143 Biochimica et Biophysica Acta, 498 (1977) 143--153 © Elsevier/North-Holland Biomedical Press BBA 28258 KINETICS OF ERYTHROCYTE LYSIS BY SNAKE VENOM CARDIOTOXINS * ABRAHAM I. LOUW and LEON VISSER Molecular Biochemistry Division, National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria, 0001 (Republic of South Africa) (Received December 29th, 1976) Summary Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 pg) could-be conveniently and rapidly assayed with high reproducibility in a recording spec- trophotometer, and reliable kinetic data were accumulated. Cardiotoxins from two different genera (Hemachatus haemachates and Naja rnossarnbica mossambica) displayed virtually identical hemolytic properties. Hemolysis increased linearly with time, in contrast with a sigmoidal pattern when phospholipase was present as an impurity. Low concentrations of Ca 2÷ (<1 mM) stimulated cardiotoxin action. A limiting plateau rate of hemolysis reached during concentration dependence experiments in which the level of either cardiotoxin or of erythrocytes was varied, suggested that the interaction of cardiotoxin with erythrocyte membranes is a saturation phenomenon only at a high ratio of cardiotoxin : erythrocytes. No hemolysis was observed with an homologous neurotoxin or S-methylated cardiotoxin, providing evidence for specificity. The linear Arrhenius plots obtained for the temperature depen- dence of cardiotoxin-induced hemolysis strengthened the conclusion that its action involves more than a detergent-like effect on membrane phospholipids. Introduction Cardiotoxin (direct lytic factor, cytotoxin, cobramine) [1--3] is one of the major constituents of the venom from snakes of the genera Na]a and Hemacha- tus. Currently, the primary structures of more than 30 of these toxins are known. The cardiotoxins are a family of small proteins, exhibiting a relatively low intravenous toxicity (LDso of 1--3 pg/g mouse), when compared to snake * Dedicated to Prof. Dan Potgieter~ eminent teacher of biochemistry for 21 years at the University of Pretoria.