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Insight into the Binding Mechanism of Imipenem to Human Serum Albumin by Spectroscopic and Computational Approaches
journal contribution
posted on 2014-06-02, 00:00 authored by Md Tabish Rehman, Hira Shamsi, Asad U. KhanThe
mechanism of interaction between imipenem and HSA was investigated
by various techniques like fluorescence, UV–vis absorbance,
FRET, circular dichroism, urea denaturation, enzyme kinetics, ITC,
and molecular docking. We found that imipenem binds to HSA at a high
affinity site located in subdomain IIIA (Sudlow’s site I) and
a low affinity site located in subdomain IIA–IIB. Electrostatic
interactions played a vital role along with hydrogen bonding and hydrophobic
interactions in stabilizing the imipenem–HSA complex at subdomain
IIIA, while only electrostatic and hydrophobic interactions were present
at subdomain IIA–IIB. The binding and thermodynamic parameters
obtained by ITC showed that the binding of imipenem to HSA was a spontaneous
process (ΔGD° = −32.31 kJ mol–1 for high affinity site and ΔGD° = −23.02
kJ mol–1 for low affinity site) with binding constants
in the range of 104–105 M–1. Spectroscopic investigation revealed only one binding site of imipenem
on HSA (Ka ∼ 104 M–1). FRET analysis showed that the binding distance
between imipenem and HSA (Trp-214) was optimal (r = 4.32 nm) for quenching to occur. Decrease in esterase-like activity
of HSA in the presence of imipenem showed that Arg-410 and Tyr-411
of subdomain IIIA (Sudlow’s site II) were directly involved
in the binding process. CD spectral analysis showed altered conformation
of HSA upon imipenem binding. Moreover, the binding of imipenem to
subdomain IIIA (Sudlow’s site II) of HSA also affected its
folding pathway as clear from urea-induced denaturation studies.