Observations on the Cytolytic Activity of Lactoperoxidase Using a Continuous Assay

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Abstract
The combination of myeloperoxidase, hydrogen peroxide and a halide ion forms a cytotoxic mixture that effectively kills bacteria, fungi and viruses. A turbidometric assay that allows continuous monitoring of the cytolytic activity of toxic agents toward various target cells was developed. This assay monitored 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, was expressed as the number of cells lysed per minute, which facilitated the determination of kinetic constants. Using this procedure the cytolytic activity exerted by various peroxidases in the presence of hydrogen peroxide and a halide ion proceeded in at least 2 stages. During the 1st stage no lysis occurred, but scanning electron microscopy showed that there were alterations in the target cell membrane. During the 2nd stage the target cells lysed, releasing metabolites and macromolecules. The lytic action of peroxidases is directed toward the target cell membrane, which appears to acquire an increased rigidity and subsequently disintegrates.