Kinetics and thermal stability of two peroxidase isozymes from Eupatorium odoratum.
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 (deltaH*), free energy of inactivation (deltaG*) and activation entropy (deltaS*), 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 105/s.M, respectively, when 2,2'-azino-bis-(3-ethylbenz-thiazoline-6 sulfonic acid) was used as the substrate. Maximum affinity (i.e., lowest Km value) to H2O2 was shown by E5 and E7 along with Pyrogallol and was 0.02 and 0.05/s.M, respectively.
Rani
D Nisha
DN
Polymer Science, Chemical Sciences Division, Regional Research Laboratory (CSIR), Trivandrum 695 019, India.
Abraham
T Emilia
TE
D052160:Benzothiazoles; D002384:Catalysis; D036604:Chromolaena / Q000201:enzymology*; D019277:Entropy; D004795:Enzyme Stability; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009195:Peroxidase / Q000378:metabolism*; D011748:Pyrogallol / Q000378:metabolism; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000737:chemistry; D013997:Time Factors
D052160:Benzothiazoles; D007527:Isoenzymes; D013451:Sulfonic Acids; D011748:Pyrogallol; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006861:Hydrogen Peroxide; D009195:Peroxidase