Study of binding of 12(S)-hydroxy 5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid to bovine serum albumin using dynamic surface tension measurements

In a recent paper,(1) we demonstrated that molecular interactions between biopolymers and other smaller molecules can be detected by means of dynamic surface tension measurements. In the present paper, we demonstrate that the same methodology can be employed for investigating dose effects and specificity of molecular interactions. Three similar lipids were chosen for this study: 12(S)hydroxy-5(Z),8(Z),1 0(E),14(Z)-eicosatetraenoic acid (12(S)-HETE-free acid), methyl 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoate (12(S)-HETE-methyl ester), and 5(Z),8(Z),11(Z),14(Z)-eicosatetraenoic acid (arachidonic acid-free acid). These substances were added to a fatty acid free bovine serum albumin (BSA) aqueous solution at different lipid concentrations. The characteristic tension response indicates that molecular interactions between 12(S)-HETE-free acid and BSA exist. The detected interactions are concentration dependent: at a molecular ratio of lipid to protein of 1:1, the binding of 12(S)-HETE-free acid to BSA is hydrophobic in nature; at the molecular ratio of lipid to protein of 10:1, a secondary binding occurs and is hydrophilic in nature. Similar molecular interactions were not detected between 12(S)-HETE-methyl ester or arachidonic acid-free acid and BSA, indicating that the interactions between 12(5)-HETE-free acid and BSA are specific. As an independent means, surface elasticity is used to probe the molecular interactions at the interface. In the case of 12(S)-HETE-free acid but not its methyl ester or arachidonid acid, distinct higher surface elasticities were observed at lipid concentrations in excess of a molecular ratio of lipid to protein of 1:1. This finding reinforces the above stipulations.