Abstract:
The Eupatorium odoratum leaf peroxidase exists as at least seven distinct isozymes (three cationic, three anionic, and one neutral). These isozymes were identified and separated by preparative iso-electric focusing. Thermal stability, including the activation enthalpy (Delta H*), free energy of inactivation (Delta G*) and activation entropy (Delta S*), and kinetic studies of two isozymes, one having a pI of 5.0 (E5) and another one having a pI of 7.0 (E7) with mol mass of 43 and 50 kD, respectively, were studied in detail. Of the molecular weight of E5 and E7, 25 and 32% correspond to the carbohydrate content of the isozymes. Optimal pH was in the acidic range of 3.6-3.8 for E5 and 3.8 for E7 with the oxidation of ABTS. E7 and E5 showed activation energy for inactivation, 194.8 and 145.4 kJ/mol, respectively. Both the isozymes showed distinct substrate specificity. The catalytic specificity constant for E5 and E7 were 112 x 105 and 124 x 10(5)/s(.)M, respectively, when 2,2'-azino-bis-(3-ethylbenz-thiazoline-6 sulfonic acid) was used as the substrate. Maximum affinity (i.e., lowest K(m) value) to H(2)O(2) was shown by E5 and E7 along with Pyrogallol and was 0.02 and 0.05/s(.)M, respectively.
