尖吻蝮(Agkistrodon acutus)蛇毒凝血酶样成分的研究Ⅲ:蛇毒凝血酶止血效应的研究

将纯化的蛇毒凝血酶(TLE_(3)、TLE_(4)), 经家免试验表明, 2—3凝血酶单位/kg体 重剂量能显著地使家兔全血凝血时间短1/3—1/2。药后1h即有促凝作用, 以2—4h凝血(止血)效应最强, 12h已消失。经家兔及家犬实验性创伤止血实验表明, 对创伤出血有止血作用。表2参11

Fluorescence spectra of hemorrhagin III from the snake venom of Agkistrodon acutus

Hemorrhagin III (AaH III) was separated and purified from the crude snake venom of Agkistrodon acutus, and its molecule weight was determined accurately to be 23; 284.4 +/- 0.1 by LDI1700-MALDI-TOF-MS. Emission spectra of AaH III showed that Trp residues were located by a great degree in the hydrophobic area. Addition of SDS and guanidine-HCl led to change of the molecular conformation of AaH III, and caused the fluorescence quenching of Trp residues. The red-shifted emission band of AaH III after adding guanidine-HCl showed that Trp residues exposed in polar solvents. The effects of pH, EDTA and metal ions on the fluorescence spectra of AaH III were also investigated.

皖南尖吻蝮蛇(Agkistrodon acutus)蛇毒出血毒素Ⅲ(AaHⅢ)的荧光淬灭研究

研究了几种淬灭剂对皖南尖吻蝮蛇蛇毒出血毒素Ⅲ（ＡａＨⅢ）内源荧光的影响。溴代琥珀酰亚胺（ＮＢＳ）法测得每个ＡａＨⅢ分子中含有３个色氨酸（Ｔｒｐ）残基。实验得到丙烯酰胺（Ａｃｒ）和Ｉ－的碰撞淬灭常数Ｋｓｖ分别为３８．３和４．２７Ｌ／ｍｏｌ，ｆａ分别为０．９６和０．９９，Ａｃｒ和Ｉ－均可以淬灭ＡａＨⅢ分子中几乎全部Ｔｒｐ残基的荧光，初步验证Ａｃｒ与ＡａＨⅢ分子间无明显的相互作用。利用荧光淬灭方法，结合ＡａＨⅢ的荧光发射光谱可以推断，ＡａＨⅢ中的Ｔｒｐ残基较大程度位于分子表面的亲水区。

Characterization, crystallization and preliminary X—ray diffraction analysis of acutohaemolysin, a haemolytic toxin from Agkistrodon acutus venom

Acutohaemolysin, a phospholipase A2 (PLA2) from the venom of the snake Agkistrodon acutus, has been isolated and purified to homogeneity by anion-exchange chromatography on a DEAE-Sepharose column followed by cation-exchange chromatography on a CM-Sepharose column. It is an alkaline protein with an isoelectric point of 10.5 and is comprised of a single polypeptide chain of 13 938 Da. Its N-terminal amino-acid sequence shows very high similarity to Lys49-type PLA2 proteins from other snake venoms. Although its PLA2 enzymatic activity is very low, acutohaemolysin has a strong indirect haemolytic activity and anticoagulant activity. Acutohaemolysin crystals with a diffraction limit of 1.60 Å were obtained by the hanging-drop vapour-diffusion method. The crystals belong to the space group C2, with unit-cell parameters a = 45.30, b = 59.55, c = 46.13 Å, [beta] = 117.69°. The asymmetric unit contains one molecule

尖吻蝮蛇凝血酶进入国家一类新药临床实验阶段

由中国科学院昆明动物研究所肖昌华研究员主持的“注射用尖吻蝮蛇凝血酶”研究项目, 经国家药品监督管理局审查, 符合国家一类新药审批的有关条件, 批准于2003年4月进入Ⅰ期临床实验 (批件号: 2003L01430). 尖吻蝮蛇凝血酶为我国特产的尖吻蝮(Agkistrodon acutus) 蛇毒中分离纯化的蛇毒凝血酶(Hemocoagulase, 止血酵素), 其生理作用与从矛头蝮 (Bothrope jararoca)蛇毒中得到的蛇毒凝血酶 (Hemocoagnalse, Reptilase. Batroxobin)相似, 但化学结构则完全不同, 是一种新型结构的蛇毒止血酶, 属一类生化药品制剂。

