Potency of Bactericidal Proteins Purified from the Large Granules of Bovine Neutrophils

The novel population of large granules of bovine neutrophils, which is the cell store of bactericidal activity independent of O2 derivatives, was extracted with an acid medium. Several fractions were resolved from the extract by ion-exchange chromatography (with carboxymethyl-cellulose) and gel filtration (with Sephadex G-50). Some of these fractions contained only a very limited number of major components, as detected by polyacrylamide gel electrophoresis. The purified bactericidal proteins exhibited their activity at 0.1 to 10 micrograms/0.3 ml of assay mixture containing 1 X 10(6) to 2 X 10(6) CFU of Staphylococcus aureus or Escherichia coli in media with physiological concentrations of Na+, K+, Mg2+, and Ca2+. Two fractions, containing polypeptides with apparent molecular weights ranging from 28,000 to less than 12,000, caused rather selective and rapid (5 to 20 min) killing of S. aureus. Their action was accompanied by significant binding to the gram-positive bacteria of some low (less than 12,000)-molecular-weight components. Other Sephadex G-50 fractions, containing the first emerging proteins with relatively high molecular weights, were more active on E. coli than on S. aureus. With the gram-negative bacteria there was a 10-min delay in the onset of bactericidal activity, which thereafter developed very fast. On the basis of the in vitro potency of the large-granule bactericidal proteins, we suggest that even partial discharge of granule content into the phagosomes may supply the phagocytic vacuoles of bovine neutrophils with a very efficient nonoxidative bactericidal system acting on both gram-positive and gram-negative microorganisms.