Rapid isolation of a venom protein by affinity chromatography on immobilized antibodies

International Society on Toxinology: European Meeting USE OF SYNTHETIC

PEPTIDES

ACTIVITY

FOR

THE

RELATIONSHIPS

STUDY

419

OF STRUCTURE-

OF APAMIN

C. GRANIER, E. PEDROSO MULLER and J. VAN RIETSCHOTEN Laboratoire de Biochimie, Facult6 de M6decine Secteur Nord, Boulevard Pierre Dramard, 13326 Marseille Cedex 3, France Six analogs of the bee venom neurotoxin apamin have been designed. The modifications concern either the number or the chemical nature of the cationic side chains of the residues 13-14. Three peptides ([Lys 13, Lys~q-apamin ; [Lys13]-apamin and [Lysla]-apamin) were obtained by solid phase peptide synthesis and have been extensively characterized. Their subsequent chemical modifications have afforded three new derivatives. Neurotoxicity of these six structural analogs of apamin was measured as their LDs0 in the mouse. It was found that activity is observed only if the region 13-14 bears two positive charges. The chemical nature of cationic groups strongly influences neurotoxicity. [Lys~3-Lys14]-apamin has a low activity which is increased when the peptide is modified into [Har 4, Har a3, Har~4]-apamin (Har = Homoarginine). The two analogs [Lys~3]-apamin and [Lys~4]-apamin have a much higher neurotoxicity and are almost equipotent to apamin.

ISOLATION VENOM

AND

CHARACTERIZATION

OF MIDDLE-ASIAN

OF TOXINS

SCORPION

BUTHUS

FROM

THE

EUPEUS

E. V. GRISHIN,N. M. SOLDATOV,L. N. SOLDATOVAand Yu. A. OVCHINNIKOV Shemyakin Institute of Bioorganik Chemistry, USSR Academy of Sciences, Moscow, U.S.S.R. The separation of the scorpion Buthus eupeus venom was undertaken. Twelve different toxins were isolated from scorpion venom by gel filtration and ion exchange chromatography. Finally eight polypeptides toxic to mice and four insect toxins were obtained. All the isolated scorpion toxins are basic polypeptides crosslinked by four disulfide bridges and they have a tool. wt of 4000-9000. Insect toxins I1, 13 and 14 belong to the novel structural type of scorpion toxins. The essential features of these insect toxins are low mol. wt (4000) and the presence of two or three methionines which are absent in the known scorpion toxins. The total amino acid sequence of the insect toxin I1 and partial structure of the insect toxin I2 (tool wt. 7000) were determined. It was shown that insect toxins I1, I3 and I4 in contrast to I~ have no structural similarity with known scorpion toxins.

RAPID

ISOLATION

OF A VENOM

CHROMATOGRAPHY

PROTEIN

ON IMMOBILIZED

BY AFFINITY ANTIBODIES

F. GUBEN~EK and D. ~UN]~ Department of Biochemistry, J. Stefan Institute, University of Ljubljana, Ljubljana, Yugoslavia Isolation of the phospholipase A2 from the venom of Vipera ammodytes requires at least three chromatographic separations and two concentrations of the eluted peaks followed by dialysis in order to match the starting conditions for the next chromatographic separation. These are all time consuming procedures, so that isolation of a particular protein, in the above case phospholipase A~, takes at least 7-10 days. Affinity chromatography was shown to be an efficient isolation method providing that a convenient biospecific reaction between protein and ligand takes place. Commercial antisera contains antibodies against all proteins present in the venom. By the use of immobilized pure protein that has been isolated from the venom by the lengthy classical procedure one can obtain highly specific antibodies that do not bind other venom proteins. By immobilization of these highly specific gamma globulins and subsequent affinity chromatography of crude venom, we were able to obtain electrophoretically pure phospholipase A2 fraction and phospholipase-free Vipera ammodytes venom in one step.