Collis R. Geren

Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom.

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead (Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 +/- 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide power azure and had an intraperitoneal LD50 value in mice of less than 5.1 microgram/g body weight. The enzyme rapidly destroyed fibrinogens ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the alpha-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzymes activity was restored by incubation with ZnCl2.

Proteolytic activity in the digestive fluid of larvae of Trichoplusia ni

The digestive fluid of Trichoplusia ni larvae has caseinolytic activity over a wide alkaline range, with greatest activity at pH 9.8. Activity was linear with respect to time and concentration. The temperature optimum is 45°C. Tryptic and chymotryptic activities were demonstrated using the substrates TAME and BTEE and the inhibitors TLCK, TPCK, and PMSF.

Enzymatic and hemolytic properties of brown recluse spider (Loxosceles reclusa) toxin and extracts of venom apparatus, cephalothorax and abdomen

Loxosceles reclusa venom apparatus extract, toxin, cephalothorax and abdomen extracts were tested for six activities. Protease, lipase, nonspecific hydrolase and direct hemolytic activities were found primarily in abdomen extracts while sphingomyelinase activity appeared predominantly in the toxin. Appreciable complement-mediated hemolysis was not observed.

Pathogenesis of hemorrhage induced by hemorrhagic proteinase IV from timber rattlesnake (Crotalus horridus horridus) venom

The effects on the vascular system of a purified toxin, hemorrhagic proteinase IV, from Crotalus horridus horridus venom were studied with emphasis on the pathogenesis of hemorrhage. White mice were injected intramuscularly with sublethal doses of the hemorrhagic toxin, and tissue samples were obtained at 5 and 30 min, 3 and 24 hr after the injection. There was a good correlation between amount of toxin injected and amount of hemorrhage. Microscopically, extensive areas of hemorrhage around muscle and adipose cells were observed just 5 min after injection. At later time periods the changes were similar, but the hemorrhage was more extensive. Many vessels were plugged by aggregations of platelets. Electron microscopy showed that endothelial cells of capillaries were affected to various degrees. Some were swollen and had plasma membranes that formed large blebs; others were thin and disrupted. In vessels where the intercellular junctions could be observed, they were intact even when the endothelial cells were damaged or ruptured, indicating hemorrhage per rhexis instead of per diapedesis. Basal lamina were often disorganized or absent. Both intravascular and extravascular fibrin deposition were commonly observed. Hemorrhagic proteinase IV from C. h. horridus venom induces hemorrhage per rhexis and platelet aggregation within 5 min of intramuscular injection into mice, and marked fibrin deposition within 30 min of injection.

Purification and characterization of a toxin from brown recluse spider (Loxosceles reclusa) venom gland extracts

Abstract A Toxic protein has been purified from brown recluse spider (Loxosceles reclusa) venom gland extracts. Cation-exchange chromatography at pH 4.0 was used as a high-yield method to purify the toxin to apparent homogeneity, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activities associated with the toxin included lethality to mice, induction of necrosis in rabbits, calcium-dependent hemolysis of human erythrocytes and a decrease in the calcium-induced coagulation time of human plasma. All four activities were destroyed when the toxin was subjected to chemical modification of disulfide bonds. The toxin had an apparent mol. wt of 31,000 in the nonreduced conformation and 37,000 in the disulfide-modified form, as determined by sodium dodecyl sulfate gel electrophoresis. Iodination of the toxin did not destroy any of the four associated activities. Discontinuous polyacrylamide gel electrophoresis of the purified toxin revealed three apparent components. Collection and assay of these components showed that only two of these retained their activities, and the total recovery of activity was less than 40%.

Systemic effect in mice of venom apparatus extract and toxin from the brown recluse spider (Loxosceles reclusa)

Abstract Brown recluse spider ( Loxosceles reclusa ) venom toxin causes death but no necrosis in mice. Six hours after challenge with toxin isolated from venom apparatus extract, mice show elevations of a number of serum enzyme activities indicating damage to the liver and possibly to other tissues including those of the kidney, heart and central nervous system. No obvious changes were apparent in plasma Na + , K + and Ca 2+ concentrations after venom challenge. The hematocrit of venom-challenged animals was not significantly different from controls. Plasma glucose decreased slightly. Urine K + and Ca 2+ concentration increased while Na + decreased. No evidence for in vivo hemolysis was observed, although the venom did cause in vitro hemolysis of mouse erythrocytes.

