The Binding of Divalent Cations to Escherichia coli α‐Haemolysin

α‐haemolysin, an extracellular protein toxin of Escherichia coli , is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca 2+ ‐binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca 2+ ‐binding sites have not been characterized to date in the native protein. The results in this paper show that when Ca 2+ levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. Ca 2+ and, at higher concentrations, Sr 2+ and Ba 2+ , support the lytic activity, but Mg 2+ , Mn 2+ , Zn 2+ and Cd 2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of α‐haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of 45 Ca 2+ binding reveals three equivalent, independent sites, with K d ≈ 0.11 mM. No 45 Ca 2+ binding is observed when the protein is incubated with Zn 2+ ; conversely, incubation with Ca 2+ prevents subsequent binding of 65 Zn 2+ . In the light of three‐dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosa [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12 , 3357–3364] it is suggested that α‐haemolysin may bind a larger number of Ca 2+ than the three that are more easily exchangeable and are thus detected in the 45 Ca 2+ ‐binding experiments. In addition, structural similarities and conservation of ion‐binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn 2+ and Ca 2+ .