Proteolytic activity of Bitis ssp venoms.

<p><b>[A]</b> Gelatinolytic activity analyzed by zymography: Bitis spp venoms (30 μg) were incubated in the absence (buffer) or presence of 5 mM EDTA (EDTA), 5 mM 1,10-phenanthroline (PHE) or 5 mM PMSF (PMSF), submitted to electrophoresis and subsequently incubated overnight at 37°C, in 50 mM Tris-HCl, pH 8.3. Following incubation, the gel was stained with 0.1% Coomassie Brilliant Blue and the gelatinolytic activity was detected as unstained bands on a dark background. <b>[B]</b> Fibrinogenolytic activity was assessed using a SDS-PAGE (10%) gel. Thirty micrograms of human purified fibrinogen (Fn) were incubated with 1 μg of Bitis spp venoms pre-incubated or not with inhibitors of serine–(PMSF) or metallo–(PHE) proteinases and ran under reducing condition. The digestion of gelatin and the cleavage of fibrinogen were visualized by Coomassie Brilliant Blue staining.</p

Summary of the biological activities of <i>Micrurus</i> spp venoms, cross-reactivity and neutralization potential of the Brazilian coral snake antivenom.

<p><b>PLA₂:</b> Phospholipase A₂; <b>P:</b> Protease; <b>H:</b> Hyaluronidase;</p><p>High (+++); Intermediate (++); Low (+); Very low (+/−); Absent: (−); not determined (nd).</p

Quality of horse F(ab′)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

<div><p>Abstract Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab′)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results Horse F(ab′)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab′)2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab′)2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.</p></div

Biochemical characterization of <i>Bitis</i> ssp venoms.

<p><b>[A]</b> Venoms samples (30 μg) from <i>B</i>. <i>arietans</i> (Ba), <i>B</i>. <i>g</i>. <i>rhinoceros</i> (Br) and <i>B</i>. <i>nasicornis</i> (Bn) snakes were separated by SDS-PAGE (8–16% gel) under non-reducing and reducing conditions and silver stained. <b>[B]</b> Venom samples (10 μg) from <i>Bitis</i> ssp were separated by SDS-PAGE and electrotransfered into nitrocellulose membranes to analyze the presence of oligosaccharide residues. Blots were probed with WGA-HRPO (WGA) or Con A-HRPO (Con A) and the reactions developed with DAB.</p

Inhibition of the proteolytic activity of <i>Bitis</i> ssp venoms by the experimental antivenoms.

<p><b>[A]</b><i>Bitis</i> spp venoms (20 μg) were pre-incubated (30’ at RT) with F(ab’)₂ Ba (10 μL neat) or F(ab’)₂ Br+ Bn (10 μL neat) antivenoms and ran under non-reducing conditions on a 10% polyacrylamide gel containing 1% gelatin type A. <b>[B]</b> One microgram of <i>B</i>. <i>arietans</i>, <i>B</i>. <i>g</i>. <i>rhinoceros</i> or <i>B</i>. <i>nasicornis</i> venoms were pre-incubated with 10 μL of neat sera prior incubation with 30 μg of human purified fibrinogen (Fn) then, ran under reducing condition in a 10% SDS-PAGE. The digestion of gelatin and the cleavage of fibrinogen were visualized by Coomassie Brilliant Blue staining.</p

Cleavage of Angiotensin I by <i>Bitis</i> ssp venoms and identification of the cleavage sites on the primary sequence.

<p><b>[A]</b> Angiotensin I (65 μM) was incubated at 37°C with 1 μg of <i>Bitis arietans</i> venom (1 h/37°C) or 5 μg of <i>B</i>. <i>nasicornis</i> or <i>B</i>. <i>g</i>. <i>rhinoceros</i> venoms (2 h/37°C) in phosphate buffer (50 mM sodium phosphate, 20 mM NaCl, pH 7.4). In parallel, the venoms were pre-incubated, 30 min prior the addition of angiotensin I, with PHE (5 mM), PMSF (5 mM) or EDTA (100 mM). <b>[B]</b> The hydrolysis products, collected during the reverse-phase chromatography, were submitted to mass spectrometry analysis. All venoms cleaved angiotensin I in different cleavage sites. The lines indicate the cleavage sites on angiotensin I primary sequence after treatment with the different <i>Bitis</i> venoms. The bold solid line indicates the cleavage point for angiotensin 1–7 generation and the solid lines indicate the other cleavage points observed after the treatment with the venoms. The dashed line indicates the sites on angiotensin I primary sequence cleaved only by <i>B</i>. <i>g</i>. <i>rhinoceros</i> and <i>B</i>. <i>nasicornis</i> venoms.</p

