Adenosine 5′-triphosphate binding to bovine serum albumin

Binding of ATP to bovine serum albumin was shown by ultrafiltration and NMR. The binding was pH dependent. Scatchard analysis revealed that at pH 5.4, 6.4 and 7.4, dissociation constant K-d was 13, 40 and 120 mu M, respectively, and no binding was observed at pH 8.4. The binding stoichiometry was 1:1 for all pH. Dimer of BSA did not bind ATP. From chemical shifts of P-31-NMR, K-d was estimated to be 15 mu M at pH 5.4, which is very close to that determined by ultrafiltration. While adenosine did not interfere with the binding, GTP, dCTP, ADP, UTP, AMP, phosphate and pyrophosphate were competitive inhibitors and their inhibition constants K-i were 25, 32, 36, 50, 130, 1000 and 186 mu M, respectively. Fatty acids such as lauric acid and palmitic acid did not interfere with the binding. Warfarin was a non-competitive inhibitor. Cl- competitively inhibited the binding, and the inhibition constant was 20 mM. The dissociation constants of the Cl- binding were reported to be 0.42 mM for the first binding site, 10-5 mM for the second and 303-143 mM for the third [G. Scatchard, W.T. Yap, J. Am. Chem. Sec., 86 (1964) 3434; G. Scatchard et al., J. Am. Chem. Sec. 79 (1957) 12]. This suggests that the ATP binding site may be the second Cl- binding site. (C) 1997 Elsevier Science B.V.