Purification of a 76-kDa iron-binding protein from human seminal plasma byaffinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 mi of seminal plasma. Despite its ability to bi nd a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yea st RNA as substrate. It showed an apparent molecular weight of 76 kDa and r esulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting w ith Arg-3 was identical to that of one of the N-terminally truncated lactof errin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence , has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recogni tion by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and hum an seminal protein were recognized by antibodies anti-milk lactoferrin. Whe n tested for its iron binding capacity, with Fe-NTA as iron donor, the prot ein purified was able to bind iron up to 100% saturation, as judged by abso rbance at 465 nm. (C) 1999 Elsevier Science B.V. All rights reserved.