Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes.

Abstract

A potently bactericidal cationic protein that increases the envelope permeability of susceptible Gram-negative bacteria has been isolated from human leukocytes and purified to near homogeneity. The active fraction was extracted with 0.2 M sodium acetate (pH 4.0) from the granules of polymorphonuclear leukocytes obtained from normal individuals or from patients with chronic myelocytic leukemia. Mononuclear cells contained less than 30% of the activity extracted from polymorphonuclear cells. Successive chromatography of acid extracts on Sephadex G-75 and Bio-Rex 70 purified 40-fold a protein fraction that appeared to be at least 95 to 98% pure as judged by rechromatography on Sephadex G100, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein has an isoelectric point at pH 9.8 and an apparent molecular weight of 58,000 to 60,000, estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein exhibits no protease, lysozyme, myeloperoxidase, or phospholipase A activity and may, therefore, be a noncatalytic bactericidal protein. Killing by the purified protein of several strains of Escherichia coli and Salmonella typhimurium occurs with only slight inhibition of bacterial protein synthesis, but is accompanied by an almost immediate increase in permeability of the envelope of susceptible bacteria to normally impermeant actinomycin D. Entry of actinomycin D can be measured by the inhibitory effect of the antibiotic on protein (or RNA) synthesis. Both bactericidal and permeability increasing activities are maximal at pH 7.0, inhibited by Mg2+, Ca”., or heparin, and inactivated by heating at 80” for 10 min. “Rough” strains of E. coli and S. typhimurium are most sensitive to the biological effects (0.5 to 2.0 pg of protein (10 to 40 nM) produce >90% bactericidal and permeability effects on 10’ bacteria/ml); “smooth’‘-strains are less sensitive (2 to 20 pg), and several species of Gram-positive bacteria as