Antibacterial peptide from normal rabbit serum. 2. Compositional microanalysis.

The composition and homogeneity of the primary bactericide (PC-III) isolated from normal rabbit serum were examined by microanalytical techniques. Dansylated acid hydrolysates of PC-III were analyzed by two-dimensional thin-layer chromatography on polyamide layers. Quantitation of the separated dansyl amino acids was achieved by elution into methanol, followed by determination of fluorescence in a filter fluorometer. The method proved inexpensive and reliable, allowing accurate analysis of peptide samples containing greater than 20 pmol of each amino acid. By use of this technique, an amino acid composition for PC-III is presented which suggests that the rabbit serum bactericide contains a single, 2000-dalton peptide composed of 17 amino acid residues, 24% basic and 35% nonpolar. One of the basic residues appears to be a modified amino acid, tentatively identified as N epsilon-methyllysine. Although small amounts of carbohydrate and lipid were also detected in PC-III by fluorescent and isotopic techniques, gel filtration or extraction with organic solvents removed much of this material without significantly affecting biological activity.