/ . Biochem., 79, 393-405 (1976)

Interaction of Cyclodextrins with Fluorescent Probes and Its Application to Kinetic Studies of Amylase Hitoshi KONDO, Hiroshi NAKATANI, and Keitaro HIROMI Laboratory of Enzyme Chemistry, Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, September 26, 1975

1. It was found that 6-/>-toluidinyInaphthalene-2-sulfonate (TNS) showed pronounced fluorescence enhancement when it was added to a-, ft-, and f-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) a-Cyclodextrin forms a 1 : 1 complex with TNS. ii) /3- and /-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 a-cyclodextrin, 0.65 mM for /3-cyclodextrin and 0.66 mM for /-cyclodextrin in the 1 : 1 complex, and the secondary dissociation constants in the 2 : 1 complex were 71.4 mM for /3-cyclodextrin and 32.6 mM for f-cyclodextrin. iv) The apparent quantum yields of cyclodextrin-TNS complexes were also determined, using quinine sulfate as a standard, to be 0.040 for a-cyclodextrin, 0.024 for /3cyclodextrin, and 0.004 for r-cyclodextrin in the 1 : 1 complex, and the apparent quantum yields attributable to secondary binding in the 2 : 1 complex were 0.136 for /3-cyclodextrin and 0.074 for /-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 /3-cyclodextrin hydrolysis by Taka-amylase A [EC 3.2.1.1] at pH 5.3 and 25°. The Michaelis constant Km and molecular activity fh in the hydrolysis of /3cyclodextrin 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 a-cyclodextrin of the Taka-amylase A-catalyzed hydrolysis of ^-cyclodextrin.

Abbreviations : ANS, 8-anilinonaphthalene-l-sulfonate ; TNS, 6-/>-toluidinylnaphthalene-2-suIfonate. Vol. 79, No. 2, 1976

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Cyclodextrins, which are produced from starch by transglucosidation catalyzed by Bacillus macerans amylase (1, 2), are cyclic molecules in which 6, 7, and 8 D-glucopyranose units are bonded through a-1,4-glucosidic linkages. They are referred to as a-, /3-, and r-cyclodextrins, respectively. Their structures are schematically represented in Fig. 1, together with their internal diameters. One of the most noteworthy properties of cyclodextrins is their ability to form inclusion complexes with a variety of organic and inorganic compounds (7, 2). These complexes have been studied by a variety of physicochemical techniques such as absorption spectrometry (3), X-ray diffraction (4—8), microcalorimetry (9), optical rotatory dispersion (10, 11), circular dichroism (11), nuclear magnetic resonance (12), solubility measurements (13), etc. Cramer et al. (3) have shown that the fluorescence of 8-anilinonaphthalene-l-sulfonate (ANS) (Fig. 1) is greatly increased upon addition of cyclodextrins, and attributed this effect to the formation of inclusion complexes of the dye. Recently, we found that 6-/Koluidinylnaphthalene-2-sulfonate (TNS) (Fig. 1) shows

TNS(6-p-totukfinylruphthatene-2-sulfonate)

ANS(8-anilinonaphlhalene- 1-sulfonate) Fig. 1. Schematic representation of cyclodextrins and structural formulae of TNS and ANS. For cyclodextrins, O denotes a glucopyranose unit and denotes an a-1,4-glucosidic linkage. Internal diameters of cyclodextrins are shown.

even greater enhancement of fluorescence than ANS on addition of cyclodextrins. The interaction between the dye and cyclodextrins seemed worthy of detailed quantitative investigation to elucidate the nature of the interaction. We also found that non-cyclic dextrins show much smaller fluorescence enhancement than cyclodextrins, though the effect is appreciable for longer chain amyloses. This property may be used very effectively to detect the hydrolytic ring opening of cyclodextrins catalyzed by acids or enzymes. In this paper, the interaction of TNS with a-, /3-, and 7-cyclodextrins was studied quantitatively by following the enhanced fluorescence upon interaction. Moreover, a new method was developed for following the hydrolysis of cyclodextrins by amylases, using TNS as a probe. EXPERIMENTAL Materials—a-Cyclodextrin was purchased from Hayashibara Biochemical Laboratories Inc. and Sigma Chemical Co. and was recrystallized successively from 60% w-propyl alcohol and distilled water (13). /3-Cyclodextrin was purchased from Hayashibara Biochemical Laboratories Inc. and recrystallized from distilled water (13). ?--Cyclodextrin was a generous gift from Prof. T. Kuge and Dr. K. Takeo of Kyoto Prefectural University. TNS, purchased from Sigma Chemical Co., was recrystallized twice from distilled water before use (14). The purity of TNS was examined by ascending thin layer chromatography (Kiesel gel 60 Ft54, Merck) using tso-butyl alcohol saturated with 3% aqueous ammonia as a solvent (14). The crystalline TNS sample moved as a single spot, indicating its homogeneity. Stock solutions were made up with 0.08M sodium acetate buffer and were prepared freshly. The concentration of TNS was determined spectrophotometrically, taking the molecular absorptivity £TNS=4,300 at 366

nm (75). The sodium salt of ANS was purchased from Tokyo Kasei Co., Ltd. and recrystallized as the magnesium salt. The concentration of ANS was determined spectrophotometrically, / . Biochem.

