Valoti M, Della Corte L, Tipton K F, Sgaragli G
Centro di Ricerca Interdipartimentale sul Metabolismo, Università di Siena, Italy.
Biochem J. 1988 Mar 1;250(2):501-7. doi: 10.1042/bj2500501.
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.
已表明大鼠肠道过氧化物酶的不纯制剂在低离子强度下会聚集，而在较高离子强度下会解聚。这种聚集伴随着比活性的降低，这可能导致反应进程曲线出现滞后行为。利用这种可逆聚集获得了相对纯的提取物，随后通过在伴刀豆球蛋白A - 琼脂糖上进行亲和色谱，再进行疏水色谱，将其纯化至表观均一性。纯化后的酶未表现出离子强度依赖性聚集行为，以50,000的相对分子质量单体形式存在。已表明纯化后的酶能催化常用抗氧化剂2 - 叔丁基 - 4 - 甲氧基苯酚（丁基化羟基茴香醚，BHA）的过氧化物转化反应，生成3,3'-二叔丁基 - 2,2'-二羟基 - 5,5'-二甲氧基联苯，其米氏常数（Km）值为176微摩尔，最大反应速度为每毫克8微摩尔/分钟。该底物的特异性常数kcat./Km与对底物愈创木酚的相似。
