Fluid from a post-operative
wound, six
leg ulcers and a large
blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous
acetonitrile containing 1%
trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the
blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a
lysozyme assay). The material corresponding to HPLC peaks of the
wound fluid extract was identified as:
histone H2B fragments 1-11,1-15 and 1-16, intact
thymosin beta-4,
defensins HNP1, 2 and 3,
lysozyme and the
peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated
blister fluid was extremely rich in anti-Bm activity (mainly
defensins) and
lysozyme. It may also contain factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in
lysozyme, factors which together with
thymosin could be active in wound healing. It is concluded that analysis of
wound fluid yields
peptide and activity patterns with novel fragments of important
peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.