Ibrahim F. Heneine

Detoxification of the T2 fraction from a scorpion (Tityus serrulatus, Lutz and Mello) venom by iodination and some immunogenic properties of the derivatives

The iodination of the T2 fraction abolished its lethal capacity, and doses up to 30 times the LD50 were injected i.p. in mice without noticeable toxic effects. The modified fraction retained its immunological properties. Antibodies generated against the iodinated T2 fraction were also reactive toward the native T2 fraction, T1 fraction and the whole soluble venom.

The effect of stepwise iodination on biological properties of Bothrops jararaca venom

By titrating 5 mg of native venom with aliquots of a 2 x 10(-2) M iodine monochloride solution, neutralization of lethality by the incorporation of iodine was found with 200 +/- 5 microliters of solution, and above, up to 310 +/- 10 microliters, when saturation with iodine was attained. Doses up to 1500 micrograms (equivalent to 32 LD50 of native venom), where injected i.p. in mice without lethal effects. Proteolytic, phospholipase A2 and esterolytic activities were greatly reduced, but a low activity persisted even in fully iodinated samples. Direct hemolysis was markedly inhibited, and incapacity to coagulate fibrinogen and horse plasma was also observed in the iodinated samples. Hemorrhage and necrosis in rat skin, caused by 20 micrograms of iodinated venom were not elicited by doses up to 120 micrograms of iodinated anavenom. In mice, the myonecrosis that resulted from direct i.m. injection of native venom, and the massive hemorrhage caused by 5 LD50 doses injected i.p. were abolished by venom iodination. Blood congestion in liver, spleen, kidneys, and lungs, almost disappeared with iodination to the level of neutralization, and was barely seen with venom samples iodinated to saturation. The clinical signs of impaired physical activity, appearing in mice injected with 700 to 1500 micrograms of the iodinated anavenom were intensified by captopril and attenuated by epinephrine.

Reversion by polyclonal antibodies of α effects of Tityus serrulatus venom on frog sciatic nerve

Fraction T2 from Tityus serrulatus venom produced a marked lengthening of action potentials recorded with the single sucrose-gap technique, a characteristic effect of alpha scorpion toxins. This effect was not reversed by thorough washing of the nerve. On the other hand, T2 fraction deactivated by complete iodination did not cause any alteration of the compound action potential, even if applied in concentration as high as 4000 times the half saturation dose of the unmodified T2 fraction. This high dose of deactivated T2 did not hinder the onset of the full effect of a single dose of T2 fraction applied subsequently. Polyclonal antibodies against native or against iodinated venom reverted the action of T2 fraction, restoring normal electrical response. We conclude that both types of antibodies may remove the effect of alpha toxins on sodium channel.

The hemoglobin of the snail Biomphalaria glabrata. The absence of sulfhydryl groups (SH), presence of disulfide bonds (SS), and their relation to ligand properties☆

1. 1. The hemoglobin of Biomphalaria glabrata (Mollusca, Planorbidae), was devoid of SH groups, its two cysteinil residues forming an SS bridge for each 3.4 × 104 daltons of assumed molecular mass. 2. 2. The molecule dissociated incompletely, and no evidence of chain separation was obtained. 3. 3. The natural osmolality of the laboratory-reared snails hemolymph was 0.085 ± 0.003 osmolal. 4. 4. The anaerobic pH of hemolymph was 7.22 ± 0.02. 5. 5. The O2 affinity was related to both concentration and pH, and decreases sharply with SS breaking. 6. 6. The Bohr effect was concentration-dependent, and increases slightly with SS rupture. 7. 7. The cooperativity n was 1.6–1.7 at the natural conditions of pH and osmolality. It was related to pH and concentration, decreasing to near 1 with rupture of SS bonds.

The effect of dithiothreitol on the activity of tityustoxin from the scorpion Tityus serrulatus venom

The acetylcholine (ACh) liberating effect on rat brain slices of tityustoxin, an alpha toxin from the scorpion Tityus serrulatus venom, was measured in the absence and presence of dithiothreitol (DTT). The rate of net ACh liberation by toxin concentrations of 2 nmol in 5 ml of organ bath, was 7.5 +/- 0.09 nmol g-1 min-1. If DTT at a final concentration of 1 mM was added after a 10-min incubation period with toxin alone, inhibition of tityustoxin activity was 94%. With DTT 0.1 or 0.01 mmolar inhibition was 74% and 57%, respectively. The intense secretagogue effect, both in salivary and pancreatic glands of adult rats, induced by sublethal doses of tityustoxin was not affected by i.v. injection 10 min later of DTT 1 mumol g-1 of rat weight. When tityustoxin was injected i.p. at a dose 3 times the LD50 in mice, death ensued in 40 to 60 min. If toxin inoculation in mice was followed 10 min later by DTT 1 mumol g-1 of mouse weight, injected i.p. or i.v., deaths were delayed to 90 to 110 min, but no survival was observed. At necropsy, none of the mice treated with DTT showed any signs of pulmonary edema.

