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., Della Corte, L., Tipton, K.F., Sgaragli, G.P. (1988). Purification and characterization of rat intestinal peroxidase. Its activity towards 2-t-butyl-4-methoxyphenol (BHA). BIOCHEMICAL JOURNAL, 250(2), 501-507 [10.1042/bj2500501].
Purification and characterization of rat intestinal peroxidase. Its activity towards 2-t-butyl-4-methoxyphenol (BHA)
Valoti, Massimo;Sgaragli, G. P.
1988-01-01
Abstract
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.File | Dimensione | Formato | |
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https://hdl.handle.net/11365/38561
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