RESUMO
No larger molecular weight component appeared in the high-performance gel chromatogram when calcium alone was added to the riboflavin-binding protein solution (RfBP), indicating that calcium alone did not aggregate it. RfBP bound calcium, but the amount of bound calcium decreased markedly upon dephosphorylation. Cross-linked RfBP, which was detected by high-performance gel chromatography using simulated milk ultrafiltrate as the effluent, was formed when calcium and phosphate were added. Cross-linking of RfBP was confirmed by ultracentrifugal analysis. RfBP was found to be cross-linked through its phosphate groups, since no cross-linked fraction was detected when calcium and phosphate were added to a dephosphorylated RfBP solution. RfBP cross-linked by calcium phosphate formed at 12-20 mM calcium, 13-17 mM phosphate, and 10 mM citrate was a dimer, since its retention time was consistent with that of the dimer cross-linked by glyceraldehyde. The physiological function of phosphate groups of RfBP was also discussed.