Lactoferrin (LF), a principal glycoprotein component of secretions that bathe mucosal surfaces and of the specific granules of neutrophils (PMN), is capable of high affinity coordinate binding of Fe3+ and CO3 2- It has been shown to have bacteriostatic proper- ties related to its ability to with-hold iron and is capable of a potent bactericidal effect that is independent of this iron deprivation. The overall objectives of these continuing studies are to characterize the mechanisms of LF killing and determine the nature and biological significance of IgA-LF interactions. Specifically, the distribution and quantity of IgA-LF complex in human milk and saliva will be determined using ELISA procedures specific for this complex. IgA-LF complex will be isolated and characterized as to the physicochemical nature of the association, its antibody specificity, and its bactericidal activity as it relates to antibody specificity. An in vitro model for IgA-LF complexity will be examined using various IgA preparations. The kinetics and nature of this association will be examined using methods similar to that used for determining SC binding to IgA. Determination of the active sites of the LF molecule responsible for bactericidal activity will be pursued using proteolytic and chemical digestion and isolation of the defined fragments. Their biological activity and the nature of their binding to target sites will be examined. The possibility that proteolytic cleavage of Lf may be important in the sequence of events leading to expression of cidal activity will be approached using various protease inhibitors in the cidal reaction mixtures. LF target sites on the surface of bacteria will be examined using LF that has been labeled using mild reductive methylation. The influence of surface modification of bacteria on kinetics of binding of LF will be examined and the relationship of binding kinetics to bactericidal activity will be determined. The possible involvement of radical mechanisms in LF-mediated toxicity will be investigated by determining the nature of damage to target microorganisms determining the effects of anaerobiosis and various radical scavengers in LF cidal activity and by comparing LF effects with those of known radical generators. LF will be purified from both PMN and saliva and the biochemistry and biological activity compared with that of milk LF. The role of LF in the bacteria dal activity of the PMN will be examined using LF depletion/reconstitution studies with neutrophils and a dual staining procedure for the simultaneous determination of phagocytosis and killing of target microorganisms.
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