The binding of bilirubin to albumin. A study using spin-labelled bilirubin

Binding between human serum albumin and a spin-labelled derivative of bilirubin was investigated by circular dichroism, fluorescence quenching, electron spin resonance and visible spectroscopy. The orders of magnitude of the binding constants obtained by flurorescence quenching and electron spin resonance spectroscopies were 10(7) and 10(3) 1 . mol-1, respectively. These data suggest that most spin-labelled bilirubin interacts with human serum albumin at the side not holding the spin-labelled side-arm. CD measurements showed the presence of at least two sites, associated with opposite Cotton effects. It is worthy of note that the Cotton sign of the first site is inverted with respect to the corresponding one of bilirubin. CD measurements on mixed systems (spin-labelled bilirubin/human serum albumin/bilirubin) were also performed. The decomposition of the ternary curves shows that the rotatory power of bilirubin bound to human serum albumin is higher in the ternary system than in the binary (bilirubin/human serum albumin). The corresponding CD measurements for the binding between spin-labelled bilirubin and bovine serum albumin are also reported and discussed.