Phototherapy-induced covalent binding of bilirubin to serum albumin.

Bilirubin displays a detectable fluorescence emission only when it is complexed with serum albumin, whereas free bilirubin has a very low fluorescence yield. Actually, nearly complete disappearance of bilirubin emission was obtained when the unirradiated human serum albumin-bilirubin complex was precipitated with acetone to extract the pigment; complete removal of protein-bound bilirubin (as monitored by fluorescence spectroscopy) was achieved by repeating the acetone extraction after incubation of the complex in the phosphate buffer, pH 7.4, containing 7 M guanidinium chloride; the latter compound causes on extensive unfolding of protein molecules. On the other hand, in the case of irradiated solutions, even after denaturation of the protein with 7 M guanidinium chloride, a detectable amount of bilirubin-type fluorescent material was found to be associated with albumin. This finding clearly shows that bilirubin and/or some photoproduct underwent in part a photoinduced covalent binding with human serum albumin. Fragmentation of the bovine albumin polypeptide chain according to the procedure detailed in the experimental section yielded only one peptide-containing material fluorescent in the 530 nm region. This fact underlines the selective nature of the photobinding reaction. The amino acid composition of the isolated peptide is shown in Table 2; the composition is closely similar with that found for peptide 187-397 of native bovine serum albumin. In the case of the jaundiced babies who were subjected to phototherapy, we were able to demonstrate that only after 7 to 9 hr of exposure to light a detectable amount of bilirubin-type fluorescent material was present even at the end of the serum treatment with acetone and guanidinium chloride (see Fig. 1; Table 1). Fractional precipitation of the serum proteins by addition of controlled amounts of ammonium sulphate showed that the fluorescent material was present only in the albumin fraction. The photoadduct disappeared about 15 to 20 days after the phototherapy had been discontinued. This period of time represents the natural turnover period of human serum albumin.