A STABLE WATER-SOLUBLE AMETHYLBENZIDINE-2-HYDROXYPROPYL-BETA-CYCLODEXTRIN INCLUSION COMPLEX AND ITS APPLICATIONS IN ENZYME ASSAYS

3,3',5,5'-Tetramethylbenzidine (TMB), a hydrophobic and noncarcinogeni c chromogen with a high absorption coefficient widely used in solid-ph ase assays involving labeled horseradish peroxidase was rendered solub le (up to 40 mM) and more stable for at least 2 months at 22-24 degree s C by forming a water-soluble inclusion complex with 2-hydroxypropyl- beta-cyclodextrin (hp-beta-CyD). Cyclic voltammetry and absorbency mea surement were employed to characterize the TMB-hp-beta-CyD complex. We ll-defined cyclic voltammograms of TMB exhibited two oxidation waves w hich merged into a single wave with increasing hp-beta-CyD concentrati ons. Cyclic voltammetry was then used to examine the effect of complex ation with hp-beta-CyD on the oxidation potential of TMB and provided evidence of a 1:1 complex between TMB and the cyclodextrin molecule wi th a formation constant of 1.6 M(-1). Enzyme assays for D-glucose, lac tate, and glutamate were performed by coupling the TMB-hp-beta-CyD/hor seradish peroxidase system to the respective oxidase enzymes with the formation of either a blue (absorption coefficient of 35,800 M(-1)cm(- 1) at 650 nm) or a yellow color (absorption co-efficient of 67,300 M(- 1) cm(-1) at 450 nm) as an indication of the metabolite concentration. These assays possessed a sensitivity limit below 10 mu M and the resu lts obtained were in excellent agreement with standard enzymatic assay s when tested in various food and clinical samples. (C) 1994 Academic Press, Inc.