PURIFICATION OF CYSTEINE-RICH BIOACTIVE PEPTIDES FROM LEUKOCYTES BY CONTINUOUS ACID-UREA POLYACRYLAMIDE-GEL ELECTROPHORESIS

A new continuous acid-urea-polyacrylamide gel electrophoresis (CAU-PAGE) preparative method was developed and used to purify rabbit and human defensins. With it, we identified two post-translationally modified forms of rabbit defensins NP-1 and NP-2, and purified a processed RANTES (β-intercrine) peptide from leukophoresed human leukocytes. CAU-PAGE afforded approximately 70% recovery of rabbit defensin NP-5. The recovered defensins were not N-terminally modified, and their in vitro antimicrobial activity was equivalent to that of defensins purified by previously described chromatographic methods. Since CAU-PAGE is performed under nonreducing conditions, it should be especially useful for purifying cationic peptides with intramolecular disulfide bonds, such as defensins and α or β-intercrines. © 1994 Academic Press, Inc. All rights reserved.