Lysine 190 is a covalent bilirubin-binding amino acid in human delta bilirubin

In this study, a covalent bilirubin-binding amino acid residue of the albumin molecule in δ bilirubin was identified. Icteric human serum was treated with a diazo reagent and bilirubin was converted to a stable azodipyrrole. Then, an albumin fraction containing an azodipryrrole-albumin covalent conjugate was obtained by affinity chromatography using Blue Sepharose CL-6B, and it was further purified by reverse phase high performance liquid chromatography. To identify the covalently bound azodipyrrole amino acid residue in albumin, the azodipyrrole-albumin conjugate was cleaved to fragmented peptides by chemical and enzymatical methods, and three main peptide fractions which had the absorption characteristics of azodipyrrole at 530 nm were isolated. Their amino acid sequence analysis revealed that all of the peptides corresponded to residues 187–191 in albumin, where lysine residue 190 was identified as the covalent-binding site of albumin to bilirubin in δ bilirubin.