Purification and characterization of rat intestinal peroxidase. Its activity towards 2-t-butyl-4-methoxyphenol (BHA).
Impure preparations of rat intestinal peroxidase were shown to aggregate at low ionic strengths and to disaggregate at higher values. This aggregation was accompanied by a decrease in specific activity, which could lead to hysteretic behaviour of reaction progress curves. Advantage was taken of this reversible aggregation to obtain a relatively pure extract, which was subsequently purified to apparent homogeneity by affinity chromatography on concanavalin A-Sepharose followed by hydrophobic chromatography. The purified enzyme did not show the ionic-strength-dependent aggregation behaviour, behaving as a monomer of Mr 50,000. The purified enzyme was shown to catalyse the peroxidatic conversion of the commonly used antioxidant 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA) to form 3,3'-di-t-butyl-2,2'-dihydroxy-5,5'-dimethoxybiphenyl, with a Km value of 176 microM and a maximum velocity of 8 mumol/min per mg. The specificity constant, kcat./Km, for this substrate was similar to that shown towards the substrate guaiacol.
Valoti
M
M
Centro di Ricerca Interdipartimentale sul Metabolismo, Università di Siena, Italy.
Della Corte
L
L
Tipton
K F
KF
Sgaragli
G
G
D000818:Animals; D002083:Butylated Hydroxyanisole / Q000378:metabolism; D002850:Chromatography, Gel; D006139:Guaiacol / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry
D046911:Macromolecular Substances; D002083:Butylated Hydroxyanisole; D006139:Guaiacol; D006861:Hydrogen Peroxide; D010544:Peroxidases