Characterizing the binding of nucleotide ATP on serum albumin by ³¹P NMR diffusion

The pulsed-field-gradient (PFG) ³¹P NMR diffusion spectra were measured under varied sample conditions to characterize the low-affinity binding of adenosine 5′-triphosphate (ATP) on human serum albumin (HSA) or bovine serum albumin (BSA). The NMR diffusion constants of ATP, ATP-HSA, or ATP-BSA were illustrated as function of ATP concentrations. The binding curves of ATP-HSA and ATP-BSA were identical but strikingly different from the ATP curve. Using a "Scatchard plot", the apparent binding constant (K) and number of ATP binding sites (n) on serum albumin were evaluated as K = 75.25 (mol/L) ^-1 and n = 10, respectively. At a pH < 5.0 and a pH > 9.0 or a temperature > 45 °C, the diffusion data of ATP-HSA were found to increase remarkably, suggesting that the dissociation of ATP from HSA was largely enhanced, probably because of pH-or heat-induced protein structural change, degradation, or aggregation. In addition, our data indicated that ADP was strongly competitive with ATP for the low-affinity binding to HSA, but heptanone and Cl ^- were essentially noncompetitive. These results are important for further elucidating the interaction of ATP with serum albumin and its possible effect on related bioprocesses. The method can be well applied to study the binding of other nucleotides/nucleosides on proteins.