purification and the biochemical (proteolytic, kinetic, and structural) and pharmacological characterization (fibrinogenolytic and platelet aggregation) of a new serine protease named TLBbar from B. barnetti snake venom.…”