EFFECTS OF IONIC-STRENGTH AND PH ON THE BINDING OF MEDIUM-CHAIN FATTY-ACIDS TO HUMAN SERUM-ALBUMIN

Binding equilibria for the interactions of the medium-chain fatty acid anions, laurate and myristate, with defatted human serum albumin have been investigated under varying environmental conditions such as ionic strength and pH. Since these ligands bind strongly to albumin (K-ass approximate to 10(7) M(=1)), conventional equilibrium dialysis is not a feasible method for these investigations. Consequently, we employed a dialysis method, allowing determination of very low concentrations of unbound ligand by measuring the rate of exchange of labelled ligand across a dialysis membrane under conditions of chemical equilibrium. Over a range of ionic strength, 8-68 mM, the binding of the first few molecules of laurate to albumin was weakened with increasing ionic strength, whereas the binding of subsequent molecules seemed to proceed independently of ionic strength. The binding of myristate by albumin, however, appeared to be independent of ionic strength in the observed range of concentrations. The influence of pH in the range 5.1-9.0 on the binding of the two fatty acid anions by albumin was more complicated. The first molecule of laurate appeared to bind with a slightly weaker affinity to pH, while the trends for the following molecules varied. The binding of myristate (irrespective of concentration) seemed ro strengthen monotonously with pH, but this conclusion depends critically on the interpretation of the kinetic behaviour of the myristate anion. We have previously shown [Pedersen, A. O., Honore, B. & Brodersen, R. (1990) Eur. J. Biochem. 190, 497-502] that the strength of binding of the first few molecules of the two fatty acid anions to albumin decreases with increasing temperature, whereas binding of subsequent molecules seems to proceed independently of temperature. We explain these findings as follows. The binding of the first few (3 or 4) molecules of the C-12 laurate anion is clearly driven by formation of ionic bonds between the fatty acid anion and positively charged groups, such as lysine residues, in the albumin molecule, whereas the binding of subsequent molecules of laurate seems to depend more on hydrophobic interactions. In the case of the C-14 myristate anion, the of the first few (only 1 or 2) molecules may depend on ionic forces, but binding of the following molecules of myristate seems to depend on hydrophobic interactions only. Thus, multiple binding of both ligands is considered to be an association between fatty acid anions stacked on the protein molecule and/ or it may take place in hydrophobic regions of the albumin molecule. This type of binding will contribute entropy rather than enthalpy to the total binding energy.