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 Cunitrogen chelate and Cu salts involving the COOH groups.
J. R. Ferraro, J.-G. Wu, R. D. Soloway, W.-H. Li, Y.-Z. Xu, D.-F. Xu, and G.-R. Shen, "Copper Binding to Bilirubin as Determined by FT-IR and EPR Spectroscopy," Appl. Spectrosc. 50, 922-927 (1996)