Multi-Spectroscopic Characterization of Human Serum Albumin Binding with Cyclobenzaprine Hydrochloride: Insights from Biophysical and In Silico Approaches
Abstract
:1. Introduction
2. Results and Discussion
2.1. UV-Visible Spectroscopy of HSA-CBH Complex
2.2. Alterations in Secondary Structure of HSA
2.3. Fluorescence Quenching Analysis of HSA in the Presence of CBH
2.4. Thermodynamic Parameters and Forces Involved in HSA-CBH Binding
2.5. Energy Transfer Efficiency and HSA and Binding Distance
2.6. Denaturation of HSA with Guanidine Hydrochloride (GdmCl)
2.7. Enzyme Activity
2.8. Molecular Docking
2.9. Molecular Dynamics Simulation
3. Materials and Methods
3.1. Reagents
3.2. UV-Visible Absorption Spectra Measurements
3.3. Fluorescence Quenching and Data Analysis
3.4. Thermodynamic Analysis of the Binding Process
3.5. Far-UV Circular Dichroism (CD) Spectropolarimetry for Structural Change Determination
3.6. Guanidine Hydrochloride-Induced Unfolding Studies in the Absence and Presence of CBH
3.7. Esterase-Like Activity of HSA
3.8. Molecular Docking
3.9. Molecular Dynamics
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
CD | Circular dichroism |
CBH | Cyclobenzaprine hydrochloride |
∆H | Enthalpy |
HSA | Human serum albumin |
Km | Michaelis-Menten constant |
λmax | Wavelength maxima |
MRE | mean residue ellipticity |
Vmax | maximum velocity |
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Parameter | 25 °C | 30 °C | 37 °C |
---|---|---|---|
n (binding stoichiometry, HSA:CBH) | 0.99 ± 0.11 | 1.01 ± 0.13 | 1.101 ± 0.12 |
KSV (Stern-Volmer constant, M−1) | 1.7 ± 0.21 × 104 | 1.3 ± 0.24 × 104 | 1.0 ± 0.31 × 104 |
Kb (binding constant, M−1) | 1.6 ± 0.22 × 104 | 1.4 ± 0.19 × 104 | 1.2 ± 0.23 × 104 |
kq (bimolecular quenching rate constant, M−1 s−1) | 2.6 ± 0.12×1012 | 2.2 ± 0.15 × 1012 | 1.7 ± 0.19 × 1012 |
ΔH° (binding enthalpy, kcal mol−1) | - | −4.44±0.16 | - |
TΔS° (entropy change, kcal mol-1) | 1.28 ± 0.18 | 1.30 ± 0.15 | 1.33 ± 0.16 |
ΔG° (Gibbs free energy change, kcal mol-1) | −5.72 ± 0.12 | −5.75 ± 0.15 | −5.78 ± 0.14 |
Parameter | Value |
---|---|
J (cm−3 M−1) | 3.08 × 10−15 |
Ro (nm) | 2.18 |
r (nm) | 2.34 |
EFRET | 0.39 |
Fo | 2546 |
F | 1829 |
Protein | ΔGDo, kcal mol−1 | mg, kcal mol−1 M−1 | Cm, M |
---|---|---|---|
0.0 μM CBH | 4.92 ± 0.24 | 1.98 ± 0.14 | 2.48 |
100 μM CBH | 5.42 ± 0.19 | 2.01 ± 0.18 | 2.69 |
HSA:CBH | Vmax (mM min−1) | Km (mM) | kcat (min−1) | kcat/Km (mM−1 min−1) |
---|---|---|---|---|
1:0 | 10.2 × 10−4 | 34.8 × 10−2 | 12.8 × 10−2 | 0.36 |
1:5 | 10.2 × 10−4 | 50.6 × 10−2 | 12.8 × 10−2 | 0.25 |
1:10 | 10.0 × 10−4 | 60.0 × 10−2 | 12.5 × 10−2 | 0.20 |
Complex | Binding Free Energy (ΔG) (kcal mol−1) | Residues Involved | |
---|---|---|---|
Hydrogen Bond | Hydrophobic Interaction | ||
HSA-CBH | −7.55 | S192 | Y150, E153, S192, K195, Q196, K199, L238, R257, H288, A291, E292 |
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Baig, M.H.; Rahman, S.; Rabbani, G.; Imran, M.; Ahmad, K.; Choi, I. Multi-Spectroscopic Characterization of Human Serum Albumin Binding with Cyclobenzaprine Hydrochloride: Insights from Biophysical and In Silico Approaches. Int. J. Mol. Sci. 2019, 20, 662. https://doi.org/10.3390/ijms20030662
Baig MH, Rahman S, Rabbani G, Imran M, Ahmad K, Choi I. Multi-Spectroscopic Characterization of Human Serum Albumin Binding with Cyclobenzaprine Hydrochloride: Insights from Biophysical and In Silico Approaches. International Journal of Molecular Sciences. 2019; 20(3):662. https://doi.org/10.3390/ijms20030662
Chicago/Turabian StyleBaig, Mohammad Hassan, Safikur Rahman, Gulam Rabbani, Mohd Imran, Khurshid Ahmad, and Inho Choi. 2019. "Multi-Spectroscopic Characterization of Human Serum Albumin Binding with Cyclobenzaprine Hydrochloride: Insights from Biophysical and In Silico Approaches" International Journal of Molecular Sciences 20, no. 3: 662. https://doi.org/10.3390/ijms20030662