Binding of [3H]palmitate to BSA.
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) 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 microM) 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.
Faculty of Pharmacy and Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
B M
BM
Elmadhoun
G Q
GQ
Wang
J F
JF
Templeton
F J
FJ
Burczynski
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D005260:Female; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012588:Scintillation Counting; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D014316:Tritium
D014316:Tritium; D012710:Serum Albumin, Bovine; D019308:Palmitic Acid