Enzymatic characterization of <i>Bitis</i> ssp venoms and the blockage of activities by antivenoms raised against <i>B</i>. <i>arietans</i> or <i>B</i>. <i>g</i>. <i>rhinoceros</i> plus <i>B</i>. <i>nasicornis</i>.

<p><b>[A]</b><i>Hyaluronidase activity</i>: <i>B</i>. <i>arietans</i>, <i>B</i>. <i>g</i>. <i>rhinoceros</i> and <i>B</i>. <i>nasicornis</i> venoms samples (30 μg) pre-incubated with hyaluronic acid at 37°C for 30 min. Alternatively, venom samples (30 μg) were pre-incubated with 10 μL of antivenoms raised against <i>B</i>. <i>arietans</i> venom (α-Ba) or <i>B</i>. <i>g</i>. <i>rhinoceros</i> plus <i>B</i>. <i>nasicornis</i> venoms (α-Br + Bn) for 30 min at RT, prior incubation with hyaluronic acid. The turbidity of the mixture was measured in a spectrophotometer at λ_em 405 nm. The results are representative of three individual experiments and expressed in units of turbidity reduction (UTR) <i>per</i> mg of venom. Statistical analyses were performed using two way Anova analysis (*<i>P</i>< 0.05). <b>[B]</b><i>Phospholipase A</i>_<i>2</i><i>activity</i>: venom samples (2.5 μg) were added to the phospholipid FRET substrate and the fluorescence was immediately measured in a fluorimeter. Alternatively, venom samples (2.5 μg) were pre-incubated with 10 μL of antivenoms raised against <i>B</i>. <i>arietans</i> venom (α-Ba) or <i>B</i>. <i>g</i>. <i>rhinoceros</i> plus <i>B</i>. <i>nasicornis</i> venoms (α-Br + Bn) for 30 min at RT, prior incubation with phospholipid FRET substrate. The specific activity was expressed as UF/min/μg. Results are representative of 3 independent experiments performed in quadruplicate. Statistical analyses were performed using two way Anova (*<i>P</i>< 0.05).</p

Antivenom neutralization of <i>Micrurus</i> venoms lethal toxicity.

<p>Samples corresponding to 2 LD₅₀, of each <i>Micrurus</i> venom, were mixed with serial dilutions of the coral snake horse antivenom. The mixtures were incubated for 30 min at 37°C and the animals were i.p. inoculated. The effective dose (ED₅₀) was calculated, from the number of deaths within 48 h of injection of the venom/antivenom mixture, using probit analysis and expressed as mL of antivenom <i>per</i> µg of venom.</p

SDS-polyacrylamide gel eletrophoresis.

<p>Samples (20 µg) of <i>M. ibiboboca</i>, <i>M. lemniscatus</i>, <i>M. fulvius</i>, <i>M. altirostris</i>, <i>M. spixii</i>, <i>M. surinamensis</i>, <i>M. corallinus</i>, <i>M. frontalis</i> and <i>M. hemprichii</i> venoms were analyzed by SDS-PAGE in a gradient gel (7.5% to 15%) and silver stained.</p

Determination of the phospholipase A₂ activity.

<p>Samples of individual <i>Micrurus</i> venoms (4 µg), or a mixture of <i>M. frontalis</i> (2 µg) and <i>M. corallinus</i> (2 µg), venoms were incubated for 20 min at 37°C with 180 µL of a mixture containing 5 mM Triton X-100, 5 mM phosphatidylcholine, 2 mM HEPES, 10 mM calcium chloride and 0.124% (wt./vol) bromothymol blue dye in water. Results are representative for three separate experiments and expressed as nanomoles acid <i>per</i> minute <i>per</i> µg of venom.</p