Studying the interactions between tea polyphenols and α-amylase

Abstract

Dixon, Cornish-Bowden and Lineweaver-Burk plot analyses were applied to study the detailed kinetics of inhibition of porcine pancreatic a-amylase (PPA) by tea polyphenols. Fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) were combined with the kinetics of inhibition to elucidate the mechanism of binding interactions of tea polyphenols and PPA. Then, the reciprocal of competitive inhibition constant (1/Kic), fluorescence quenching constant (KFQ) and binding constant (Kitc) obtained from these measurements were compared and correlated to analyse the relations between PPA inhibition and binding behaviour. The role of the galloyl moiety in binding of catechins and theaflavins with PPA was highlighted as well. In addition, the effects of three soluble polysaccharides (citrus pectin (CP), wheat arabinoxylan (WAX) and oat b-glucan (OBG)) on the inhibition of PPA by tea polyphenols were studied through PPA inhibition, IC50 value, inhibition kinetics and fluorescence quenching methods. Furthermore, the effects of tea polyphenols on binding of PPA with normal maize starch granules were studied in terms of binding ratio, dissociation constant (Kd) and binding rate. Then, the association constant (1/Kd), 1/Kic and KFQ of tea polyphenols were correlated to analyse the effects of PPA inhibition on the binding of the enzyme with starch. The results show that green, oolong and black tea extracts, epigallocatechin gallate, theaflavin-3, 3r-digallate and tannic acid were competitive inhibitors of PPA, whereas epicatechin gallate, theaflavin-3r-gallate and theaflavin were mixed-type inhibitors with both competitive and uncompetitive inhibitory characteristics. Only catechins with a galloyl substituent at the 3-position showed measurable inhibition. Kic values were lower for theaflavins than catechins, with the lowest value for theaflavin-3, 3r-digallate. The lower Kic than the uncompetitive inhibition constant for the mixed-type inhibitors suggests that they bind more tightly with free PPA than with the PPA-starch complex. A 3 and/or 3r-galloyl moiety in catechin and theaflavin structures was consistently found to increase inhibition of PPA through enhanced association with the enzyme active site. A higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red-shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also supported by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between 1/Kic, KFQ and Kitc. Therefore, the combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA. Interestingly, CP, WAX and OBG could each increase the IC50 values and Kic, and decrease KFQ of tea polyphenols interacting with PPA. The data show a competitive interaction equilibrium between polysaccharides, polyphenols and PPA. For individual polyphenols, there were negative linear correlations between both the values of 1/Kic and KFQ and that of IC50 with and without polysaccharides, indicating that the decreased inhibitory activity of polyphenols induced by the polysaccharides was caused by the reduced binding of polyphenols with PPA. Additionally, the slopes of the linear relationships between IC50 and Kic and between KFQ and 1/Kic remained stable with and without polysaccharides, indicating that the three constants may be combined to characterize the effects of soluble polysaccharides on the PPA inhibition by polyphenols. Furthermore, polyphenols which have inhibitory activity against PPA increased binding of the enzyme with starch in a polyphenol concentration-dependent manner, while polyphenols without the inhibitory activity did not affect the binding of PPA with starch. The results are consistent with a binding equilibrium between polyphenols, starch granules and PPA. The Kd value for PPA binding with starch was decreased by tea polyphenols, with the effects greater for theaflavins than catechins and for galloylated compared with non-galloylated polyphenols. Tea polyphenols were also shown to increase the binding rate of the enzyme by increasing the observed rate constant and decreasing the half-lives. Tea polyphenols were shown to be adsorbed onto starch granules as well. Additionally, there were positive linear correlations between 1/Kd and 1/Kic and between 1/Kd and KFQ. Taken together the data suggest that binding of polyphenols with PPA promotes PPA binding to starch granules. Despite the greater amount of PPA on the granules, starch hydrolysis is reduced because the polyphenol inhibition of PPA persists after binding to starch.