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Reactions of hydroxyl radicals with horseradish peroxidase (HRP) have been studied by means of pulse radiolysis technique in the absence and presence of oxygen. Hydroxyl radicals, produced in excess towards enzyme, react exclusively with the protein part of HRP with the rate constant k = 1.1 x 10(11) M-1 s-1. Activity loss induced by OH. is connected with such an enzyme modification that causes both the interference with substrate binding and partial blocking of the channel used by peroxide. It is shown that in the presence of oxygen the loss of activity is ca 10% higher, mainly due to restrictions in the formation of compound I, ie ferryl [Fe(IV) = O] pi-radical cation.

Some properties of catalase and horseradish peroxidase entrapped into reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane with those in an aqueous solution of AOT are compared. Secondary structure of catalase significantly changes and activity is completely lost in the presence of aqueous micellar solution of AOT. In AOT/n-heptane reverse micelles secondary structure of catalase does not change. AOT has no effect on horseradish peroxidase in aqueous solution. On the other hand slight changes in secondary structure of horseradish peroxidase in AOT/n-heptane reverse micelles appear. It is concluded that electrostatic interactions between catalase or horseradish peroxidase with AOT molecule at neutral pH are not the main factor determining reactivity of investigated enzymes in reverse micelles.

The reactions of two heme peroxidases, horseradish peroxidase and lactoperoxidase and their compounds II (oxoferryl heme intermediates, Fe(IV) = O or ferric protein radical Fe(III)R.) and compounds III (resonance hybrids [Fe(III)-O2-.--Fe(II)-O2] with superoxide radical anion generated enzymatically or radiolytically, and with hydroxyl radicals generated radiolytically, were investigated. It is suggested that only the protein radical form of compound II of lactoperoxidase reacts with superoxide, whereas compound II of horseradish peroxidase, which exists only in oxoferryl form, is unreactive towards superoxide. Compound III of the investigated peroxidases does not react with superoxide. The lactoperoxidase activity loss induced by hydroxyl radicals is closely related to the loss of the ability to form compound I (oxoferryl porphyrin pi-cation radical, Fe(IV) = O(Por+.) or oxoferryl protein radical Fe(IV) = O(R.)). On the other hand, the modification of horseradish peroxidase induced by hydroxyl radicals has been reported to cause also restrictions in substrate binding (Gebicka, L.Gebicki, J.L., 1996, Biochimie 78, 62-65). Nevertheless, it has been found that only a small fraction of hydroxyl radicals generated homogeneously does inactivate the enzymes.

It has been shown that dimethyl sulfoxide competes with the enzyme substrate towards horseradish peroxidase compound I, but probably not towards horseradish peroxidase compound II. Dimethyl sulfoxide may hinder the reaction of superoxide with horseradish peroxidase compound II by modifying heme center. In this connection dimethyl sulfoxide is not recommended as a solvent in catalytic studies of horseradish peroxidase.

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