acid-phosphatase and 2-2--azobis(2-amidinopropane)

We assessed the antioxidative effects of quercetin-feeding on ddY strain male mice by measuring luminol-amplified chemiluminescence that was enhanced by a hydrophilic free radical initiator 2, 2'-azobis(2-amidinopropane) dihydrochloride. Quercetin suppressed chemiluminescent intensity in a dose-dependent manner in vitro with a half-inhibition concentration (IC(50)) of 3x10(-8) M, which was lower than the value of synthetic antioxidant 2, 6-di-tert-butyl-p-cresol (IC(50): 7x10(-7) M). Lysosomal (12000xg pellet) and supernatant fractions obtained from the livers of mice fed a diet containing 0.2% quercetin for 7 days showed more inhibition of chemiluminescent intensity than those from non-treated mice. Quercetin feeding also resulted in the inhibition of lipid peroxidation as determined by a thiobarbituric acid reactive substance test which detected suppression of the release of lysosomal enzymes induced by lipophilic free radical initiator 2, 2'-azobis(2,4-dimethylvaleronitrile). These results probably reflect radical quenching activity of quercetin, indicating that the measurement of chemiluminescence is a useful tool for the assessment of total antioxidant activity in biological systems.

Topics: Acetylglucosaminidase; Acid Phosphatase; Administration, Oral; Amidines; Animals; Azo Compounds; Butylated Hydroxytoluene; Dose-Response Relationship, Drug; Lipid Peroxidation; Liver; Luminescent Measurements; Luminol; Lysosomes; Male; Mice; Mice, Inbred Strains; Nitriles; Oxidants; Phenols; Quercetin; Subcellular Fractions; Thiobarbituric Acid Reactive Substances

Acid phosphatase (AP) is readily inactivated when exposed to the free radicals generated in the pyrolysis of 2, 2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions. On average, a large number of tryptophan groups are modified by each protein molecule that loses its catalytic activity. In spite of this, the enzyme inactivation takes place without induction times, a result that indicates either that damage is progressive or that damage of a critical target is needed to inactivate the enzyme (all-or-nothing mechanism). A Lineweaver-Burk plot of the enzyme activity measured at pH 4.8 is not compatible with an all-or-nothing mechanism, showing that after exposure of the native protein ensemble to the free radical source there are partially damaged molecules whose affinity for the substrate is widely different from that of the native molecules. On the other hand, the partially damaged ensemble shows a normal Michaelis-Menten behavior when the activity is measured at pH 7.0, with only a reduced value of V(M), relative to that of the unmodified ensemble. These results show that the native protein and modified proteins that remain active constitute different populations, with different responses to pH changes. Comparative heat denaturation studies of the native and pretreated proteins support this proposal.

Topics: Acid Phosphatase; Amidines; Catalysis; Enzyme Stability; Fluorescence; Free Radicals; Hydrogen-Ion Concentration; Kinetics; Oxidants; Peroxides; Protein Denaturation; Solanum tuberosum; Temperature; Tryptophan; Urea