眼镜蛇毒纤维蛋白原溶解因子的纯化及理化性质的研究

经Heparin-SepharoseCL-6B亲和层析和SephadexG-150分子筛层析,从中国眼镜蛇毒(Najanajaatra)中纯化出一种可水解纤维蛋白原Aα链的蛋白酶——组分F.SDS-PAGE测得分子量为47100,为一含2%中性己糖的单肽链糖蛋白;它对纤维蛋白、酪蛋白和苯甲酰-L-精氨酸乙酯(BAEE)均无水解作用,也无激活纤溶酶原的作用;100μg皮下或皮内注射,未见出血反应。组分F对酸和热均不稳定。EDTA,EGTA,PMSF和肝素等可抑制其纤维蛋白原溶解活性,benzami-dine,aprotinin和大豆胰蛋白酶抑制因子(SBTI)则无抑制作用。     

Isolation and characterization of nerve growth factor from the venom of Naja naja atra.

Nerve growth factor was isolated from the venom of Naja naja atra by ion exchange and gel permeation chromatography and was found to be homogeneous by disc gel electrophoresis. The molecular weight was estimated to be approximately 20,000 by gel filtration and 22,000 by ultracentrifugation. This protein, which showed an isoelectric point of pH 7.02, probably consists of two subunits of equal molecular weight which are held together or interact with each other noncovalently. The biological activity survives treatment by a number of proteolytic enzymes, such as trypsin [EC 3.4.21.4], chymotrypsin [EC 3.4.21.1], and pepsin [EC 3.4.23.1].     

Amino acid sequence of nerve growth factor purified from the venom of the Formosan cobra Naja naja atra

Nerve growth factor (NGF) was isolated from the venom of the Formosan cobra (Naja naja atra). The amino acid sequence was determined by a combination of conventional methods. The total number of amino acid residues was 116, giving a molecular mass of 13,057 Da. The sequence was identical with that deduced from the nucleotide sequence of an NGF cDNA from the venom gland of Naja naja siamensis, reported by Selby et al. [J. Neurosci. Res., 18, 293-298 (1987)].     

眼镜蛇毒中降压因子的提取及其降血压作用的研究

目的：从广西眼镜蛇蛇毒中分离纯化血管紧张素转换酶抑制剂(Angiotensin Converting Enzyme Inhibitor,ACEI),命名为降压因子（Hypotensive Factor,HF）,并测定其生物活性。方法：采用Sephacryl S-100凝胶过滤,CM Sepharose F.F.离子交换层析分离纯化HF,高效液相鉴定纯度,SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定其分子量,紫外分光光度法测定HF对血管紧张素转换酶（ACE）的抑制活性。用离体兔子十二指肠平滑肌测定HF增强缓激肽(Bradykinin,BK)的效应。结果：纯化的广西眼镜蛇蛇毒HF经SDS-PAGE检测显示单一条带,测得其相对分子量约为8.2kD,由十二种氨基酸组成,蛋白回收率为5.70%。HF对ACE有明显的抑制作用,其抑制作用与剂量呈正相关。IC50为1.02?g/ml。HF能增强BK对离体兔子十二指肠平滑肌的收缩效应。结论：本方法成功地从广西眼镜蛇蛇毒中纯化出降血压成分。该成分与血管紧张素转换酶抑制剂作用相似,对血管紧张素转换酶有明显的抑制作用。     

Circular dichroism study of the unfolding-refolding of a cardiotoxin from Taiwan cobra (Naja naja atra) venom

Circular dichroism spectroscopy has been used to study the unfolding-refolding process of a cardiotoxin from Taiwan cobra (Naja naja atra) venom upon addition of fluoroalcohols or sodium dodecyl sulfate (SDS) to its aqueous solution. In these experiments, the disulfide bridges remained intact. The unfolding process has been found to be reversible both for fluoroalcohols and for SDS unfolding. The reversibility of the unfolding-refolding process of cardiotoxin in aqueous mixtures of fluoroalcohols was dependent on the volume per volume ratio of alcohol to water. SDS did not unfold the secondary structures of cardiotoxin whereas its tertiary structure was affected. If the SDS concentration in aqueous solution exceeded the critical micelle concentration value of SDS, a quasi-refolded state of cardiotoxin was observed. The mechanism of unfolding-refolding is discussed in terms of molecular interactions which might govern the protein conformation in solution.     

Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja)

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits.     

