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 (beta-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 alpha or beta-intercrines.
Harwig
S S
SS
Department of Medicine, UCLA School of Medicine 90024.
Chen
N P
NP
Park
A S
AS
Lehrer
R I
RI
D000595:Amino Acid Sequence; D000818:Animals; D001770:Blood Bactericidal Activity; D001797:Blood Protein Electrophoresis / Q000379:methods*; D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003545:Cysteine / Q000737:chemistry; D023082:Defensins; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005069:Evaluation Studies as Topic; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000737:chemistry; D008969:Molecular Sequence Data; D011817:Rabbits; D014508:Urea
D001798:Blood Proteins; D023082:Defensins; D014508:Urea; D003545:Cysteine