Studies on horseradish peroxidase in dimethyl sulphoxide/water mixtures. The activation of hydrogen peroxide and the binding of fluoride

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Abstract
The variation was studied in spectra and in reactivity towards H2O2 of solutions of horseradish peroxidase [EC 1.11.1.7] in dimethyl sulfoxide/water mixtures, obtained by diluting stock solutions of the enzyme activity was assayed and the binding of F- by the peroxidase in 65% (vol/vol) dimethyl sulfoxide was studied. A broadly similar pattern of changes is observed whether one starts from water or from dimethyl sulfoxide; the changes are essentially reversible, though hysteresis is observed. When the dimethyl sulfoxide content of the solvent mixture is increased, the peroxidase retains its ability to activate H2O2 up to 74% (vol/vol) dimethyl sulfoxide. The peroxidase in 65% (vol/vol) dimethyl sulfoxide binds F- together with proton (or the equivalent loss of HO-), as already established for aqueous solutions. The occurrence in such solutions of both the ability to activate H2O2 and the inability to bind F- without taking up H+ or losing HO- supports the proposed mechanism for activating H2O2, whereby the protein binds the substrate in the form of the more reactive HO2-.