Urea inhibits the activity of alkaline phosphatase during the reaction course. The
inactivation is progressively stronger for the placental, intestinal and renal subforms.
Influence of reaction temperature, pH, type and molarity of buffer, magnesium chloride, albumin
and enzyme concentration on the inactivation mechanism is evaluated. In all experimental
conditions the process follows pseudofirst-order kinetics and the inactivation profiles are
distinct and typical for each enzymatic subform. With a simple graphical analysis, a single
inactivation curve in controlled experimental conditions, allows the identification of each
isoenzyme from the slope and the calculation of the respective fractional amount from the
intercept of the time-activity plot.
Moderate concentrations of guanidinium chloride induce both instantaneous and time-dependent modifications of the catalytic and optical properties of intestinal alkaline phosphatase, which undergoes consecutive conformational transitions at about 0.05 M, 0.25 M and 1.0 M denaturant. A paradoxical activation is observed up to 1.0 M-guanidine, with a maximum at 0.25 M- and a mid-point around 0.5 M-guanidine. Difference absorbance and fluorescence spectra imply a change in the state of ionization of the protein residues, with variation in molecular size suggested by light-scattering. Random-coil formation is indicated by a lower fluorescence yield, a more polar environment of the aromatic residues and another separate tryptophan emission. Iodide quenching confirms the alterations of conformation. Deprotonation favours the loss of the intramolecular constraints and the enhancement of the structure disruption by guanidine.
Ascorbic acid is found strikingly to decrease the activity of bovine kidney alkaline
phosphatase in vitro. The inhibition of alkaline phosphatase is a function of ascorbic acid
concentration and is time and temperature dependent. The presence of the substrate protects
the enzyme against the inhibitory action of the vitamin.
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