Detection of galactosyltransferase using chemically modified piezoelectric quartz

Abstract In this report a piezoelectric crystal (quartz crystal microbalance) was used to detect the enzyme galactosyltransferase. A circular hole was etched in the piezoelectrics gold electrode and glucosamine covalently attached to the surface of the exposed quartz. Separate colorimetric testing showed that the glucosamine surface densities were in agreement with those found in the affinity chromatography literature and were estimated at 1.0 μmol/m2. The modified surface was exposed to a drop of test solution containing excess galactosyltransferase and cofactors sufficient to trap analyte in an enzyme—substrate complex on the surface of the crystal. The piezoelectric was removed from solution, dried and the frequency changes caused by the attraction of the enzyme recorded. Shifts in the resonant frequency of the crystal indicated that a complex between surface-bound glucosamine, Mn(II), UDP (uridine 5-diphosphate) and enzyme formed when the crystal was exposed to the liquid phase, but a complicating background signal existed from non-specific adsorption of enzyme, buffer and cofactors. It was also discovered that etching the piezoelectric electrode caused a degradation in crystal Q. In addition, an environmental chamber was constructed for the determination of crystal frequencies. An average drift rate of 0.05 Hz/h was achieved with a reproducibility of frequency measurement for an individual crystal at ±5 Hz.

A thrombin-like enzyme from timber rattlesnake venom.

The procoagulant component has been purified from timber rattlesnake (Crotalus horridus horridus) venom by DEAE-cellulose ion-exchange chromatography followed by affinity chromatography on immobilized p-aminobenzamidine and a final DEAE-Sepharose chromatography. As obtained, the procoagulant gave a single band of Mr 29 500 +/- 2000 on SDS-polyacrylamide gel electrophoresis whether or not the sample was reduced prior to electrophoresis. Schiffs stain indicated the presence of some carbohydrate. The procoagulant showed one predominant and four minor bands on discontinuous gel electrophoresis. All caused fibrinogen solutions to clot. Treatment of the preparation with neuraminidase did not cause the minor bands to comigrate with the major band. Amino acid analysis revealed the presence of eight half-cystines, all of which were present as cystines in the intact molecule. The procoagulant has 13 tryptophans per molecule and an extinction coefficient for a 1% solution at 280 nm of 26.3. This venom procoagulant was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the A alpha-chain of fibrinogen. It was not inhibited by protein trypsin inhibitors, N-ethylmaleimide or dithiothreitol, but it was inactivated by phenylmethylsulfonyl fluoride, indicating an active-center serine. The procoagulant catalyzed the release of negligible acid-soluble peptides from bovine serum albumin, casein and hemoglobin.

Sphingomyelinase D activity of brown recluse spider (Loxosceles reclusa) venom as studied by 31P-NMR: effects on the time-course of sphingomyelin hydrolysis

The time-course for the hydrolysis of the D linkage of chicken egg yolk sphingomyelin in a Triton X-100 mixed micelle and of lysophosphotidylcholine micelles, as catalyzed by brown recluse spider venom and brown recluse spider toxin, was followed by phosphorous-31 nuclear magnetic resonance spectroscopy. The overall rate of hydrolysis of sphingomyelin in mixed micelles was found to be an order of magnitude faster than the hydrolysis of lysophosphotidylcholine. Incorporation of lysophosphotidylcholine into mixed micelles with Triton X-100 inhibited the lipase activity of brown recluse spider venom and brown recluse spider venom toxin. The effects of increased rates of overall reaction were observed with increased temperature and also with decreased ionic strength. The presence of divalent calcium ions was found to be necessary for hydrolytic activity, but only in catalytic amounts (less than 1 mM).

Fractionation and partial characterization of toxic components of timber rattlesnake venom

Abstract The venom of the timber rattlesnake, Crotalus horridus horridus, has been separated into five distinct fractions using diethylaminoethyl cellulose ion-exchange chromatography with elution by an increasing sodium acetate gradient and pH adjustment. Three of these five fractions are lethal and hemorrhage-inducing in mice and also have the ability to catalyze the hydrolysis of hide powder. Of these three, one (fraction Ia) is not retained by the diethylaminoethyl cellulose. This fraction has been further separated into five fractions (A, B, C, D and E) by ion-exchange chromatography on carboxymethyl cellulose. Two distinct lethal activities are apparent in these five fractions. The most basic of these (fraction E) appears to contain only a single protein component of mol. wt 17,000 ± 1000 as determined by SDS gel electrophoresis. In mice it is lethal with paralysis of limbs and respiratory distress apparent prior to death. It has none of the hemorrhagic or enzymatic activities of the whole venom. The other lethal activity is present mainly in fraction B. It consists of, at least, seven protein components and contains the phosphodiesterase and hemorrhagic activities of fraction Ia. It also contains a very weak hide powder hydrolyzing activity. The location of a number of previously reported activities among the various fractions obtained from the two types of chromatographies has been determined.