myotoxin-a and Muscular-Diseases

Many rattlesnake venoms (family Crotalidae) contain small, highly basic toxins which cause contracture and necrosis of skeletal muscle in experimental animals. Isolation of these myotoxins requires several chromatographic steps taking several days to complete. We report the isolation of crotamine, myotoxin a, and myotoxin a-like molecules from whole venom of three species of rattlesnakes using a Hydrazide Avidchrom Cartridge. Milligram amounts of highly purified and biologically active myotoxin can be obtained in 30 min to 2 hr.

Topics: Animals; Antibodies; Chromatography, DEAE-Cellulose; Crotalid Venoms; Electrophoresis, Polyacrylamide Gel; Female; Immunoblotting; Mice; Muscular Diseases; Toxins, Biological

Antiserum against myotoxin a was purified using affinity chromatography. Myotoxin a was conjugated to an Affi-Gel agarose gel bead support and crude antiserum applied to the column. Antibody was eluted with distilled water, acetic acid and phosphate-buffered saline, and the protein concentration in the effluent was estimated by the absorbance at 280 nm. Antibody eluted with distilled water was used to develop an ELISA to detect antibodies to myotoxin in the bloodstream. Mice were injected with either 0.10, 0.15 or 0.20 ml of crude antiserum, and blood samples were taken during a four-week period. Samples were assayed for antimyotoxin using the antibody detection ELISA. Blood levels of antimyotoxin decreased significantly (P less than 0.05) within 1 hr after mice received 0.10 ml of crude antiserum (i.v.). Levels of antimyotoxin in mice given 0.15 and 0.20 ml of antiserum decreased significantly at 3 hr after the injection. Mice given 0.20 ml antiserum had significantly (P less than 0.05) higher amounts of antimyotoxin than mice receiving 0.10 ml antiserum during the 24-hr period after injection. However, after 24 hr all three treatment groups had less than 100 ng antimyotoxin/ml and did not differ significantly from one another. Measurement of antivenom in the bloodstream of snakebite patients might help determine if and when additional antivenom should be administered.

Topics: Animals; Antibodies; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Crotalid Venoms; Enzyme-Linked Immunosorbent Assay; Immunodiffusion; Male; Mice; Muscular Diseases; Necrosis; Neutralization Tests; Rabbits

Mixtures containing polyvalent (Crotalidae) antivenin and antimyotoxin a serum were tested for their ability to neutralize the myotoxic, hemorrhagic and lethal activities of crude C. v. viridis venom when mixed with the venom prior to injection into white mice. A light microscopic method was used to measure the local myotoxic activity of the venom, i.e. myonecrosis index. The results show that the neutralizing ability of a 1:1 mixture of antisera for myonecrosis was 16 times that of antimyotoxin a serum alone and 63 times that of antivenin alone. There was no difference in neutralizing ability of the three ratios (2:1, 1:1, 1:2) of antivenin: antimyotoxin serum tested. Hemorrhage was measured by a new method in which the amount of hemoglobin in a muscle extract was measured after i.m. injection of test solution. The results show that the ability of a 1:1 mixture to neutralize hemorrhage was comparable to that of antivenin alone. There was no difference in hemorrhage neutralizing ability of the three ratios tested. In its ability to neutralize lethality, the 1:1 mixture was again comparable to antivenin. However, when the three different ratios of antisera were tested for neutralization of lethality the 2:1 and 1:1 ratios were as effective as antivenin alone, whereas the 1:2 ratio (antivenin: antimyotoxin serum) was less effective than antivenin alone. Thus the addition of antimyotoxin a serum to antivenin in equal proportions greatly improves the neutralization of the myotoxic activity of C. v. viridis venom and does not decrease the ability of antivenin to neutralize hemorrhage and lethality.

Topics: Animals; Antivenins; Crotalid Venoms; Female; Hemorrhage; Immune Sera; Lethal Dose 50; Mice; Muscular Diseases; Necrosis

Antiserum to myotoxin a was tested for its ability to prevent local myonecrosis induced by myotoxin a and C. v. viridis venom. Antiserum was injected i.v. either 5 min before or immediately, 15 min, 30 min, 1 hr or 3 hr after i.m. injection of toxin or venom. A light microscopic method was used to measure the effects of myotoxin a, i.e. vacuolation index, and whole venom, i.e. myonecrosis index. The results show that antimyotoxin a serum neutralizes the myotoxicity of a sublethal amount of myotoxin a if injected 56 min before or immediately after toxin, but not if injected 15 min after the toxin. Its neutralizing ability for crude C. v. viridis venom was considerably better, neutralizing a dose of 0.75 microgram/g even if injection of antiserum was delayed for 30 min after venom injection. Thus, antimyotoxin a serum might be useful in treating myonecrosis resulting from prairie rattlesnake (C. v. viridis) venom poisoning.

Topics: Animals; Antivenins; Crotalid Venoms; Female; Mice; Muscular Diseases; Necrosis; Neutralization Tests; Time Factors

A previously unknown polypeptide myotoxin, designated myotoxin a, was isolated for the first time from prairie rattlesnake (Crotalus viridis viridis) venom. Electrophoretic homogeneity of myotoxin a was shown in beta-alanine disc gel polyacrylamide gel electrophoresis and in isoelectric focusing gel electrophoresis. Molecular weight and isoelectric point estimates of 4100 and 9.6 were obtained by gel filtration and isoelectric focusing gel electrophoresis, respectively. Amino acid composition showed a total of 39 amino acid residues, with 10 lysine residues and two disulfide bridges. When the two disulfide brides were reduced and alkylated, the myotoxic activity was abolished, indicating that the disulfide bridges of myotoxin a are essential for its biological activity. The loss of the biological activity is probably due to a marked change in secondary structure. The circular dichroic spectrum indicates that the chemically modified, inactive myotoxin exhibits typical random-coil conformation.

Topics: Amino Acids; Animals; Chemical Phenomena; Chemistry; Circular Dichroism; Crotalid Venoms; Disulfides; Dogs; Electrophoresis, Disc; Hemolysis; Hemorrhage; In Vitro Techniques; Isoelectric Focusing; Isoelectric Point; Mice; Molecular Weight; Muscular Diseases; Oxidation-Reduction; Snake Venoms; Structure-Activity Relationship