pH-dependence of the binding constant of Ca2+ to beta-bungarotoxin from Bungarus multicinctus venom

pH-dependence of the binding constant of Ca2+ to beta-bungarotoxin was studied at 25 degrees C and ionic strength 0.2 by the tryptophyl fluorescence and ANS-fluorescence methods. The results were compared with those of other phospholipases A2. The binding constant at neutral pH value was in good agreement with that reported by Abe et al. [1977) Eur. J. Biochem. 80, 1-12). The pK value of an ionizable group was perturbed from 7.25 to 5.90 by the Ca2+ binding. Competition with the Ca2+ binding was observed in the protonation of another group with a pK value of 5.05. The former group was assigned as His 48 in the A chain of the protein and the latter as Asp 49 to which Ca2+ ion can coordinate. No participation of the alpha-amino group was observed. The pK value (7.25) of His 48, determined in the present experiment, was very close to that (6.9) estimated from the pH-dependence of the chemical reaction of p-bromophenacyl bromide with His 48 (Kondo et al. (1978) J. Biochem. 84, 1291-1300). The binding constant of Ca2+, KEM = 6.76 X 10(3) M-1, when all the ionizable groups participating in the Ca2+ binding are completely deprotonated, was found to be very similar to those for all the phospholipases A2 reported previously, indicating similarity in the conformation of the Ca2+ binding site. Inhibitory metal ions, Ba2+ and Sr2+, exhibited binding constants similar to that of Ca2+ at neutral pH value.