AN in vivo METHOD FOR ESTIMATING THE CONCENTRATION OF CROTAMINE IN A SOLUTION

Abstract

very sensitive method for estimating the concentration of crotamine in a solution was developed. This method was based on the time required for the appearance of permanent hyperextension of the rear legs of mice as a function of the dose administered. This method can be used to determine toxin doses as low as 0.32 mg/kg<img SRC="http:/img/fbpe/jvat/v3n1/image2875.gif"> . Its high specificity for crotamine means that it can be used to measure toxin concentrations in the presence of other proteins and polypeptides.

Crotalus durissus terrificus venom; crotamine; method to estimate the concentration

Short communication

AN in vivo METHOD FOR ESTIMATING THE CONCENTRATION OF CROTAMINE IN A SOLUTION

O. G. HAMPE

, A. A. BELLÓ

1 Department of Biophysics and 2 Department of Physiology, Institute of Biosciences, Federal University of Rio Grande do Sul, Porto Alegre, State of Rio Grande do Sul, Brazil.

ABSTRACT. A very sensitive method for estimating the concentration of crotamine in a solution was developed. This method was based on the time required for the appearance of permanent hyperextension of the rear legs of mice as a function of the dose administered. This method can be used to determine toxin doses as low as 0.32 mg/kg. Its high specificity for crotamine means that it can be used to measure toxin concentrations in the presence of other proteins and polypeptides.

KEY WORDS: Crotalus durissus terrificus venom, crotamine, method to estimate the concentration.

INTRODUCTION

Crotamine, a toxin found in the venom of the southern Brazilian rattlesnake Crotalus durissus terrificus, produces a variety of effects when injected into small animals(1,2,3,4,7,8). The most typical of these responses is the development of a spastic paralysis of the rear legs(1,7). The measurement of crotamine concentrations based on physical and chemical methods is only possible using pure toxin samples. Since methods based on biological activity have been used to quantify small amounts of hormones and amines in solution, we tried to develop a simple, fast and highly specific biological procedure for estimating the concentration of crotamine in solution.

Crotamine was purified from a desiccated pool of venom as previously described(5). Only samples homogeneous by polyacrylamide gel electrophoresis at acid and alkaline pH or in the presence of sodium dodecyl sulfate, and having the same amino acid composition as that described earlier(6) were used. The protein concentration was measured spectrophotometrically at 280 nm Male and female adult white Swiss mice (18 to 25 g) were obtained from the Central Animal Facility of the Universidade Federal do Rio Grande do Sul. The assay for crotamine activity consisted of measuring the time necessary for the appearance of hyperextension (mantained at least during 5 min.) of the rear legs of the mice after i.p. injection of the toxin. At doses lower than 25 mg/kg the hyperextension was intermittent until its establishment. Figure 1shows the linear relationship between the log of the time, (t) required for the manifestation of hyperextension and the log of amount of crotamine (mg/kg) injected, (D). Each point represents the data obtained with one dose of toxin injected into 39 mice. For the dose-response curve, doses of crotamine ranging from 0.137 to 40.0 mg/kg were used. Only amounts > 0.32 mg kgproduced hyperextension. The expression below could be used to determine the amount of toxin injected since the weight of the mice used and the time required for the establishment of permanent hyperextension of the rear legs are known.

log t = 3.20 - 0.80 log D (± 0.48)

FIGURE 1.
Relationship between the time elapsed (seconds, t) before the appearance of permanent hyperextension of the rear legs of mice and the dose (D) of crotamine administered. The regression line shown has a slope of - 0.80, a y-axis intercept of 3.20 and an r value of -0.93.

To verify whether the method was reproducible in the presence of other proteins and polypeptides, samples of pure crotamine were added to rattlesnake venom solutions containing a known concentration of toxin and aliquots of the mixture were then i.p. injected in mice. The paralysis time obtained with this mixture corresponded to the total crotamine injected.

The in vivo method described above has certain advantages over the physical and chemical methods, including: 1) its high specificity for crotamine; 2) its applicability to both pure toxin samples and to mixtures containing other proteins and polypeptides, and 3) its sensitivity which allows the accurate determination of toxin doses as low as 0.32 mg/kg. This method can also be used to quantify the remaining activity of crotamine after chemical modification of its amino acid residues.

In order to improve the accuracy of the results, we recommend that each determination of an unknown concentration be performed in triplicate.

08 MOUSSATCHÉ H., GONÇALVES J.M., VIEIRA G.D., HASSON A.V. Pharmacological actions of two proteins from Brazilian rattlesnake venom. In: BUCKLEY EE., PORJES N. Eds. Venoms. Washington: A.A.A.S., 1956: 275-9.

Received 20 May 1996.

Accepted 03 July 1996.

CORRESPONDENCE TO:

O. G. HAMPE - Departamento de Biofísica, Instituto de Biociências Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, CEP 90050-170, Porto Alegre, RS, Brasil.

• ¹
  01 BARRIO A., VITAL-BRAZIL C. Neuromuscular action of the Crotalus durissus terrificus (Laurenti) poisons. Acta Physiol. Latinamer., 1951, 1, 291-308.
• 02 CHEYMOL J., GONÇALVES J.M., BOURILLET F., ROCH-ARVEILLER M. Action neuromusculaire comparée de la crotamine et du venin de Crotalus durissus terrificus Var. crotaminicus-I. Toxicon, 1971, 9, 279-86.
• 03 GONÇALVES J.M., VIEIRA, L.C. Estudo sobre venenos de serpentes brasileiras. I. Análise eletroforética. An. Acad. Bras. Ciênc., 1950, 22, 141-50.
• 04 HAMPE O.G., BELLÓ A.A. Avaliação in vivo da atividade da crotamina. In: CONGRESSO BRASILEIRO DE BIOFÍSICA, 9, São Paulo, 1984. Resumos.. São Paulo, 1984: 271.
• 05 HAMPE O.G., VOZÁRI-HAMPE M..M., GONÇALVES J.M. Crotamine conformation: Effect of pH and temperature. Toxicon, 1978, 16, 453-60.
• 06 LAURE L.C. Die primärstruktur des crotamins. Hoppe-Seiler's Physiol. Chem., 1975, 356, 213-5.
• 07 MOUSSATCHÉ H., VIEIRA G.D. Sobre o mecanismo da contratura produzida pelo veneno da cascavel (Crotalus durissus terrificus). An. Acad. Bras. Ciênc., 1953, 25, 249-58.
• 08 MOUSSATCHÉ H., GONÇALVES J.M., VIEIRA G.D., HASSON A.V. Pharmacological actions of two proteins from Brazilian rattlesnake venom. In: BUCKLEY EE., PORJES N. Eds. Venoms Washington: A.A.A.S., 1956: 275-9.  

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1997

History

• Accepted
  03 July 1996
• Received
  20 May 1996