Interactions of bilirubin with bovine serum albumin in aqueous solution.

The interaction of bllirubin at concentrations less than 25 PM with bovine serum albumin (BSA) in aqueous solutions ranging from pH 3.5 to 8.6 was studied at room temperature by using the methods of optical rotatory dispersion (ORD) and absorption spectrophotometry between 230 and 600 mp. The binding of bilirubin by BSA at pH 5 caused a red shift of an absorption maximum from about 440 to 457 rnp, the latter having a millhnolar extinction coefficient of 58. This bilirubin-BSA complex exhibited probably the largest Cotton effect ever reported in the visible region, with an amplitude of approximately 1.5 x lo6 degrees*cm2-decimole-l, expressed as molar rotation of bilirubin (protein rotations were deducted), with a peak at 435 rnp and a trough at 485 to 487 ml.c. In addition, relatively smaller anomalous rotations were observed in the region between 250 and 400 mp. The ORD behavior of the bilirubin-BSA complex was dependent upon the pH and buffer in the system. The amplitude of the main Cotton effect decreased greatly at either side of pH 5, with significant changes in the absorption spectra. At pH 5, low concentrations of sodium acetateacetic acid buffer (0.2 mM ionic strength) decreased the amplitude to about one-tenth of that observed in the absence of acetate buffer when the order of addition was: bilirubin, buffer, and finally excess BSA. Sodium chloride, 50 mM, added last did not affect the ORD profile at pH 5 in the presence of excess BSA; however, at pH 7.5, 0.1 M NaCl with 10 mM Tris-HCI buffer added last reduced the Cotton effect significantly. ORD as well as spectrophotometric titrations gave a molar ratio of unity for the bilirubin-BSA complex at both pH 5 and 7.4. At pH 5 and 25” i 2’, the apparent association (binding) constant was estimated to be (5.7 f 1.5) X lo6 M-l. The remarkably large Cotton effects observed at pH 5 were interpreted by the formation of a dissymmetric conformation of the bilirubin molecule upon binding to BSA in a specific manner which would permit dipole-dipole coupling between the juxtaposed dipyrrylmethene chromophores. This conformation was considered to be modified by pH, through changes in the ionization of binding groups and by