Purification and characterization of a fibrinogenase from the venom of Western Diamondback rattlesnake (Crotalus atrox)

A fibrinogenolytic enzyme was isolated from the venom of Western Diamondback rattlesnake (Crotalus atrox) by a three-step procedure involving gel filtration and anion-exchange chromatography. The molecular weight was estimated as 22 900 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 4.65. The enzyme rapidly destroyed the ability of bovine fibrinogen to form a clot on incubation with thrombin. Incubation of fibrinogen with the fibrinogenolytic enzyme for 5 min resulted in the disappearance of the beta-chain of fibrinogen and the appearance of lower molecular weight fragments. Thus the enzyme can be classified as a beta-fibrinogenase. However, on prolonged incubation of the fibrinogen there was also a partial digestion of the alpha-chain. The fibrinogenase showed no activity towards fibrin or casein or arginine esters. The fibrinogenolytic activity was inhibited by phenylmethanesulphonyl fluoride (PMSF) but was unaffected by EDTA.     

Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox.

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.     

A novel high molecular weight fibrinogenase from the venom of Bitis arietans

A fibrinogenase (Ba100) with an apparent molecular mass of 100 kDa under non-reducing conditions and a pI of 5.4 was purified from the venom of the African puff adder (Bitis arietans) by fibrinogen affinity chromatography. Under reducing conditions the protease dissociates into subunits of 21 kDa and 16 kDa. N-Terminal amino acid sequencing showed these two chains to have 66.7% homology and homology to C-type lectins. The fibrinogenase activity of Ba100 cleaves the Aalpha and Bbeta chain of fibrinogen rendering the molecule unable to polymerise into fibrin clots. Ba100 inhibited platelet aggregation in platelet rich plasma, and clot formation in whole blood, in a concentration dependent manner.     

A novel alpha-type fibrinogenase from Agkistrodon rhodostoma snake venom.

By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21,800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved A alpha- and subsequently the gamma-chain of fibrinogen, leaving the B beta-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +/- 4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.     

Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake (Agkistrodon contortrix contortrix)

A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom has been purified by combination of CM-cellulose chromatography, molecular sieve chromatography on Sephadex G-100, p-aminobenzamidine-agarose affinity chromatography, and DEAE-cellulose chromatography. The enzyme, fibrolase, has a molecular weight of 23,000-24,000 and an isoelectric point of pH 6.8. It is composed of approximately 200 amino acids, possesses a blocked NH2-terminus and contains little or no carbohydrate. The enzyme shows no activity against a series of chromogenic p-nitroanilide substrates and is not inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, Trasylol, or p-chloromercuribenzoate. However, the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator). Metal analysis revealed 1 mol of zinc/mol of protein. Study of cleavage site preference of the fibrinolytic enzyme using the oxidized B chain of insulin revealed that specificity is similar to other snake venom metalloproteinases with cleavage primarily directed to an X-Leu bond. Interestingly, unlike some other venom fibrinolytic metalloproteinases, fibrolase exhibits little if any hemorrhagic activity. The enzyme exhibits direct fibrinolytic activity and does not activate plasminogen. In vitro studies revealed that fibrolase dissolves clots made either from purified fibrinogen or from whole blood.     

Properties of glutathione peroxidase isolated from human plasma

Human plasma glutathione peroxidase (GPx) was purified to homogeneity by ammonium sulfate fractionation, gel filtration on Sephadex G-150, chromatography on DEAE Sephacel, chromatofocusing with polybuffer, and gel filtration with Sephadex G-75. This isolation resulted in about 5,400-fold purification of the enzyme with a 32% yield in enzyme activity. The final preparation had a specific activity of about 28 units (mmoles NADPH oxidized) per milligram of protein. Determination of selenium on the purified enzyme revealed a content of 3.8 g atoms per mole GPx. Gel electrophoresis using SDS with standard proteins revealed a molecular weight of about 23,000 for the subunits, which would indicate a molecular weight of about 92,000 for the native enzyme. Amino acid analyses of the purified GPx indicated aspartate, glutamate, proline, glycine, alanine, and leucine as the predominant amino acids and cysteine, methionine, tryptophan, and histidine as the minor amino acids.     

