Interaction of Cyclodextrins with Fluorescent Probes and Its Application to Kinetic Studies of Amylase

Abstract

1. It was found that 6-p-toluidinyInaphthalene-2-sulfonate (TNS) showed pronounced fluorescence enhancement when it was added to α-, β-, and γ-cyclodextrin solutions. 2. The following results were obtained by quantitative study of the interactions of three kinds of cyclodextrins with TNS by following TNS fluorescence at pH 5.3 and 25°. i) α-Cyclodextrin forms a 1: 1 complex with TNS. ii) β- and γ-Cyclodextrins form 1: 1 and also 2: 1 complexes; in the latter two cyclodextrin molecules bind to one TNS molecule, iii) The dissociation constants of cyclodextrin-TNS complexes were determined to be 54.9 mM for α-cyclodextrin, 0.65 mM for β-cyclodextrin and 0.66 mM for γ-cyclodextrin in the 1: 1 complex, and the secondary dissociation constants in the 2: 1 complex were 71.4 mM for β-cyclodextrin and 32.6 mM for γ-cyclodextrin. iv) The apparent quantum yields of cyclodextrin-TNS complexes were also determined, using quinine sulfate as a standard, to be 0.040 for α-cyclodextrin, 0.024 for β-cyclodextrin, and 0.004 for γ-cyclodextrin in the 1: 1 complex, and the apparent quantum yields attributable to secondary binding in the 2: 1 complex were 0.136 for β-cyclodextrin and 0.074 for γ-cyclodextrin. v) Pronounced TNS fluorescence enhancement seems to be due to the formation of inclusion complexes of cyclodextrins with TNS. 3. The pronounced fluorescence enhancement of TNS accompanying the formation of cyclodextrin-TNS inclusion complexes was effectively utilized to selectively monitor the rate of β-cyclodextrin hydrolysis by Taka-amylase A [EC 3.2.1.1] at pH 5.3 and 25°. The Michaelis constant K_m and molecular activity k_o in the hydrolysis of β-cyclodextrin catalyzed by Taka-amylase A were determined to be 10.0±0.5 mM and 224.1±11.1 min⁻¹, respectively. Furthermore, this method was applied to the kinetic study of inhibition by maltose and α-cyclodextrin of the Taka-amylase A-catalyzed hydrolysis of β-cyclodextrin.