Binding of [3H]palmitate to BSA

Determination of the BSA-palmitate high-affinity binding constant (K-a) traditionally relied on the heptane-water partitioning technique. We used this technique to calculate K-a for the BSA-[H-3]palmitate complex, to determine if K-a was independent of protein concentration, and to determine if the unbound [H-3]palmitate concentration is constant at different BSA concentrations using constant BSA-to-palmitate molar ratios (range 1:1 to 1:4). After extensive extraction of non-[H-3]palmitate radiolabeled substances, the heptane-to-buffer partition ratio, in the absence of BSA, was 702 +/- 19 (mean +/- SD, n = 6). This value was much lower than the predicted value of 1,376 and was highly dependent on which phase (organic or aqueous) initially contained the [H-3]palmitic acid. The data were consistent with the notion of self-association of [H-3]palmitate in the aqueous phase. K-a for the BSA-[H-3]palmitate complex was determined to be similar (2.2 +/- 0.1) x 10(8) M-1 (mean +/- SD, P > 0.05) at all BSA concentrations studied. At each BSA-to-palmitate molar ratio, the equilibrium unbound ligand concentration was constant only at low BSA concentrations (<10 mu M) and at low BSA-to-palmitate molar ratios (i.e., 1:1 and 1:2). At higher BSA concentrations and molar ratios, the unbound ligand concentration increased with an increase in protein concentration. Hepatocyte uptake using the manufacturer-supplied radiolabeled product was significantly higher than with the purified product, suggesting that a non-[H-3]palmitate radiolabel is also a substrate for the uptake process.