title: A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C.
abstract: Horseradish peroxidase (HRP) is a commercially important enzyme that is available from a number of supply houses in a variety of grades of purity and isoenzymic combinations. The present article describes a comparative study made on nine HRP preparations. Six of these samples were 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 mMABTS and 0.2 mM H(2)O(2) was around 950 s(-1) (about 770 s(-1) for the less pure samples) and with a 5 mM guaiacol and 0.6 mM H(2)O(2) was about 180 s(-1) for all the samples. A similar value (approximately 1000 s(-1)) was observed for acidic HRP but only at higher concentrations of ABTS (20 mM). With 20 mM guaiacol the molar catalytic activity of the acid isoenzyme was 65 s(-1). The apparent K(M) for ABTS of the acidic isoenzyme was 4 mM whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H(2)O(2) 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), apparent dissociation constant (K(l)), and apparent rate constant of inactivation (k(inact)) were about twice as large for the acidic samples (1350, 2.6 mM, 9 x 10(-3) s(-1)) as for the basic (650, 1.3 mM, 5 x 10(-3) s(-1)). The apparent catalytic constant (k(cat)) was 3-4 times larger, and the efficiency of catalysis (k(cat)/K(l)) was double for the acidic isoenzyme, but the efficiency of inactivation (k(inact)/K(l)) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bioreactors, or assays).
authors.affiliation: Departamento de Biología Vegetal, Fisiología Vegetal, Universidad de Murcia, 30001 Murcia. Spain.
authors.forename: A N
authors.initials: AN
authors.lastname: Hiner
authors.forename: J
authors.initials: J
authors.lastname: Hernández-Ruíz
authors.forename: M B
authors.initials: MB
authors.lastname: Arnao
authors.forename: F
authors.initials: F
authors.lastname: García-Cánovas
authors.forename: M
authors.initials: M
authors.lastname: Acosta