尖吻蝮蛇毒凝血酶及其生产方法

本发明提供一种具有止血功能的尖吻蝮蛇毒凝血酶Ⅰ及其生产方法。经阴离子交换剂柱层析法分离纯化和快速蛋白纯化工作站程序再纯化，本发明从我国特产尖吻蝮(Agkistrodon acutus)的蛇毒中得到纯度在97％以上的尖吻蝮蛇毒凝血酶Ⅰ；经测定，该酶Ⅰ由A、B两个亚基(链)组成，其中，A亚基含有135个氨基酸，B亚基含有126个氨基酸。与现有蛇毒凝血酶(如Reptilase)相比，它是一个全新的新型酶止血剂；经药理、毒理、药代等研究表明，本发明的尖吻蝮蛇毒凝血酶Ⅰ是一个见效快、副作用小、安全有效的新型止血剂。

一种尖吻蝮蛇毒凝血酶及其生产方法

本发明经阴离子交换剂柱层析法分离纯化及快速蛋白纯化工作站程序再纯化，从我国特产尖吻蝮(Agkistrodon acutus)蛇毒中得到纯度在97％以上的尖吻蝮蛇毒凝血酶Ⅱ；经测定，该酶Ⅱ由A、B两个亚基(链)组成，其中，A亚基含有125个氨基酸，B亚基含有123个氨基酸；与现有蛇毒凝血酶(如Reptilase)相比，它是一个全新的新型酶止血剂。经药理、毒理、药代等研究表明，它是一个见效快，副作用小的、安全有效的新型止血剂。

五步蛇出血毒结构和功能的研究

通过Sephadex-G-100分子筛层析，QAE-Sephadex-A-50阴离子交换层析和两次Poly-buffer-exchanger离子交换层析，分得两个出血毒蛋白，分别称之为HF-1和HF-2。通过碱性PAGE，等电聚焦（IEF），SDS-PAGE和线性密度梯度PAGE，证实两出血蛋白为电泳均一。它们均为三聚体蛋白，分别含有三个相同亚基。HF-1含3 x 113个氨基酸位点，而HF-2含3 x 104个氨基酸位点。EDTA可抑制两者的活性，而碘代乙酸部份抑制HF-2的活性而不抑制HF-1的活性。从氨基酸组成分析发现，HF-1不含有Cys，而HF-1的激光喇曼光谱也没有Cys的特征峰，这在出血毒中是少见的。HF-1和HF-2均属#alpha#－纤溶酶类，同时兼有出血活性和蛋白水解酶活性。还实验了出血HF-2对血小板聚焦的抑制活性，证实HF-2抑制血小板聚集依赖于其对血浆纤原的降解。

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Para) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMP alpha-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 mu g, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both a and beta-chains of the fibrinogen molecule, and it can be inhibited by EDTA. EGTA and beta-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. Published by Elsevier Ltd.

Milkshark (Rhizoprionodon acutus) microsatellite genotype data (six loci) from samples collected from five locations in eastern Australia and central Indonesia.

Biodiversity of sharks in the tropical Indo-Pacific is high, but species-specific information to assist sustainable resource exploitation is scarce. The null hypothesis of population genetic homogeneity was tested for scalloped hammerhead shark (Sphyrna lewini, n=244) and the milkshark (Rhizoprionodon acutus, n=209) from northern and eastern Australia, using nuclear (S. lewini, eight microsatellite loci; R. acutus, six loci) and mitochondrial gene markers (873 base pairs of NADH dehydrogenase subunit 4). We were unable to reject genetic homogeneity for S. lewini, which was as expected based on previous studies of this species. Less expected were similar results for R. acutus, which is more benthic and less vagile than S. lewini. These features are probably driving the genetic break found between Australian and central Indonesian R. acutus (F-statistics; mtDNA, 0.751 to 0.903; microsatellite loci, 0.038 to 0.047). Our results support the spatially-homogeneous management plan for shark species in Queensland, but caution is advised for species yet to be studied.

Negligible evidence for regional genetic population structure for two shark species Rhizoprionodon acutus (Rüppell, 1837) and Sphyrna lewini (Griffith & Smith, 1834) with contrasting biology

Biodiversity of sharks in the tropical Indo-Pacific is high, but species-specific information to assist sustainable resource exploitation is scarce. The null hypothesis of population genetic homogeneity was tested for scalloped hammerhead shark (Sphyrna lewini, n = 237) and the milk shark (Rhizoprionodon acutus, n = 207) from northern and eastern Australia, using nuclear (S. lewini, eight microsatellite loci; R. acutus, six loci) and mitochondrial gene markers (873 base pairs of NADH dehydrogenase subunit 4). We were unable to reject genetic homogeneity for S. lewini, which was as expected based on previous studies of this species. Less expected were similar results for R. acutus, which is more benthic and less vagile than S. lewini. These features are probably driving the genetic break found between Australian and central Indonesian R. acutus (F-statistics; mtDNA, 0.751–0.903, respectively; microsatellite loci, 0.038–0.047 respectively). Our results support the spatially homogeneous monitoring and management plan for shark species in Queensland, Australia.