CYCLODEXTRIN-TNS INTERACTION AND ITS APPLICATION

taking the molecular absorptivity £ANS=4,950 at 350 nm (16). Quinine sulfate was purchased from Nakarai Chemical Co., Ltd. and used without further purification. Crystalline Taka-amylase A [EC 3.2.1.1] was prepared from "Taka-diastase Sankyo" according to the method of Akabori et al. (17) with a minor modification (Matsushima, Y., personal communication). The concentration of Taka-amylase A was determined spectrophotometrically at 280 nm, assuming E\%n = 22.1 (18) and a molecular weight of 51,000 (19). Maltoheptaose was prepared from /S-cyclodextrin according to the method of Nitta et al. (20). Glucose, maltose, maltotriose, and amylose EX-I (DP.=17) were purchased from Hayashibara Biochemical Laboratories Inc. and potato amylose (Lot No. MOR4819, DP.=600) was purchased from Sigma Chemical Co. These materials and other chemical reagents of guaranteed grade were used without further purification. Methods — Fluorescence measurements were performed in 0.08 M sodium acetate buffer (pH 5.3) at 25° with a Hitachi MPF-2A Spectrofluorometer. The excitation wavelengths of the TNS-cyclodextrin system, quinine sulfate, and the ANS-cyclodextrin system were 366 nm, 366 nm, and 360 nm, respectively. The wavelengths for emission measurement were 460 nm for the TNS-cyclodextrin system and 510 nm for the ANS-cyclodextrin system. The fluorescence titration of ANS and TNS with cyclodextrins was carried out at constant concentrations of each dye by the successive dilution method, as follows: a definite amount of •dye-cyclodextrin mixture was withdrawn from a cuvette after the first measurement had been made, and the same volume of the dye solution was added to the cuvette to give a lower concentration of the cyclodextrin. The initial sample was used as a standard to correct for instrumental signal drift with time, and its fluorescence intensity was recorded together with that of the sample in each measurement. For calculation of the quantum yield, quinine sulfate was used as a standard. Absorption spectra were measured at 25° Vol. 79, No. 2, 1976

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using a Shimadzu UV-200 Spectrophotometer with an expanded scale (0—0.1 absorbance unit). The dependence of the fluorescence intensity of the TNS-cyclodextrin system on ionic strength was studied in 0.048 M sodium acetate buffer (pH 5.4) by varying the concentration of sodium chloride from 0.04 to 0.64 M. Buffers employed for measurement of the pH dependence of the fluorescence intensity of the )S-cyclodextrin-TNS system were as follows (all 0.1 M): pH 3-4, CH,COONa-HCl; pH 5, CH,COONa-CHsCOOH; pH 6-7, NaH,PCv Na 2 HPO,; and pH 9.1-13, glycine-NaOH. The quantum yield (0,) of a sample was calculated from the observed absorbance (A) at 366 nm (the excitation wavelength of TNS) and the area enclosed by the emission spectrum, according to following equation (14, 21). (Area), (Area),

(l-10" A «) (1-K)- A ')

(1)

where the subscripts s and q indicate the sample and the standard quinine sulfate, respectively. The quantum yield of quinine sulfate in 0.5 N HtSO< was taken as 0.55 (22). The concentration of TNS was maintained between 9.73xlO"«M and 8.10xl0-»M, where the absorbance at the wavelength of excitation was 0.041 or less. The absorbance of the quinine sulfate solution was also kept below 0.003. The areas of emission spectra were measured by weighing the chart paper under each curve. Hydrolysis of cyclodextrins catalyzed by Taka-amylase A was carried out in 0.08 M sodium acetate buffer, pH 5.3, at 25°, adding the enzyme solution with a micropipette to the substrate solution in a test tube. The enzyme concentration employed was 2.62xlO~7M, and the substrate concentration ranged from 1.0— 12.5 mM. At appropriate intervals, 2 ml aliquots of reaction mixture were withdrawn and dropped into 2 ml of 0.08 N NaOH solution. Then 4 ml of TNS solution (dissolved in 0.08 M sodium acetate buffer, pH 5.3, (8.61-9.96) x 10"4 M) was added to the stopped reaction mixture (final pH 11.8) and its fluorescence intensity was measured using cyclodextrin solution as a reference; their fluorescence intensities were recorded together for each measurement. At high concentrations of cyclodextrin, the

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stopped reaction mixtures were appropriately diluted with 0.08 M sodium acetate buffer (pH 5.3) to the concentration range where linearity holds for the fluorescence intensity standard curve. A stopped-flow apparatus (Union Giken SF 70) was used to check the rate of interaction between cyclodextrins and TNS. The fluorescence emission was observed through a cutoff filter (transmitting above 430 nm) at right angles to the excitation beam (366 nm) from a tungsten lamp (50 W) (23). RESULTS Dissociation Constants and Quantum Yields of TNS Included in Cyclodextrins—Figure 2 shows the effect of various concentrations of a- and ^-cyclodextrins upon the fluorescence intensities of TNS and ANS. Obviously TNS not only has much higher fluorescence intensity than ANS, but also there is a larger difference in the sensitivity towards a- and ^-cyclodextrins. Therefore, we focused our attention on TNS as a fluorescent dye for studying the

interaction with cyclodextrins. A typical example of the fluorescence titrations is shown in Fig. 3 for the y9-cyclodextrinTNS system. The enhancement of TNS fluorescence upon interaction can reasonably be assumed to be due to the formation of an inclusion complex. The stoichiometry and the dissociation constants of cyclodextrin-TNS complexes were determined as follows. If a cyclodextrin molecule forms an inclusion complex with a TNS molecule, as shown in Eq. 2, the ' dissociation constant of the cyclodextrin-TNS complex (KA) is defined by Eq. 3. CD + T ^ ^ CD-T Co—x T0—x Ki x

[CD] [T]

(2)

(C0-x) ( T . - J : )

[CD-T]

(3)

where Co, To, and x represent the total concentrations of cyclodextrin and TNS and the concentration of cyclodextrin-TNS complex, respectively, and [CD], [T], and [CD-T] the equilibrium concentrations of these species, re-

100 •

10"4 (CD)(M)

10"

Fig. 2. Effects of a- and /9