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Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride
Author(s) -
Hung HuiChih,
Chang GuGang
Publication year - 1998
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(19981001)33:1<49::aid-prot5>3.0.co;2-g
Subject(s) - guanidinium chloride , chemistry , denaturation (fissile materials) , fluorescence , dissociation (chemistry) , guanidine , chromatography , enzyme , biochemistry , nuclear chemistry , organic chemistry , physics , quantum mechanics
Human placental alkaline phosphatase is a membrane‐anchored dimeric protein. Unfolding of the enzyme by guanidinium chloride (GdmCl) caused a decrease of the fluorescence intensity and a large red‐shifting of the protein fluorescence maximum wavelength from 332 to 346 nm. The fluorescence changes were completely reversible upon dilution. GdmCl induced a clear biphasic fluorescence spectrum change, suggesting that a three‐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. The 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 completely active. The enzymatic activity lost started at 2.0 M GdmCl and was monophasic but coincided with the second‐phase denaturation with [GdmCl] 0.5 = 2.46 M. Inorganic phosphate provides substantial protection 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 dimers after dilution of the GdmCl concentration. Refolding kinetics for the first‐phase denaturation is first‐order but not second‐order. The biphasic phenomenon thereby was a mixed dissociation‐denaturation process. A completely folded monomer never existed during the GdmCl denaturation. The biphasic denaturation curve thereby clearly demonstrates an enzymatically fully active intermediate state, which could represent an active‐site structure intact and other structure domains partially melted intermediate state. Proteins 33:49–61, 1998. © 1998 Wiley‐Liss, Inc.