MODIFICATION OF BIOLOGICAL PROPERTIES OF PROTEIN TOXINS BY STEPWISE IODINATION

By gradual incorporation of stable iodine into toxins and whole venoms it is possible to abolish completely the physiological, lesion and lethal properties of the native components. The properties of iodinated antigens and from antibodies generated by these detoxified derivatives, are presented. The hapten is incorporated into tyrosyl and histidyl residues. The derivatives can be obtained in less than one hour. Within the same batch of protein, there is a determinable stoichiometric ratio hapten/protein to achieve the desired modified properties of the derivative. The iodinating solutions are easy to prepare, can be accurately standardized and have unlimited shelf lives. The cost of the whole procedure is very low. No side-effects, local or systemic were observed, even with prolonged use of the derivatives. The method was applied to toxic components and whole venom of the scorpion Tityus serrulatus, and the hypertensive, bradipneic, oliguric, lesional, lethal and cytotoxic effects were completely abolished. Polyclonal antibodies generated by these iodinated antigens neutralized the virulent effects of native components and reversed the α effects of the whole venom in frog sciatic nerves. They conferred active immunization in mice, rats, guinea pigs, goats, horses and pigeons. Crotoxin and whole venom of Crotalus durrissus terrificus lost the lesional and lethal activity, but conserving the immunogenic capacity. They produced antibodies against the native components, giving also vaccinal protection. While the virulent crotalic antigens had a cytotoxic activity, the iodinated antigens were highly mitogenic with human white cells. Five bothropic venoms were neutralized in the hemorrhagic, tissue lesion and lethal capacity, the derivatives were immunogenic. Repetitive sublethal doses of scorpionic, crotalic and bothropic venoms lead invariably to an amyloid-like deposit in tissues, whereas the iodinated samples were ineffective. Allergenic extracts of Schistosoma mansoni can be transformed into anallergic derivatives that retains antigenic properties. Violently allergenic extracts of Ascaris lumbricoides suum can be completely deactivated with iodination, but conserved immunological competence. Cholera, tetanus and botulinum toxins, as iodinated toxoids, had its lesional and lethal capacity completely avoided. Physiological proteins with strong biological activity can also be rendered innocuous. Iodinated insulin lost its capacity to lower blood glucose levels, but induced high avidity antibodies in guinea-pigs and rabbits. By iodination, kallikrein can be turned unable to contract rat uterus, and to liberate kinins from kinninogen. Modified tonin do not increase the blood pressure in rats. Aqueous extracts of Leptospira canis and L. icterohaemorrhagiae after iodination, were innocuous to hatched eggs, and immunogenic in mice and rabbits. A lectin from Macrotylema axillare, lost the hemaglutination capacity with only 75% of iodine saturation. The derivative was highly immunogenic in rabbits. Heavy iodination can transform self-antigens in non-self, generating antibodies in same species animals. All derivatives obtained were stable, did not show any reversion to toxicity, generated antibodies against the native antigens and gave active protection when injected in animals. The injections were also apparently painless. The time gap between the accident and the administration of antibodies is discussed for systemic and local effects. A new schedule for immunization, only feasible with toxoided venoms is presented. It is based on a clonal expansion induced by a small dose, followed by an exponential saturation dose of the same toxoid. The attainment of higher levels of protecting antibodies against the native antigen in the generated sera, is unmatched by other procedures. Data for practical use of iodination is presented.

Detoxified Venom from Crotalus Durissus Terrificus is Devoid of Cytotoxic Activity and Induces Mitogenesis

The venom from the snake Crotalus durissus terrificus was detoxified by stepwise incorporation of stable cationic iodine. The venoid, in a dosage equivalent to 100 LD50 of the lethal venom, was injected in mice without lethal exits. The native venom (NAT), or its toxoided (TXD) derivative were incubated in presence or absence of mitogens, with human mononuclear (MN), B and T cells, with a pulse of [3H]Thymidine. No synergistic or antagonistic effects were observed in the combined activity of the mitogens and NAT or TXD. In the direct action of NAT the incorporation of radioactivity into MN and T cells diminished with venom increase in concentration indicating that the cytotoxicity of the native venom was correlated with the amount added. With B cells, the native venom exercised an initial mitogenic activity, declining in the higher concentration. On the other hand, the TXD showed a consistent effect, increasing the thymidine uptake in a manner related to concentration. This stimulation by TXD was observed with all groups of cells. The results indicate that, by abolishing direct cytotoxic activity with toxoiding of this venom, a derivative that enhances mitogenesis in these white cells can be obtained.

Antiserum for insulin radioimmunoassay generated by an insulin derivative devoid of hypoglycemic activity

A tetraiodinated derivative of bovine insulin, prepared at pH 1 with stable iodine, was unable to cause signs of hypoglycemia in doses up to 2.4 micrograms/g in fasting mice, when native insulin caused 100% mortality. In neutral and acidic solutions, in absence of chaotropic agents, it behaved as the monomer, and could be separated from less iodinated, active species, that appeared as dimers, by conventional gel filtration. To generate antibodies in guinea-pigs, the tetraiodinated insulin was injected in doses three times higher than native insulin, without any harm to recipient animals. The induced antiserum was compared with antiserum generated by conventional methods in radioimmunoassay (RIA) of native insulin, and parallel curves were obtained.