Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride

✍ Scribed by Hui-Chih Hung; Gu-Gang Chang

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 289 KB
• Volume: 33
• Category: Article
• ISSN: 0887-3585
• DOI: 10.1002/(sici)1097-0134(19981001)33:1<49::aid-prot5>3.0.co;2-g

✦ Synopsis

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] Ͼ 3 M. The dissociated monomers reassociated to dimers after dilution of the GdmCl concentration. Refolding kinetics for the first-phase denaturation is firstorder but not second-order. The biphasic phenomenon thereby was a mixed dissociationdenaturation 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.