A BRADYKININ‐POTENTIATING FACTOR (BPF) PRESENT IN THE VENOM OF BOTHROPS JARARACA

Abstract

Corrado & Rocha e Silva, 1962) it was shown that some metal-binding agents, like dimer- caprol (BAL) and thioglycollic acid, were able to potentiate in vitro and in vivo some of the pharmacological actions of synthetic bradykinin.While determining whether dimercaprol could potentiate in situ the slowly reacting substances released by the venom of Bothrops jararaca when acting on the guinea-pig isolated ileum, it was found that the venom itself had a strong potentiating action upon the concentrations elicited by bradykinin.The present report deals with the chemical nature and biological actions of this bradykinin-potentiating factor (BPF). METHODS Partial purification of BPFPreliminary experiments (Ferreira & Rocha e Silva, 1963) led to the following partial purification of BPF: 2 g of Bothrops venom were suspended in 200 ml. of distilled water and heated for 5 min in a boilingwater bath.Absolute ethanol (1.5 1.) was added.The mixture was centrifuged (2,000 revs/min) during 60 min and the supernatant fluid was evaporated under reduced pressure.The factor was extracted from dry powder with three successive additions of 20 ml. of 90% ethanol.To the alcoholic solution four volumes of ethyl ether were added.The precipitate was separated by centrifugation and dissolved in 50 ml. of distilled water.The aqueous solution was then lyophilized.The resulting powders will be referred to in this paper as " bradykinin-potentiating factor " (BPF). Physicochemical properties ofBPFSolubility tests.These were performed by shaking for 1 hr, 100 ,kg of BPF (previously dried in glass tubes) with 2 ml. of the following solvents: absolute ethanol and acetone, ethyl ether, chloroform and water-saturated n-butanol.The activity of the supernatant fluid collected by centrifugation and dried under reduced pressure was estimated as a percentage of a control value determined with distilled water.Dialysis.BPF (1.5 mg) was dissolved in 1.5 ml. of distilled water and dialysed (dialyser tubing i to j in., Thomas Co., U.S.A.) against thirty volumes of distilled water with constant agitation by a magneto-stir system.After 20 hr the potentiating activity was estimated inside and outside the dialysis bag.Acid hydrolysis.This was performed at 1000 C in sealed tubes by incubating 250 ,tg of BPF with 6 N-hydro- chloric acid or 200 spg of BPF with 1.5 N-hydrochloric acid.After a definite time interval the hydrolysis was stopped in the first experiment by evaporation under reduced pressure or in the latter by neutralization with a sufficient amount of N-sodium hydroxide solution.One sample was set apart for ninhydrin reaction (Troll & Cannan, 1963) and another, to which tris buffer, pH 7.8, was added, for biological estimation.Enzymatic hydrolysis.This was tested by incubating BPF with trypsin or chymotrypsin at 370 C. The concentrations of the reagents in the incubation mixtures were: (1) Trypsin, 100 Jzg/ml.;BPF, 500 jtg/ml.;