Purification and biochemical characterization of atroxase, a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom

Crotalus atrox venom contains a variety of proteases which render fibrinogen incoagulable and solubilize fibrin. One of these proteases was purified by using ion-exchange and gel permeation liquid chromatography. The protease, called atroxase, consists of a single nonglycosylated polypeptide chain with a molecular weight of 23,500 and an isoelectric point of pH 9.6. Amino acid analysis indicates atroxase to contain 206 residues with no sulfhydryl groups. Metal analysis found zinc and potassium at 1 mol/mol of protein, and calcium at 0.3 mol/mol of protein. Proteolytic activity is inhibited by ethylenediaminetetraacetate and alpha 2-macroglobulin. Maximal proteolytic activity occurs at pH 9.0 and 55 degrees C. Proteolytic specificity, using oxidized insulin B chain, is similar to that of several hemorrhagic toxins found within the same venom, yet atroxase shows no hemorrhagic activity and exhibits low lethality when tested on Swiss Webster mice. Atroxase, an A alpha, B beta fibrinogenase, cleaves the A alpha chain of fibrinogen first followed by the B beta chain and shows no effect on the gamma chain. The nonspecific action of the enzyme results in the extensive hydrolysis of fibrinogen which releases a variety of fibrinopeptides. Fibrin solubilization appears to occur primarily from the hydrolysis of alpha-polymer and unpolymerized alpha and beta chains. Although crude venom induces platelet aggregation, atroxase demonstrated no ability to induce or inhibit aggregation.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom

By Q-sepharose column ion-exchange chromatography, alkyl-sepharose column hydrophobic chromatography the purified fibrinogenolytic enzyme was obtained from Agkistrodon halys halys venom. It is a single peptide-chain with molecular weight about 28 kDa. It was founded that this enzyme cleaved A alpha-chain of fibrinogen, pH-optimum was determined in the range of 7.5-8.0. Its fibrinogenolytic activity was estimated 15.6 mM fibrinogen/min per mg protein; caseinolytic activity was estimated 7.5 c.u., and amidolytic activity was 0.325 mM pNA/min/mg and 0.175 mM pNA/min/mg for S2238 and S2251 respectively; K(m) was 5.6 mM. The enzyme activity was inhibited by DFP and benzamidine. These results suggest that the enzyme is serine protease. It inhibited the platelet-aggregation.     

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.     

Characterisation and inhibition effect of cetrimide on collagenase produced byAspergillus flavus, isolated from mycotic ulcers

Collagenase was found to be the most important enzyme, produced by mycotic keratitis fungi. Therefore,Aspergillus flavus collagenase enzyme has been purified by ammonium sulphate precipitation, Sephadex G25 and DEAE-cellulose chromatography. Electrophoretic analysis for the purified enzyme indicated one subunit of molecular weight of 70–90 KDa when examining on SDS-PAGE. Cetrimide (cetyl trimethyl ammonium bromide) has been tested against the purified collagenase enzyme and indicated reversible competitive inhibitor (kis=0.15 mg/ml) with high promising activity. Cetrimide might be used to inhibit mycotic keratitis fungi.     

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.     

Ornithine decarboxylase from calf liver. Purification and properties

Ornithine decarboxylase (ODC) was purified 25000-fold from calf liver to apparent homogeneity by methods developed to circumvent the lability of the enzyme. Appropriate ratios of sample protein applied to column size and/or gradient size were derived for each purification procedure (ion-exchange, gel filtration ahd hydroxylapatite chromatography, electrophoresis, and thiol affinity chromatography) to maintain enzymatic activity. The enzyme was labile to dilution at all steps of the purification; the inclusion of poly(ethylene glycol) or additional protein decreased but did not eliminate the activity loss. The purified enzyme had a Stokes radius of 3.14 and a molecular weight of 54000. The Km for ornithine was 0.12 mM, and pyridoxal phosphate was 2.0 microM; the pH optimum for the decarboxylation reaction was 7.0. Analysis by sievorptive ion-exchange chromatography indicated the presence of three ionic forms. In the presence of Tris-barbital buffer containing thioglycolic acid, the ODC preparation assumed an apparent molecular weight of 100000 and a Stokes radius of 4.5 and retained full catalytic activity.     

Purification of rabbit liver guanine aminohydrolase.

