Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy

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Abstract
Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman and EPR spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin''s intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III) and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at .apprx. 1605, 1505, 1275 and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as was observed previously for serum transferrins and permit the principal absorption band (.lambda.max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. As in serum transferrin the 2 metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only 1 of the 2 sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin differs from serum transferrin in its enhanced affinity for Fe. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)-citrate is 10 times faster for apolactoferrin than for apotransferrin. The more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for Fe removal from lactoferrin causes permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). The common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) apparently has an adverse effect on the protein.