Neutrophil defensins: Purification, characterization, and antimicrobial testing

This chapter describes the large-scale purification of the four defensins, HNP-1, -2, -3, and -4, found in human neutrophils and describes procedures for testing their antimicrobial properties. It is most convenient to obtain human neutrophils from normal donors by leukaphoresis. If leukaphoresis units are not available, neutrophils can be prepared from single-unit “buffy coats” or whole blood donations and then pooled. The human leukocyte pellets are gently suspended in 100 ml of calcium and EGTA-free HBSS and transferred into a 400-ml beaker that is placed in the precooled (to 4°) Parr cell. Extraction is performed by suspending each granule pellet, typically derived from 2 to 3 x 109 neutrophils, in 5 ml of ice-cold 5% acetic acid and combining suspensions equivalent to approximately 4 x 1010 cells in a beaker. The chapter describes the procedure for the purification of defensins from granule extract. Peptide purity is assessed by 16.5% tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), using a minigel apparatus, by 12.5% AU-PAGE, and by analytical reversed-phase high-performance liquid chromatography (RP-HPLC). The individual defensins can be identified by their characteristic migration on AU-PAGE or by performing N-terminal amino acid sequencing.