Various forms of mouse lactoferrins: purification and characterization

Abstract

This work was conducted to study the microheterogeneity of mouse lactoferrin (LF). Two forms, LF 1 and LF 2, could be purified from uterine luminal fluid by ion-exchange HPLC on a Protein PAK SP 5PW column. Another form, LF 3, was purified from the epididymis homogenate by affinity chromatography on a column of Protein A-Sepharose coupled with the purified LF 2 antibody that was prepared to give no crossreaction with serum albumin. Both LF 1 and LF 2 showed a M r 74 000 band while LF 3 gave a M r 70 000 band on reducing SDS–PAGE. All of them were reduced to a M r 68 000 band after they had been digested with N-glycosidase F. The data from automated Edman degradation confirmed the completely identical 19 amino acid sequences in the N-terminal regions of these three LFs, except the lack of N-terminal Lys–Ala of LF 2/LF 3 in LF 1. LF in tissue homogenates was immunodetected by Western blot procedure using the purified LF 2 antibody. Different amounts of LF with a molecular mass of the 70 000 or 74 000 were distributed in the non-sexual organs such as kidney, spleen, lung, heart and liver and the sexual glands including epididymis, vagina, uterus, ovary and prostate. No LF was detected in stomach, intestine, testis and seminal vesicle.