The influence of fatty acid micelles on the assays for SOD activity.

The influence of fatty acid (FA) micelles on cytochrome c(cyt.c) reduction and nitroblue tetrazolium (NBT) reduction assays for SOD activity, which continue to be widely used, has been studied. In the presence of FA micelles, the use of cyt.c reduction assay was found to overestimate the real activity of SOD. This effect is attributed to the following reasons. 1. The FA micelles lead to the denaturation of cyt.c, which gives rise to suppression of the reactivity of ferricyt.c(cyt.c(ox)) towards O2-. Furthermore, this denaturation increases the reoxidation rate of ferrocyt.c, and consequently the reoxidation causes a decreased rate of cyt.c(ox) reduction. 2. Positively charged cyt.c(ox) interacts with negatively charged FA micelles, and so cyt.c(ox) on the surface of FA micelles reacts less with negatively charged O2- because of electrostatic repulsion. Also in NBT reduction assay using a positively charged probe molecule, FA micelles cause the appearance of enhancement of SOD activity, due to suppression of the reactivity of NBT towards O2- by electrostatic repulsion. However, in both chemiluminescence assay using the uncharged probe molecule and LDH-NADH assay using the negatively charged probe molecule, FA micelles cannot influence the assays of the SOD activity, because the micelles do not interact electrostatically with probe molecules.