Efficient peroxidase substrates may have a critical role in the oxidat
ion of secondary compounds by peroxidases. Hydrazines are often oxidiz
ed slowly by peroxidases due, in part, to hydrazine-dependent inactiva
tion of these enzymes. Peroxidase-catalyzed oxidation of hydrazines ma
y be dramatically affected by an efficient peroxidase substrate. We in
vestigated this hypothesis in a model system using the well-known pero
xidase substrate chlorpromazine (CPZ) and the hydrazine derivative iso
niazid. CPZ stimulated isoniazid oxidation as measured by nitroblue te
trazolium (NET) reduction and O-2 consumption. The kinetics of isoniaz
id and CPZ oxidation by horseradish peroxidase (HRP) in the presence o
f both compounds suggested CPZ was acting as an electron transfer medi
ator between HRP and isoniazid. Indeed, CPZ(.+), the product of CPZ ox
idation by HRP, was able to oxidize isoniazid. The rate constant for t
his pH-dependent reaction was (2.6 +/- 0.1) x 10(4) M(-1) s(-1) at pH
4.5. In the absence of CPZ, isoniazid-dependent irreversible inactivat
ion of HRP was observed. The inactivation process involved the formati
on of compound LII followed by accumulation of irreversibly inactivate
d HRP. CPZ completely inhibited inactivation. Thus, by acting as a red
ox mediator and preventing HRP inactivation, CPZ stimulated isoniazid
oxidation by several orders of magnitude. Similarly, other efficient p
eroxidase substrates, such as phenol and tyrosine, were also able to d
ramatically stimulate isoniazid oxidation by HRP. We suggest that the
presence of efficient peroxidase substrates may potentiate the activat
ion of isoniazid and other hydrazines. As such, these substrates may h
ave a vital role in the pharmacological and toxicological properties o
f hydrazines and other compounds.