RESUMO
Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A(2) (PLA2) to form the premicellar complexes (E(i)(#)) are characterized to discern the short-range specific interactions. Typically, E(i)(#) complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (E(i)(#), i = 1, 2, 3). In the presence of calcium, the dissociation constant K(1) for the E(1)(#) complex of PLA2 with decyl sulfate (CMC = 4500 microM) is 70 microM with a Hill coefficient n(1) = 2.1 +/- 0.2; K(2) for E(2)(#) is 750 microM with n(2) = 8 +/- 1, and K(3) for E(3)(#) is 4000 microM with an n(3) value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of E(i)(#); (c) K(i) and n(i) do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the E(i)(#) complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in E(i)(#). The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of E(i)(#). We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E and E(i)(#) aggregates is discussed.