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The binding of TQ to hsALDH changes the
The binding of TQ to hsALDH changes the characteristic enhanced green fluorescent protein sequence spectrum of the enzyme. Therefore, TQ forms a complex with hsALDH and changes its absorption properties [39]. Ksv and Kb values indicate that the binding of TQ to hsALDH is very strong and of the order of static binding (complex formation). There is approximately a 1:1 binding ratio of TQ and hsALDH [40]. Far-UV CD analysis showed that there was no significant change in the secondary structure of hsALDH upon binding to TQ. Also, the effect of temperature on the binding between the two showed that the binding constant decreases with increase in temperature. This further indicates that the quenching of fluorescence by TQ is static and there is a formation of complex between TQ and hsALDH. With increase in temperature, the hsALDH-TQ complex destabilizes and therefore binding constant decreases due to gain in molecular kinetic energy and increase in Brownian motion [41], [42]. However, the decrease in binding constant was from 2.34±0.07×105M−1 to 1.00±0.09×105M−1 when the temperature was increased from 15°C to 37°C. The binding constant is still of the order of 105 at 37°C, which is strong enough to show its activating effect. Therefore, at physiological temperature (37°C), TQ strongly binds to hsALDH and hence will activate the enzyme to a good extent. Protein-ligand interaction primarily involves the non-covalent interactions such as hydrogen bonding, electrostatic interactions, hydrophobic interactions and Van der Waals forces [43], [44]. TQ contains three methyl groups on its surface along with two ring carbonyl groups. The methyl groups along with the aromatic ring provide the hydrophobic and van der Waals forces for binding to the enzyme. Also, partial negative charge on the carbonyl oxygen may contribute to the binding through hydrogen bond formation and electrostatic interaction. A negative ΔH and a positive ΔS value (Table 4) shows that the electrostatic interactions are also involved in the hsALDH-TQ complex formation. The free energy of binding (ΔG value, Table 4) shows that there is strong binding between TQ and hsALDH resulting in complex formation. Also, negative value of ΔG (-1.26×104Jmol−1) shows that the binding of TQ to hsALDH is a spontaneous process.
FRET analysis shows that the emission spectrum of hsALDH and the absorption spectrum of TQ overlap with an r value of 4.16nm and therefore, energy transfer could take place between hsALDH and TQ. Also, it was found that 0.5Ro