A COMPARATIVE-STUDY OF THE PURITY, ENZYME-ACTIVITY, AND INACTIVATION BY HYDROGEN-PEROXIDE OF COMMERCIALLY AVAILABLE HORSERADISH-PEROXIDASE ISOENZYME-A AND ISOENZYME-C

Horseradish peroxidase (HRP) is a commercially important enzyme that i s available from a number of supply houses in a variety of grades of p urity and isoenzymic combinations. The present article describes a com parative study made on nine HRP preparations. Six of these samples wer e predominantly composed of basic HRP, pl 8.5, and three of acidic HRP , pl 3.5. Two of the basic preparations were of lower purity than the others. The apparent molar catalytic activity of basic HRP with 0.5 mM ABTS and 0.2 m MH(2)O(2) was around 950 s(-1) (about 770 s(-1) for the less pure samples) and with a 5 m M guaiacol and 0.6 m M H2O2 was abo ut 180 s(-1) for all the samples. A similar value (approximately 1000 s(-1)) was observed for acidic HRP but only at higher concentrations o f ABTS (20 m M). With 20 m Mguaiacol the molar catalytic activity of t he acidic isoenzyme was 65 s(-1). The apparent K-m for ABTS of the aci dic isoenzyme was 4 m M whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H2O2 when it was supplied as the only substrate. Under these conditions the partition ratio (r = number of catalytic cycles given by the enzyme before its inactivation), app arent dissociation constant (K;), and apparent rate constant of inacti vation (k(inact)) were about twice as large for the acidic samples (13 50, 2.6 mM, 9 . 10(-3) s(-1)) as for the basic (650, 1.3 m M, 5 . 10(- 3) s(-1)). The apparent catalytic constant (k(cat)) was 3-4 times larg er, and the efficiency of catalysis (k(cat)/K-1) was double for the ac idic isoenzyme, but the efficiency of inactivation (k(inact)/K-1) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bi oreactors, or assays). (C) 1996 John Wiley & Sons, Inc.