舟山眼镜蛇毒神经生长因子的亲和层析分离

用层析和制备SDS-PAGE法纯化舟山眼镜蛇(Najanajaatra Cantor)毒神经生长因子(NGF),免疫家兔获得抗血清。用辛酸-硫酸铵沉淀法初步纯化IgG,蛋白A-Sepharose亲和层析进一步纯化IgG,并与CNBr活化的Sepharose4B偶联,采用亲和层析法对舟山眼镜蛇毒神经生长因子进行分离纯化。产物经过SDS-PAGE检测呈一条带,并显示了良好的生物学活性。纯化NGF最大比活性为5.0×104U/mg蛋白,亲和常数为4.35×108L/mol,亲和层析分离NGF得率比传统分离方法得率提高35.2%,该方法为NGF的大量提取提供了技术支持。     

Isolation and characterization of hyaluronidase a "spreading factor" from Indian cobra (Naja naja) venom

Hyaluronidase, ubiquitous enzyme in snake venoms, known originally as "spreading factor", has not been well studied. The present study describes the purification and characterization of hyaluronidase from Indian cobra (Naja naja) venom and provides systematic evaluation of the spreading property of the enzyme. Hyaluronidase (NNH1) has been purified through gel permeation and ion exchange chromatography. The molecular mass was found to be 70.406 kDa by MALDI-TOF mass spectrometry and with the (p)i pI of 9.2. The amino acid sequence of the N-terminus was found to be NEQSTHGAYV. The enzyme shows absolute specificity for hyaluronan and belongs to the group of neutral active enzymes. Tetrasaccharides are the final product of hyaluronan digestion. The enzyme cleaves beta 1,4-glycosidic linkage and belongs to a group of endo-beta-N-acetyl hexosaminidases. Hyaluronidase indirectly potentiates the myotoxicity of VRV-PL-VIII, a phospholipolytic myotoxin, and also the hemorrhagic potency of a hemorrhagic complex-I. Localization of hyaluronan in human skin section and selective degradation by venom hyaluronidase (NNH1) corroborate the plausible in vivo degradation of hyaluronan in the extracellular matrix (ECM) resulting in easy dissemination of VRV-PL-VIII myotoxin and hemorrhagic complex-I.     

Characterization of a cytotoxin-like basic protein from the cobra (Naja naja naja) venom

A cytotoxin-like basic protein has been isolated from the venom of the nominate race of cobra (Naja naja naja from Pakistan) by a single step of high-performance liquid chromatography. The primary structure was determined and consists of 62 amino acid residues in a single polypeptide chain. It is highly similar to that of the cytotoxin-like basic proteins isolated from other Naja species, but differs in two of the SS-loop structures from that of cytotoxins.     

Comparison of the structural stability of two homologous toxins isolated from the Taiwan cobra (Naja naja atra) venom

Cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX) isolated from the Taiwan cobra venom (Naja naja atra) are structurally homologous, small molecular weight, all-beta-sheet proteins, cross-linked by four disulfide bonds at identical positions. The conformational stabilities of these toxins are compared based on temperature-dependent chemical shifts and amide proton exchange kinetics using two-dimensional NMR spectroscopy. The structure of CTX III is found to be significantly more stable than that of CBTX. In both the toxins, beta-strand III appears to constitute the stability core. In CTX III, the stability of the triple-stranded beta-sheet domain is observed to be markedly higher than the double-stranded beta-sheet segment. In contrast, in CBTX, both structural domains (double- and triple-stranded beta-sheet domains) appear to contribute equally to the stability of the protein. Estimation of the free energy of exchange (Delta G(ex)) of residues in CBTX and CTX III reveals that the enhanced stability of the structure of CTX III stems from the strong interactions among the beta-strands constituting the triple-stranded beta-sheet domain and also the molecular forces bridging the residues at the N- and C-terminal ends of the molecule.     

Preliminary crystallographic analysis of cardiotoxin V with major fusion activity from Taiwan cobra (Naja naja atra) venom

Crystals of a cardiotoxin from Taiwan cobra venom have been obtained by the vapor diffusion method using methyl pentanediol as precipitant. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 47.5 A, c = 111.3 A, alpha = beta = 90 degrees and gamma = 120 degrees and diffract to a resolution of 2.2 A. There is one molecule per asymmetric unit and the solvent content is estimated to be 53%.     

Purification and properties of the L-amino acid oxidase from monocellate cobra (Naja naja kaouthia) venom.

