Kinetics of bilirubin binding to bovine serum albumin and the effects of palmitate.

The binding of bilirubin to bovine serum albumin has been determined to be a second order process, first order in each reactant. Formation of the bilirubin-albumin complex was observed as an increase in absorbance at 480 nm by stopped flow spectrophotometry. The effect of long chain fatty acid on the binding of bilirubin was investigated at palmitic acid levels of 0 to 7 mol/mol of albumin at 37 degrees and pH 7.4. The second order rate constant is 1.5 X 10(6) M-1 S-1 when the palmitate/albumin ratio is less than 5 and drops to 0.5 X 10(6) M-1 S-1 when the palmitate/albumin ratio reaches 6. The equilibrium association constant for the bilirubin-albumin complex is 5.5 X 10(6) M-1 when the palmitate/albumin ratio is less than 1, 18 X 10(6) M-1 when the palmitate/albumin ratio is between 1 and 5, and 3 X 10(6) M-1 when the palmitate/albumin ratio is greater than 5. The mechanism of the effect of palmitate varies with the total palmitate level. At low molar ratios, addition of palmitate has no effect on the association rate but decreases the dissociation rate. At high molar ratios, addition of palmitate decreases the association rate and increases the dissociation rate. It is suggested that palmitate affects the ability of the protein to undergo the conformational changes needed to accommodate bilirubin.