Binding constant measurement by hyper-Rayleigh scattering: bilirubin-human serum albumin binding as a case study

In this paper, a new application of the hyper-Rayleigh scattering technique in determining multiple binding constants of a small molecule like bilirubin to a macromolecule like the protein human serum albumin has been demonstrated. Human serum albumin has two binding sites for bilirubin, and the binding constants have been measured by carrying out a second harmonic titration of the protein against bilirubin and vice versa. The measured binding constants K(1) = 1.5 +/- 0.43 x 10(7) M(-1) and K(2) = 1.01 +/- 0.16 x 10(6) M(-1) agree well with the reported values obtained by other methods.