The results of our assays with LPS and lipid A purified from the strains we used in the above 125I-GGPG binding studies are shown in Fig.
The full sequence of GGPG is GGGYGGRGGRLCFCRRRFCVCVGR-CONH 2 . In preliminary experiments, we determined that PG-1, GGPG, and I-GGPG exhibited virtually identical antimicrobial potency and kinetics (data not shown).
The bacteria were incubated with 125I-GGPG at 0° C for 60 min at pH 7.4 in a 10 mM Tris-acetate buffer containing 100 mM NaCl, 1% TSB, and 1% BSA.
Incubation of 125I-GGPG with intact P. aeruginosa 9027 (PG-1 sensitive) and B. cepacia 25416 (PG-1 resistant) cells demonstrated a marked difference in protegrin binding between the two strains (Fig.
BSA = bovine serum albumin; CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; C L = lethal concentration; DMSO = dimethyl sulfoxide; EC 50 = effective concentration 50%; EDTA = ethylenediamine tetraacetic acid; GGPG = modified PG-1 with a glycine-rich hexapeptide extension (GGGYGG) containing a single tyrosine residue; HEPES = N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; HPLC = high performance liquid chromatography; I-GGPG = monoiodinated variant of GGPG; LPS = lipopolysaccharide; MAC = minimal active concentration; PG-1 = protegrin-1; RU = resonance units; SDS = sodium dodecyl sulfate; SPR = surface plasmon resonance; TSB = trypticase soy broth.