The reaction of horseradish peroxidase with hydroperoxides derived from Triton X-100

Abstract

All of the commercially available Triton X-100 examined gave Compound I upon reaction with horseradish peroxidase, followed by its gradual transition into Compound II. Titration of horseradish peroxidase with Triton X-100 to form Compound I indicated that 1% ( v v ) aqueous solutions of the detergent contained 0.4 to 3.2 μ m equivalent peroxide but iodometric titration revealed 1.1 to 5.0 μ m peroxide, suggesting the occurrence of different types of peroxides, reactive and unreactive with the peroxidase. The rate constant for Compound I formation was 1.5 × 10 7 m −1 s −1 at pH 7.4 at 25°C, and for conversion into Compound II apparent firstorder rate constants were 5.2 × 10 −3 to 1.7 × 10 −2 s −1. These results indicate that the Triton peroxides are as highly reactive as hydrogen peroxide. The amount of Triton peroxides increased as aqueous solutions of the detergent were allowed to stand, but the peroxides were destroyed by treatment with sodium borohydride. Although freshly prepared aqueous solutions of sodium cholate, sodium dodecyl sulfate, Tween 20 (polyoxyethylene sorbitan monolaurate), and Emasol 1130 (an equivalent of Tween 20) did not contain any detectable amount of peroxide, aged solutions of sodium dodecyl sulfate and Emasol 1130 contained peroxides. These observations suggest the need for appropriate precautions when biologically active substances vulnerable to attack by peroxides are incubated with Triton X-100 either for their solubilization from biomembranes or for other processing.