Effect of lactoferrin on oxidative features of ceruloplasmin

In our previous report we first described a complex between lactoferrin (Lf) and ceruloplasmin (Cp) with K (d) similar to 1.8 mu M. The presence of this complex in colostrum that never contains more than 0.3 mu M Cp questions the reliability of K (d) value. We carefully studied Lf binding to Cp and investigated the enzymatic activity of the latter in the presence of Lf, which allowed obtaining a new value for K (d) of Cp-Lf complex. Lf interacting with Cp changes its oxidizing activity with various substrates, such as Fe2+, o-dianisidine (o-DA), p-phenylenediamine (p-PD) and dihydroxyphenylalanine (DOPA). The presence of at least two binding sites for Lf in Cp molecule is deduced from comparison of substrates' oxidation kinetics with and without Lf. When Lf binds to the first site affinity of Cp to Fe2+ and to o-DA increases, but it decreases towards DOPA and remains unchanged towards p-PD. Oxidation rate of Fe2+ grows, while that of o-DA, p-PD and DOPA goes down. Subsequent Lf binding to the second center has no effect on iron oxidation, hampers DOPA and o-DA oxidation, and reduces affinity towards p-PD. Scatchard plot for Lf sorbing to Cp-Sepharose allowed estimating K (d) for Lf binding to high-affinity (similar to 13.4 nM) and low-affinity (similar to 211 nM) sites. The observed effect of Lf on ferroxidase activity of Cp is likely to have physiological implications.