Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase

Abstract

The inclusion complexes of cyclomaltohexaose (α-CD), cyclomaltoheptose (β-CD), cyclomaltooctaose (γ-CD), and polymerized β-CD (β-CD n ) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD·CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants ( K) of the CD·CA equilibrium were relatively insensitive to pH for β-CD, compared to α-and γ-CDs, but were subject to substantial enhancement at low ionic strengths. The β-CD·CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25°C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical shift data indicate that the phenolic ring of CA docks within the cavity of β-CD. The Ks for β-, α-, and γ-CD determined in apple juide, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with β-CD n , did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu 2+ by CDs.