An investigation into the altered binding mode of green tea polyphenols with human serum albumin on complexation with copper

<div><p>Green tea is rich in several polyphenols, such as (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epigallocatechin-3-gallate (EGCG). The biological importance of these polyphenols led us to study the major polyphenol EGCG with human serum albumin (HSA) in an earlier study. In this report, we have compared the binding of ECG, EGC, and EGCG and the Cu(II) complexes of EGCG and ECG with HSA. We observe that the gallate moiety of the polyphenols plays a crucial role in determining the mode of interaction with HSA. The binding constants obtained for the different systems are 5.86 ± 0.72 × 10⁴ M⁻¹ (<i>K</i>_ECG-HSA), 4.22 ± 0.15 × 10⁴ M⁻¹ (<i>K</i>_{ECG-Cu(II)-HSA}), and 9.51 ± 0.31 × 10⁴ M⁻¹ (<i>K</i>_{EGCG-Cu(II)-HSA}) at 293 K. Thermodynamic parameters thus obtained suggest that apart from an initial hydrophobic association, van der Waals interactions and hydrogen bonding are the major interactions which held together the polyphenols and HSA. However, thermodynamic parameters obtained from the interactions of the copper complexes with HSA are indicative of the involvement of the hydrophobic forces. Circular dichroism and the Fourier transform infrared spectroscopic measurements reveal changes in α-helical content of HSA after binding with the ligands. Data obtained by fluorescence spectroscopy, displacement experiments along with the docking studies suggested that the ligands bind to the residues located in site 1 (subdomains IIA), whereas EGC, that lacks the gallate moiety, binds to the other hydrophobic site 2 (subdomain IIIA) of the protein.</p> </div

Prolonged Glycation of Hen Egg White Lysozyme Generates Non Amyloidal Structures

<div><p>Glycation causes severe damage to protein structure that could lead to amyloid formation in special cases. Here in this report, we have shown for the first time that hen egg white lysozyme (HEWL) does not undergo amyloid formation even after prolonged glycation in the presence of D-glucose, D-fructose and D-ribose. Cross-linked oligomers were formed in all the cases and ribose was found to be the most potent among the three sugars. Ribose mediated oligomers, however, exhibit Thioflavin T binding properties although microscopic images clearly show amorphous and globular morphology of the aggregates. Our study demonstrates that the structural damage of hen egg white lysozyme due to glycation generates unstructured aggregates.</p> </div

Variation of secondary structural components during glycation of HEWL in the presence of different sugars.

<p>Representative far UV-CD spectra of (a) HEWL-glucose, (b) HEWL-fructose and (c) HEWL-ribose solutions respectively obtained after incubation at pH 7.4 at 37 °C at different time intervals. [HEWL]=20 µM in each case.</p

Identification of attachment of sugar moieties to HEWL: Fuchsin based SDS PAGE.

<p>Representative SDS polyacrylamide gel electrophoresis of different HEWL solutions obtained after incubation at pH 7.4 at 37 °C for 31 days in the presence of different sugars using Fuchsin staining. lane 1: Horseradish peroxidase; lane 2 and 5: HEWL-glucose; lane 3 and 6: HEWL-fructose; lane 4 and 7: HEWL-ribose.</p

Tertiary structural alterations of HEWL solutions during glycation in the presence of three different sugars.

<p>Representative near UV-CD spectra of (a) HEWL-glucose, (b) HEWL-fructose and (c) HEWL-ribose solutions respectively obtained after incubation at pH 7.4 at 37 °C at different time intervals.</p

Characterization of different AGE products formed during glycation of HEWL in the presence of different sugars using fluorescence spectroscopy.

<p>Histograms represent fluorescence intensity of different HEWL solutions incubated in the presence of glucose, fructose and ribose respectively over a period of 120 days. Formation of different AGE products such as (a) other AGE products (λ_ex=350 nm), (b) pentosidine (λ_ex=335 nm) and (c) malondialdehyde (MDA) (λ_ex=370 nm). [HEWL]=5 µM in each case. Control represents native HEWL incubated in the absence of sugars at pH 7.4 at 37 °C keeping other conditions similar as that of sets in each case.</p

Densitometric analysis of SDS-PAGE.

<p>Histograms represent relative mean band intensity of different oligomeric species (dimer, trimer and tetramer) with respect to their corresponding monomer at definite time intervals (a) 1 day, (b) 20 days and (c) 31 days respectively.</p

Identification of the nature of the aggregates formed during glycation of HEWL.

<p>FESEM (a-d) and TEM images (e-h) of different HEWL solutions obtained after an incubation at pH 7.4 at 37 °C at different time intervals in the presence of different sugars. For FESEM images scale bars represent 2 µm for HEWL-glucose and HEWL-fructose; 20 µm for Control and HEWL-ribose respectively. For TEM images scale bars represent 200 nm.</p

Estimation of β-sheet content of HEWL solutions incubated in the presence of different sugars.

<p>Percentage β-sheet content of different HEWL solutions (HEWL-glucose, HEWL-fructose and HEWL-ribose) obtained after incubation at pH 7.4 at 37 °C estimated using online server DICHROWEB at different time intervals.</p

Determination of the mass of glycated HEWL.

<p>MALDI TOF spectra of different HEWL solutions obtained after an incubation of 31 days at pH 7.4 at 37 °C (a) Native HEWL (14345.30 Da) (b) HEWL-glucose (15401.22 Da) (c) HEWL-fructose (15955.81 Da) (d) HEWL-ribose (16233.48 Da).</p