Biochemical characterization of lactoferrin and a 39 kilodalton protein isolated from bovine mammary secretions during involution

Bovine lactoferrin, in quantities large enough for accurate biochemical analysis, was isolated from mammary secretions using heparin-agarose affinity chromatography. This method was more efficient and resulted in a more pure product than a method using a series of chromatographic columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis indicated that the purified lactoferrin was composed of two proteins with molecular weights of 83 and 87 kilodaltons (Kd). Endoglycosidase digestion indicated that this difference in molecular weight may not be due exclusively to carbohydrate content. Purified lactoferrin also contained several lower molecular weight proteins which were identified as proteolytic cleavage products of lactoferrin. An unidentified 39Kd protein also was isolated by heparin-agarose affinity chromatography from mammary secretions and was found to be specific to the nonlactating period. Using enzyme-linked immunosorbent assays developed for each protein, the concentrations of lactoferrin and 39Kd protein were shown to increase in parallel. Immunohistochemical localization of {dollar}alpha{dollar}-lactalbumin, {dollar}beta{dollar}-lactoglobulin and lactoferrin paralleled secretion changes of the nonlactating period. Immunohistological localization of lactoferrin occurred in the area of the basement membrane adjacent to alveoli particularly during the later stages of involution. Characterization of the mammary proteins will provide the basis for further research on the functional changes that occur in the mammary gland during the nonlactating period.