1. The L-amino acid oxidase of the monocellate cobra (Naja naja kaouthia) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 112,200 as determined by Sephadex G-200 gel filtration chromatography, and 57,400 as determined by SDS-polyacrylamide gel electrophoresis. 2. The enzyme had an isoelectric point of 8.12 and a pH optimum of 8.5. It showed remarkable thermal stability, and, unlike many venom L-amino acid oxidase, was also stable in alkaline medium. The enzyme was partially inactivated by freezing. 3. The enzyme was very active against L-phenylalanine and L-tyrosine, moderately active against L-tryptophan, L-methionine, L-leucine, L-norleucine, L-arginine and L-norvaline. Other L-amino acids were oxidized slowly or not oxidized. 4. Kinetic studies suggest the presence of a side-chain binding site in the enzyme, and that the binding site comprises of at least four hydrophobic subsites.     

Preparation of highly potent Naja naja atra (Formosan cobra) antivenin.

As the preparation of high titer antiserum against Naja naja atra venom is a time-consuming process, attempts were made to develop a immunization procedure for producing highly potent antiserums within a short period. Rabbits were immunized for 12 weeks with (1) whole venom as used routinely in our Institute, (II) whole venom adsorbed on carboxymethyl-cellulose (CMC) and mixed with Freund's complete adjuvant and (III) neurotoxin adsorbed on CMC and adjuvant mixture followed by CMC-whole venom-adjuvant mixture. The results showed that one ml of the antiserum prepared by method (III) could neutralize 445 LD50 of whole venom, in other words, its potency was 4 and 40 times higher than those prepared by methods (II) and (I), respectively.     

Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from the Indian cobra (Naja naja)

The presence of a factor immunologically related to cobra venom factor (CVF) was demonstrated in serum and plasma from the Indian cobra (Naja naja kaoutia). The factor was purified from cobra plasma by affinity chromatography on an anti-CVF gel and was found to consist of a protein composed of two polypeptide chains similar in size to those of human C3. With use of immunoblotting technique, common antigenic determinants were found in the smaller chain of the prepared material and the beta-chain of human C3; the larger chain may display antigenic determinants present in the alpha-chain of human C3. These findings suggest that this molecule represents the C3 of the cobra complement system. Common antigenic determinants were also demonstrated in the alpha-chain of CVF and the beta-chains of human and cobra C3. No reactions were observed between the beta- and gamma-chains of CVF and any antiserum against human C3 or its subunits. Upon immunodiffusion analysis, cobra serum was found to contain a factor besides C3 sharing antigens specific for CVF, while cobra C3 was antigenically deficient compared to CVF. This suggests that cobra C3 physiologically is degraded to a molecule very similar to or identical with CVF.     

Preparation and characterization of immunoglobulin yolk against the venom of Naja naja atra

Chinese Cobra (Naja naja atra) bite is one of the leading causes of snake-bite mortality in China. The traditional anti-cobra venom serum therapy was found to be expensive and with high frequency of side effects. Therefore attempts were made to generate a high titer immunoglobulin from egg yolk (IgY) of crude cobra-venom immunized Leghorn hens, and to standardize an effective method for producing avian antivenom in relatively pure form. The IgY was isolated first by water dilution method to remove the lipid, then extracted by ammonium sulfate precipitation, and purified through anion exchange chromatogram. The different purities of IgY from different isolating stages were submitted to enzyme-linked immunosorbent assay and SDS-PAGE to determine their titers. Immunoblotting showed that the purified IgY (ion exchange chromatography fraction, IECF) recognized several antigenic fractions of cobra venom, and presented with the character of polyclonal antibody. IECF on SDS-PAGE under reducing conditions migrated as a 65 kDa heavy chain and a 35 kDa light chain, respectively. The LD50 of the N. naja atra venom was 0.62 mg/kg body weight in mice. Four times the LD50 dose of venom was selected as challenge dose, and the ED50 of IgY was 3.04 mg IECF/mg venom. The results indicate that the activity of anti-snake venom IgY could be obviously elevated by ion exchange chromatography, thus possessing therapeutic significance for snakebite envenomation.     

Partial sequence of hemoglobin from cobra (Naja naja naja)

Hemoglobin from the cobra snake, Naja naja naja, was isolated and its chains separated on a CM-cellulose column. The separation profile revealed an and two chains having the molar proportions of []₂,[ ₁]₁,[ ₂]₁. The N-terminal amino acid sequence of the intact chains and of the CNBr peptides were carried out. The ₂ chain was found to be heterogeneous comprising a minor component amounting to 11%. This later showed changes at two positions 9 and 14 in the first 30 residues sequenced.