Copper binding to bilirubin as determined by FT-IR and EPR spectroscopy

Copper is known to form complexes with bilirubin (H₂BR). Such complexes have received increased attention because of their clinical significance as free-radical scavengers. The purpose of this study was to examine a series of Cu²⁺ BR complexes to ascertain the nature of the binding between Cu²⁺ and BR. Several physical mea-surements of the salts were made, such as Fourier transform infrared (FT-IR) and electron paramagnetic resonance (EPR). The complexes were prepared by dissolving protonated BR in NaOH and adding different ratios of aqueous CuCl₂. At ratios of Cu²⁺/H₂BR of 1:1 and 2:1, soluble complexes were formed. In solution, EPR spectra demonstrated nine hyperflne peaks, which, from the splitting, is indicative of Cu²⁺ coordinated to four nitrogen atoms coming from two molecules of BR. The solid obtained from the solutions demonstrated predominant infrared absorptions at 1574 and 1403 cm^-1 (previously assigned as COO vibrations, asymmetric and symmetric), whereas the 1710-cm^-1 vibration appears only as a shoulder (previously assigned as the free COOH vibration), indicative that most of the COO groups have reacted with sodium, thus accounting for the aqueous solubility. The NH stretching vibration in the pyrrole group of H₂BR has disappeared and is replaced with the OH stretching vibration in H₂O. At higher ratios of 3:1 and 5:1 (Cu²⁺/H₂BR), black precipitates are formed, which produce no EPR signals. Furthermore, the NH vibration disappears as in the soluble solution complexes. It can be concluded that the insoluble salts (higher Cu²⁺XH₂BR ratios) are mixed complexes containing the Cu-nitrogen chelate and Cu salts involving the COOH groups.