Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat.

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the -phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

Neurotoxicological effects of a thrombin-like enzyme isolated from Crotalus durissus terrificus venom (preliminary study).

A thrombin-like enzyme, purified from the venom of Crotalus durissus terrificus by gel filtration and affinity chromatography, showed a single protein band in Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 33kDa. Clear cellular morphological changes, deep ganglioside level modifications in some brain areas and behavioral alterations in pup rats injected with this protein were detected. Ganglioside composition, one of the chemical markers of brain maturation, was altered specially in the hypothalamus, hippocampus and prefrontal cortex. The most reliable behavioral effects were a delayed, maturation of the righting reflex, posture and motor response after treatment. These effects were consistent with the histological changes revealed in the cerebellum and prefrontal cortex of treated neonate rats, areas related to motor activities.     

~(125)I尖吻蝮蛇毒凝血样酶在兔体内的药物动力学

~(125)Ⅰ标记尖吻蝮蛇毒凝血样酶,放化纯95.5％和比放射性188.7 MBq/mg。 兔iv ~(125)Ⅰ-TLE后,血放射性—时间曲线符合二室模型,算出药物动力学参数。组织分布以肾、胆、肝的放射性最高。     

尖吻蝮蛇毒类凝血酶在兔体内的药代动力学研究

研究单一组份尖吻蝮蛇毒类凝血酶在兔体内的药动学过程。方法：采用放射性核素示踪动力法和聚丙烯酰胺凝胶电泳相结合,检测体液中的原形物浓度,药一时数据用３Ｐ８７程序处理。结果：兔静脉注射尖吻蝮蛇毒类凝血酶 ０．５、１、１．５Ｕ／ｋｇ三个剂量后,ｔ１／２α（快分布相半衰期）在 １２．６～ １６． ５ｍｉｎ范围内,ｔ１／２β（慢分布相半衰期）在 １３１．６～１６４．７ｍｉｎ范围内,１／２γ（消除相半衰期）在９８８～１２９２ｍｉｎ范围内。ＡＵＭ_（０→２４ｈ）（药－时曲线下面积）分别为 ２８３３±３１２、５７８０±２７５和１０７０７±１２ｎｇ／ｍｉｎ／ｍｌ比例为１：２：０４：３．７７。结论：兔静脉注射尖吻蝮蛇毒类凝血酶三个剂量后,药一时曲线经拟合符合三房室模型特征,三个时相的半衰期各剂量组之间无显著性差异,ＡＵＣ与剂量成正比,表明药物在克体内的分布和消除为一级线性动力学过程,排泄以肾脏为主。     

Synthesis of Bz-Phe(p-NO2)-Val-Arg-pNA and its specificity to thrombin-like enzymes: Similarity of a commercial product S-2160 to the nitrated compound

When Bz-Phe-Val-Arg-pNA, distributed by Kabi as S-2160, was synthesized independently in our laboratory, an unidentity was observed in the λmax values between our product and S-2160 although both compounds were indistinguishable by either thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) under ordinary conditions. Further, thrombin cleaved S-2160 faster than our product. To solve the discrepancy both products were thoroughly analyzed, and we found that S-2160 has been distributed as a modified form at the Phe residue to be Phe(p-NO₂). Bz-Phe(p-NO₂)-Val-Arg-pNA was then synthesized as an authentic sample, and the product was compared with the unmodified compound and with S-2160. From the kinetic data, it was confirmed that the nitrated compounds were more susceptible to thrombin than unmodified compound.     

‘Pergularain e I’ - a plant cysteine protease with thrombin-like activity from Pergularia extensa latex

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing ∼ 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependant and time-dependant manner. At 10 μg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of Aα and Bβ chains of fibrinogen and γ-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.     

Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom

A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr ∼29 kDa under reducing conditions and non-reducing ∼25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4 × 10⁻¹ M and the V_max 7.9 × 10⁻¹ nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 °C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF).The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database http://br.expasy.org/ with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom.TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF).In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical-scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources.     

江西蝮蛇蛇毒类凝血酶分离纯化及理化、酶学性质的鉴定

本文采用DEAE—纤维素DE—52离子交换层析及SephadexG—150凝胶过滤法,从江西蝮蛇蛇毒中分离、纯化类凝血酶。该酶为糖蛋白,分子量39,200,pI为4.96,酸性氨基酸含量较高。类凝血酶水解苯甲酰-L-精氨酸乙酯(BAEE)的最适pH为8.0,km为3.57×10~(-4)M,其活性可被二异丙基氟磷酸(DFP)和苯甲基磺酰氟(PMSF)抑制。N-溴代琥珀酰亚胺(NBS)可迅速抑制该酶的酯酶活性。该酶能直接作用于纤维蛋白原使之转变为纤维蛋白引起凝聚,但其凝固速度较牛凝血酶慢。