Observations on the cytolytic activity of lactoperoxidase using a continuous assay.

Abstract

A turbidometric assay that allows continuous monitoring of the cytolytic activity of toxic agents toward various target cells has been developed. This assay monitors the change in absorbance at 600 nm (due to light scattering) of a suspension of human red blood cells as a function of time. The rate of cell lysis, delta A600/delta t, can be expressed as the number of cells lysed per minute, which facilitates the determination of kinetic constants. Using this procedure we observed that the cytolytic activity exerted by various peroxidases in the presence of hydrogen peroxide and a halide ion proceeds in at least two stages. During the first stage no lysis occurs, but scanning electron microscopy showed that alterations in the target cell membrane take place. During the second stage the target cells lyse, resulting in a simultaneous release of metabolites and macromolecules. We conclude that the lytic action of peroxidases is directed toward the target cell membrane, which appears to acquire an increased rigidity and subsequently disintegrates.