Development of a novel probe for measuring drug binding to the F1*S variant of human alpha 1-acid glycoprotein

A novel probe was developed to measure drug association with the F1*S variant of the human serum protein alpha I-acid glycoprotein (AGP). The molecule 2-hydroxy-3,5-diiodo-N-[2(diethylamino)ethyl]benzamide (DEDIC) binds to AGP, quenching its native fluorescence. This quenching was fitted to a two-site model giving apparent dissociation constants of 0.049 +/- 0.005 and 12 +/- 2 muM (mean +/- SEM). Quenching of each of the separate variants of AGP by DEDIC was itself described by a two-site model, giving for the F1*S variant K-D(F1*s)(1)= 0.041 +/- 0.010 muM and K-D(F1*S)(2) = 29 +/- 7 muM; and for the A variant K-D(A)(1) = 0.31 +/- 0.18 muM and K-D(A)(2) = 8.8 +/- 0.7 muM. The utility of DEDIC in probing drug interactions with isolated variants was demonstrated in competition experiments with the model drugs amitriptyline and bupivacaine. In addition, the selectivity of DEDIC for variant F1*S rendered it capable of probing the binding of drugs (including the variant A-selective drug amitriptyline) to F1*S in a mixture of variants, such as occurs naturally in whole AGP. DEDIC is unique as an F1*S variant-selective probe of drug binding to whole AGP that is also sufficiently soluble to serve as a probe of drug binding to the lower affinity sites on isolated A and F1*S variants. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.