myotoxin-a and Necrosis

Myotoxin a, a muscle-necrotizing polypeptide isolated from Crotalus viridis viridis (prairie rattlesnake) venom, was nicked at Met-28 by cyanogen bromide. Amino acid analysis indicated that the methionine content was reduced to zero from the original 1 mol. Judging from circular dichroism, the nicked myotoxin a had a conformation similar to that of original myotoxin. Raman spectra indicated that the conformations of the three disulfide bonds are not affected in nicked myotoxin a. Like the original toxin, nicked myotoxin a was myotoxic and inhibited calcium ion loading activity, although the inhibitory action was slightly lower than that of the original myotoxin a. Both modified and unmodified myotoxin a showed myonecrotic activity as determined by examining histological slides. The modified toxin also inhibited the formation of decavanadate-induced two-dimensional crystalline arrays of the sarcoplasmic reticulum Ca2+-ATPase just as the original myotoxin a does.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Circular Dichroism; Crotalid Venoms; Crystallography; Cyanogen Bromide; In Vitro Techniques; Molecular Structure; Muscles; Necrosis; Peptide Fragments; Rabbits; Sarcoplasmic Reticulum; Spectrum Analysis, Raman; Structure-Activity Relationship

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

A mixture of antimyotoxin a serum and polyvalent (Crotalidae) antivenom was injected i.v. in mice either 5 min before or 5 min, 30 min, 1 hr or 3 hr after i.m. injection of venom. Neutralization of the local myotoxicity of a sublethal dose (1.5 micrograms/g) of C. v. viridis venom occurred if the antisera were injected 5 min before or 5 or 30 min after venom, but not if injected 1 or 3 hr after the venom. Hemorrhage was neutralized when the mixture was injected either 5 min before or 5 min after injection of venom, but not when injected 30 min after injection of venom. Previous results showed that the mixture of antisera neutralized the same amount of venom (1.5 micrograms/g) when mixed with the venom prior to injection. Thus it is not possible with these two antisera to neutralize myonecrosis if the time interval between injections is greater than 30 min.

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

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

Topics: Animals; Antivenins; Crotalid Venoms; Female; Immune Sera; Mice; Mice, Inbred Strains; Muscles; Necrosis; Neutralization Tests; Snake Venoms

Using a pure myotoxin (myotoxin a) isolated from prairie rattlesnake Crotalus viridis viridis) venom, a comparison between two methods for quantitating myonecrosis in mice was made. Measurement of creatine kinase (CK) levels in the plasma showed two peaks in CK levels after myotoxin injection, whereas measurement by histological assay (vacuolation index) showed only one peak. The first peak in CK levels at 3 hr after injection did not correlate with a high vacuolation index, but did correlate with contraction of muscle induced by the toxin. However, there was good correlation between the two methods at 6, 12, 24, 48 and 72 hr after injection, at which time the muscle cells were necrotic. In the case of the first CK peak this method might be measuring contraction of the muscle, but the second peak was probably measuring altered permeability of the sarcolemma since vacuolation, i.e. swelling of sarcoplasmic reticulum, did follow. High plasma CK levels usually preceded high vacuolation indexes, indicating that CK release due to altered sarcolemma permeability preceded vacuolation. Caution should be taken when using plasma CK levels to estimate the quantity of necrotic muscle cells, so as to insure that pathologic and not physiologic changes are being measured.

Topics: Animals; Creatine Kinase; Crotalid Venoms; Female; Mice; Muscle Contraction; Muscles; Necrosis; Snake Venoms