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 measurements 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 hyperfine 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⁺/H₂BR ratios) are mixed complexes containing the Cu–nitrogen chelate and Cu salts involving the COOH groups.