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Faculty of Pharmacy and Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
Determination of the BSA-palmitate high-affinity
binding constant
(Ka)
traditionally relied on the heptane-water partitioning technique. We
used this technique to calculate
Ka for the
BSA-[3H]palmitate
complex, to determine if
Ka was
independent of protein concentration, and to determine if the unbound
[3H]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-[3H]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
[3H]palmitic acid. The
data were consistent with the notion of self-association of
[3H]palmitate in the
aqueous phase. Ka
for the
BSA-[3H]palmitate
complex was determined to be similar (2.2 ± 0.1) × 108
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
µM) 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-[3H]palmitate
radiolabel is also a substrate for the uptake process.
heptane; partitioning; long-chain fatty acids; protein binding
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