EFFECT OF FREE FATTY-ACIDS ON THE BINDING-KINETICS AT THE BENZODIAZEPINE BINDING-SITE OF GLYCATED HUMAN SERUM-ALBUMIN

The effect of free fatty acids (FFA) and non-enzymatic glycation on th e binding kinetics of dansylsarcosine (DS) to human serum albumin (HSA ) was studied using the stopped-flow technique. The influence of FFA o n the binding parameters of 25% glycated HSA depended on the type of f atty acid. The addition of stearic, oleic and linoleic acids in a conc entration of 0.3 mmol/l showed no inhibitory effects on the associatio n rate constant (k2) value for DS binding to 25% glycated HSA (k2 with out FFA: 385 +/- 10 s-1, k2 with FFA greater-than-or-equal-to 385 +/-1 0 s-1). In contrast, shorter chain fatty acids (hexanoic, octanoic, de canoic, lauric and myristic acids) showed marked inhibitory effects fo r 0.3 mmol/l FFA (k2 range: 233 +/- 32 to 69 +/-5 s-1) and for 0.6 mmo l/l FFA (k2 range: 125 +/- 3 to 20 +/- 4 s-1). The association rate co nstant (k2) as well as the affinity constant (K(A)) of DS were markedl y affected by glycation: k2 was 686 +/- 61 s-1 for 7% glycated HSA, 38 5 +/- 10 s-1 for 25% glycated HSA and 209 +/- 12 s-1 for 50% glycated HSA. K(A) decreased from 6.1 +/- 2.9 x 10(5) M-1 for 7% glycated HSA, to 5.1 +/-0.1 X 10(5) M-1 for 25% glycated HSA and to 1.3 +/- 0.6 x 10 (5) M-1 for 50% glycated HSA. The results suggest that slightly increa sed concentrations of physiological long chain free fatty acids (oleic , stearic and linoleic acids) as observed in diabetic patients have no inhibitory effects additional to that of glycation on the binding kin etics of drugs to HSA at binding site II.