An in vivo method for estimating the concentration of crotamine in a solution

A very sensitive method for estimating the concentration of crotamine in a solution was developed. This method was based on the 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(-1). Its high specificity for crotamine means that it can be used to measure toxin concentrations in the presence of other proteins and polypeptides.

Polyacrylamide gel electrophoretic studies on the self-association of crotamine: characterization and molecular dimension of n-mer species.

Polyacrylamide gel electrophoresis (PAGE) at pH 4.4 was used to study the concentration dependence of absolute mobility of crotamine. Within amounts ranging from 5-65 micrograms the toxin appeared in at least three n-mer species which were characterized by their geometrical mean radius R and molecular weight Mr estimation. The R and Mr values of crotamine bands were obtained from equations described in the literature and by using standard polypeptides and proteins submitted to the same experimental conditions. When amounts of up to 20 micrograms were assayed by PAGE the bands had a monomer molecular weight value of 4650 and R was 1.08 nm. From 20-35 micrograms the toxin migrated as dimer (Mr 10,000) with an R value of 1.42 nm. However, amounts higher than 35 micrograms crotamine were mostly resolved in a "two-band" pattern with R and Mr values corresponding to higher associated species.

Model studies of crotamine self-association.

1. The self-association of crotamine in the concentration range from 10 to 40 micrograms/ml was studied at pH 6, 25 degrees C, and at low ionic strength by monitoring the effect of protein concentration on the absorbance at 196 nm. 2. Of the several mathematical models tested, an equilibrium between monomers and trimers with an association constant of 1.50 x 10(11) M-2 gives an adequate representation of the phenomenon. However, a non-ideal, two-stage model describing an equilibrium among monomers, dimers, and trimers gives the best fit of the experimental data. 3. The equilibrium constants found for dimerization and trimerization were 1.70 x 10(3) M-1 and 3.37 x 10(6) M-1, respectively. 4. This model was confirmed by polyacrylamide gel electrophoresis where a trimer band was separated from an interconverting monomer-dimer band.

Kinetic study of hemoglobin Porto Alegre [beta 9(A6)Ser----Cys] disulfide polymer reduction.

The cleavage of HbPA disulfide polymer by GSH and its indirect cleavage by yeast glutathione reductase, via reduced glutathione is obtained. Decreasing the initial proportion of GSH in the hemolysate increases the formation of HbPA disulfide polymer. In the experimental conditions used, yeast glutathione reductase is unable to perform the direct cleavage of the mixed disulfide of HbPA and GSH, using it as substrate. The reduction of HbPA polymer to tetramers by DTE is analyzed by a pseudo-first-order kinetic and two rate constants are obtained. That of 265 X 10(-3) min-1 should be concerned with one disulfide of the closed ring and one of the open ring structure of dodecamer, while that of 38 X 10(-3) min-1 is related to disulfide reduction of the octamer. The enthalpy of activation values of 8.0 kcal.mol-1 an 17.4 kcal.mol-1 obtained, from the Arrhenius plot, for the "fast" and "slow" rate disulfide reduction, respectively, are indicative that a strong conformational strain of S--S bonds in the closed ring structure is maintained. The entropy of activation values of 24 e.u. and 52 e.u. are found for the activation of disulfides from dodecamers and octamers, respectively.

A biophysical model of lysozyme self-association.

The concentration dependence of the self-association of hen egg-white lysozyme was studied spectrophotometrically at pH 6, 25 degrees C, and low ionic strength within a concentration range of 2.5-50 micrograms/ml. Of several possible mathematical models, an ideal or nearly ideal two-stage model representing an equilibrium between monomers and dimers and between dimers and trimers best describes the data. The dimerization and trimerization constants were found to be 2.5 x 10(-2) and 38 x 10(-2). Dialysis experiments confirmed that the mechanism involves three associating species. A "head-to-tail" contact between the associating sites was inferred from dialysis studies of the effect of indole and imidazole derivatives on lysozyme self-association.

Interaction of urea with lysozyme: study by the far and middle ultraviolet region absorption spectra.

The interaction of urea with lysozyme was studied in the 192-240 nm spectral region by spectrophotometry. The far and middle ultraviolet protein bands records undergo a non-linear "red shift" and "hypochromic effect" under urea titration. These spectral shifts are interpreted basically in terms of decreasing in molar absorptivity due to the binding of the denaturant with the protein chromophores. Two interaction mechanisms with different chromophores involvement are characterized. One of them is noncooperative and its is evidenced by the analysis either of the red shift or of the hypochromic effect showed by the protein far ultraviolet records in urea up to 2 M. This noncooperative effect is represented by two different and independent classes of binding sites in which the tryptophan side chain and the amide peptide bond unit are involved. The calculated stoichiometric constants give the values of 4.61 M-1 for K1 and of 0.078 M-1 for K2, while the site binding constants have the values of 0.852 M-1 for K1 and 0.086 M-1 for K2. The other mechanism which is detected by the middle U.V. band analysis of the protein in urea concentration up to 8 M shows high cooperativity (Hill coefficient of 2.56). Also in this case, tryptophan residues are involved in the binding process. No significant light-scattering influence on absorption measurements is found.