Rabbit liver guanine aminohydrolase has been purified 1250-fold by utilization of an affinity chromatographic separation on 9-(p-aminoethoxyphenyl) guanine-Sepharose with 50% recovery of activity. Polyacrylamide gel electrophoresis of the purified preparations revealed several protein bans which corresponded to regions of enzyme activity measured on gels which had been run under the same conditons. Gel concentration studies of the protein migration rate showed that the protein bans differed in molecular size. The minimum molecular weight was 100,000 from gel permeation chromatography studies. The pH optimum was near pH 8 and the Km, with guanine as substrate was 5.6 x 10-6 M. The latter values are in close agreement with partially purified preparations described in the literature.     

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.     

A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase

Leuconostoc mesenteroides NRRL B-512F has been mutated by treatment with N-nitrosoguanidine. The resulting mutant (designated as B-512FM) produces 300 times as much enzyme as the parent strain. B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m. The purified dextransucrase had a specific activity of 84 IU/mg, a 100-fold purification with 42% yield, and was shown by SDS-PAGE to have a single protein of molecular weight of 158,000 with dextransucrase activity. The procedure has been used to produce purified enzyme for sequencing. The molecular weight of 158,000 agrees with that calculated from its amino acid sequence.     

Caproyl esterase from rat testis: Purification and action on cumulus cells

Rat caproyl esterase (E.C.3.1.1.1), extracted from testis with Tween 80, was purified by cation exchange and lectin affinity chromatography. The 104-fold purified enzyme had an activity of 840 μmol/hr per mg protein. The purified esterase did not contain any hyaluronidase or N-acetyl-glucosaminidase activity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels revealed a single band of approximately 60,000 molecular weight. The esterase had an isoelectric point of 5.1. Inhibition experiments showed high sensitivity of the enzyme to sulfhydryl agents and complete inactivation by sodium aurothiomalate. The purified caproyl esterase was shown to digest the cumulus matrix from mouse ova.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

CCSV-MPase,a Novel Procoagulant Metalloproteinase from <Emphasis Type="Italic">Cerastes cerastes</Emphasis> Venom: Purification,Biochemical Characterization and Protein Identification

A procoagulant metalloproteinase called CCSV-MPase was purified from C. cerastes venom by successive chromatographic methods starting with gel-filtration through Sephadex G-75; ion-exchange DEAE-Cellulose A-50; affinity chromatography on Benzamidine Sepharose 6B and RP-HPLC on a C8 column. CCSV-MPase has been isolated to an extent of about tenfolds and its molecular mass was evaluated at 70 kDa by SDS–PAGE. CCSV-MPase hydrolyzes casein and fibrinogene as natural substrates. Its proteolytic activity was inhibited by EDTA and 1.10-phenanthroline, a chelators of bivalent cation metals and Zn²⁺ respectively. CCSV-MPase is therefore a Zn²⁺-metalloproteinase with fibrinogenolytic but not hemorrhagic activity. It greatly decreased levels of plasmatic fibrinogen when administered to rats for 24 h. This fibrinogenase hydrolyzes the Bβ chain of human fibrinogen in vitro releasing fibrinopeptide B only. LC MS/MS analysis of tryptic fragments of CCSV-MPase demonstrated that it showed some sequence similarities with four other venom metalloproteinases. CCSV-MPase could be considered as a potential therapeutic agent as it is a non-hemorrhagic enzyme and could be useful in thrombotic diseases because of its defibrinogenation of blood.     

Isolation of a protein C activator from southern copperhead venom

A protease from the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) that activates protein C was purified to homogeneity by a combination of sulfopropyl (SP)-Sephadex C-50, Sephadex G-150 and Mono-S column chromatography. The purified enzyme is a glycoprotein, and migrated as a single band in sodium dodecylsulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 37,000 under non-reducing conditions. Upon reduction with 2-mercaptoethanol, the enzyme exhibited a Mr of 40,000. The purified enzyme prolonged the clotting time of human plasma in a dose- and temperature-dependent manner. Purified bovine protein C was completely activated within 10 minutes upon incubation with the purified protease at a 1:500 enzyme: substrate ratio. This reaction was markedly inhibited by calcium ions. The purified venom protein C activator had no effect on human fibrinogen.