Abstract:
Human salivary α-amylase is non-canonical cellular interaction with Sanguinarine, an alkaloid derivative isolated from Sanguinaria canadensis, was sought through various spectroscopic and molecular docking approaches under simulated physiological conditions. Equilibrium titration by fluorescence and Uv-absorption spectroscopy was done by continuous addition of 1.3 μM of Sanguinarine (dissolved in ethanol) to a final concentration of 11.7 μM via a mixer to 2.5 ml of 2.1 μM Human salivary α-amylase in a quartz fluorescence cuvette, using 25 mM Tris-HCl buffer containing 0.1 M NaCl pH 7.4 and temperature of 15- 40 °C. Sanguinarine quenches the intrinsic fluorescence of Human salivary α-amylase through a dynamic quenching mode in a concentration dependent manner with effective quenching constant (Ksv) of 1.28 X 107 L.mol-1. The enzyme has one binding site for Sanguinarine with a binding constant (Ka) of 7.63 X 10-2 M and a dissociation constant of 5.94 X 10-5 M at physiological pH of 7.4 and at 298 K, showing a strong bonding interaction and formation of a stable Sanguinarine-Human salivary α-amylase complex. Thermodynamic parameter calculations demonstrate ΔH value of 28.37 kJmol-1, ΔS value of 72.99 Jmol-1K-1 and ΔG value of 6.62 kJmol-1 at 298 K. These indicates the bonding process was a non-spontaneous molecular interaction mechanism but entropically driven with no visible enthalpy-entropy compensation effect from the hydrophobic bonding. Synchronous fluorescence revealed conformational alteration of Human salivary α-amylase and micro-environmental change upon Sanguinarine interaction that was ‘tryptophan-gated’. The UV-visible absorption spectra of Sanguinarine-Human salivary α-amylase confirmed the interaction which may affect the physiological functions of Human salivary α-amylase. According to Forster non-radiation energy Transfer theory, the binding of Sanguinarine to Human salivary α-amylase was of great probability of less than 8 nm. The percentage binding rate of the Sanguinarine to Human salivary α-amylase increases with elevated temperature. Molecular docking showed the participation of some amino acids in the complex formation with -9.5 kcal/mol binding energy. This work established a clear relationship between Sanguinarine and Human salivary α-amylase and has helped in understanding the ligand and the enzyme properties and also their thermodynamics relationship.
