PURIFICATION AND CHARACTERIZATION OF A NOVEL CLASS-III PEROXIDASE ISOENZYME FROM TEA LEAVES

A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP ; relative molecular weight = 34,660 +/- 10, mean +/- SE) that can acc ount for a significant part of the ascorbate peroxidase activity in te a (Camellia sinensis) leaves has been purified to homogeneity. The ult raviolet/visible absorption spectrum is typical of heme-containing pla nt peroxidases, with a Soret peak at 406 nm (epsilon = 115 mM(-1) cm(- 1)) and an A(406)/A(280) value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 mu mol min(-1) mg(-1)), with a p H optimum in the range of 4.5 to 5.0. Substrate-specificity studies ha ve revealed significant differences between TP and other class III per oxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate ove r guaiacol, whereas other class III isoenzymes are characterized by 2- orders-of-magnitude higher activity for guaiacol than for ascorbate. T P also forms an unstable porphyrin pi cation radical-type compound I, which is converted to compound II within approximately 2 min in the ab sence of added reductant. Amino acid sequence data show TP to be the f irst example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.