Abstract
The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.
Highlights
IntroductionIn spite of the experimental difficulties connected with the light sensitivity and low solubility of the bile pigment in solutions of physiological pH and ionic strength, several groups have attempted to estimate the number of binding sites and the affinity constants for bilirubin binding to albumin
+ 9.4 s-’ and k:, = 3.8 f 0.5 s-l)
In spite of the experimental difficulties connected with the light sensitivity and low solubility of the bile pigment in solutions of physiological pH and ionic strength, several groups have attempted to estimate the number of binding sites and the affinity constants for bilirubin binding to albumin
Summary
In spite of the experimental difficulties connected with the light sensitivity and low solubility of the bile pigment in solutions of physiological pH and ionic strength, several groups have attempted to estimate the number of binding sites and the affinity constants for bilirubin binding to albumin. In another study carried out at pH 8.5, Beaven et al [4] presented evidence based on the circular dichroism of HSA-bound bilirubin, that at high ionic strength at 24”C, two bilirubin sites are occupied with association constants (K,,) of 3 X 10” Mm’ and 1.4 X lo M- ‘. The number of binding sites increases to three, with affinity constants in the range of 1 X lo Mm1 to 1 x lo Mm’
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
