BIPHASIC DENATURATION OF HUMAN PLACENTAL ALKALINE-PHOSPHATASE IN GUANIDINIUM CHLORIDE

Human placental alkaline phosphatase is a membrane-anchored dimeric pr otein, Unfolding of the enzyme by guanidinium chloride (GdmCl) caused a decrease of the fluorescence intensity and a large red-shifting of t he protein fluorescence maximum wavelength from 332 to 346 nm, The flu orescence changes were completely reversible upon dilution. GdmCl indu ced a clear biphasic fluorescence spectrum change, suggesting that a t hree-state unfolding mechanism with an intermediate state was involved in the denaturation process. The half unfolding GdmCl concentrations, [GdmCl](0.5), corresponding to the two phases were 1.45 M and 2.50 M, respectively. NaCl did not cause the same effect as GdmCl, indicating that the GdmCl-induced biphasic denaturation is not a salt effect. Th e decrease in fluorescence intensity was monophasic, corresponding to the first phase of the denaturation process with [GdmCl](0.5) = 1.37 M and reached a minimum at 1.5 M GdmCl, where the enzyme remained compl etely active, The enzymatic activity lost started at 2.0 M GdmCl and w as monophasic but coincided with the second-phase denaturation with [G dmCl](0.5) = 2.46 M. Inorganic phosphate provides substantial protecti on of the enzyme against GdmCl inactivation. Determining the molecular weight by sucrose-density gradient ultracentrifugation revealed that the enzyme gradually dissociates in both phases. Complete dissociation occurred at [GdmCl] > 3 M, The dissociated monomers reassociated to d imers after dilution of the GdmCl concentration. Refolding kinetics fo r the first-phase denaturation is first-order but not second-order. Th e biphasic phenomenon thereby was a mixed dissociation-denaturation pr ocess. A completely folded monomer never existed during the GdmCl dena turation, The biphasic denaturation curve thereby clearly demonstrates an enzymatically fully active intermediate state, which could represe nt an active-site structure intact and other structure domains partial ly melted intermediate state. (C) 1998 Wiley-Liss, Inc.