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In recent years research based on kaempferol (KMP) has shown its potential therapeutic applications in medicinal chemistry and clinical biology. Therefore, to understand its molecular recognition mechanism, we studied its interactions with the carrier proteins, namely, human serum albumin (HSA), bovine hemoglobin (BHb) and hen egg white lysozyme (HEWL). The ligand, KMP was able to quench the intrinsic fluorescence of these three proteins efficiently through static quenching mode. The binding constant (K b ) for the interactions of KMP with these three proteins were found in the following order: HSA-KMP > BHb-KMP > HEWL-KMP. Different non-covalent forces such as hydrogen bonding and hydrophobic forces played a major role in the binding of KMP with HSA and HEWL, whereas hydrogen bonding and van der Waals forces contribute to the complexation of BHb with KMP. KMP was able to alter the micro-environment near the Trp fluorophore of the proteins. KMP altered the secondary structural component of all three proteins. The putative binding sites and the residues surrounding the KMP molecule within the respective protein matrix were determined through molecular docking and molecular dynamics (MD) simulation studies. The conformational flexibility of the ligand KMP and the three individual proteins were also evident from the MD simulation studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this work, the interaction of a bioactive tea polyphenol (-)-epigallocatechin gallate (EGCG) with bovine hemoglobin (BHb) along with its anti-oxidative behavior and the anti-glycation property have been explored using multi-spectroscopic and computational techniques. The binding affinity for EGCG towards BHb was observed to be moderate in nature with an order of 104 M-1, and the fluorescence quenching mechanism was characterized by an unusual static quenching mechanism. The binding constant (Kb) showed a continuous enhancement with temperature from 3.468 ± 0.380 × 104 M-1 at 288 K to 6.017 ± 0.601 × 104 M-1 at 310 K. The fluorescence emission measurements along with molecular docking studies indicated that EGCG binds near the most dominant fluorophore of BHb (β2-Trp37, at the interface of α1 and β2 chains) within the pocket formed by the α1, α2 and β2 chains. The sign and magnitude of the thermodynamic parameters, changes in enthalpy (ΔH = +17.004 ± 1.007 kJ mol-1) and in entropy (ΔS = +146.213 ± 2.390 J K-1 mol-1), indicate that hydrophobic forces play a major role in stabilizing the BHb-EGCG complex. The micro-environment around the EGCG binding site showed an increase in hydrophobicity upon ligand binding. The binding of EGCG with BHb leads to a decrease in the α-helical content, whereas that of the β-sheet increased. FTIR studies also indicated that the secondary structure of BHb changed upon binding with EGCG, along with providing further support for the presence of hydrophobic forces in the complexation process. Molecular docking studies indicated that EGCG binds within the cavity of α1, α2, and β2 chains surrounded by residues such as α1- Lys99, α1-Thr134, α1-Thr137, α1-Tyr140, α2-Lys127 and β2-Trp37. Molecular dynamics simulation studies indicated that EGCG conferred additional stability to BHb. Furthermore, moving away from the binding studies, EGCG was found to prevent the glyoxal (GO)-mediated glycation process of BHb, and it was also found to act as a potent antioxidant against the photo-oxidative damage of BHb.

The binding of two bio-active flavonoids, quercetin and rutin, with bovine hemoglobin (BHb) was investigated by multi-spectroscopic and computational (molecular docking and molecular dynamics simulation) studies. The two flavonoids were found to quench the intrinsic fluorescence of BHb through a static quenching mechanism. The binding constants at 288 K were observed to be (14.023 ± 0.73) × 104 M-1 and (7.848 ± 0.20) × 104 M-1, respectively for quercetin and rutin binding with BHb. Both rutin and quercetin were observed to increase the polarity around the Trp residues of BHb as indicated by synchronous and 3D spectral studies. No significant alterations in the secondary structural components of the protein were caused during the binding of the flavonoids as studied by CD and FTIR studies. The negative molar Gibbs free energies indicated the spontaneity of the interaction processes while the binding processes were characterized by a negative enthalpy change (ΔH) and a positive entropy change (ΔS). The possibility of energy transfer from the donor (BHb) to the acceptor molecules (flavonoids) was indicated by the FRET studies. According to the fluorescence studies, the flavonoids interact near to the β2-Trp37 residue of BHb. Excellent correlations with the experimental studies were observed from the molecular docking and molecular dynamics (MD) simulation studies. Further investigations established that these flavonoids are efficient in the inhibition of glucose mediated glycation of BHb.

Flavonoids are biologically imperative compounds used as anti-oxidants, anti-cancer, anti-bacterial agents etc. The current work reports comprehensive binding studies of two important flavonoids, 6-hydroxyflavone and 5,7-dihydroxyflavone (chrysin) with bovine hemoglobin (BHb) at 298K and 308K, in aqueous medium using UV-vis spectroscopy, steady state fluorescence, circular dichroism (CD) measurements, Fourier Transform infrared spectroscopy (FT-IR) and molecular docking studies. Both 6-hydroxyflavone and chrysin can quench the intrinsic fluorescence intensity of BHb via static quenching mechanism. The values of binding constant (K b ) for BHb-chrysin complex (3.177±0.992×10 4 M -1 , at 298K) was found to be greater than that of BHb-6-hydroxyflavone complex (2.874±0.863×10 4 M -1 , at 298K) and the K b values decreased with the rise in temperature. The thermodynamic parameters indicated that hydrophobic forces and H-bonding play crucial role in BHb-6-hydroxyflavone complexation whereas electrostatic interaction plays the major role in the binding of BHb and chrysin. The binding distances from donor BHb to the acceptor ligands (6-hydroxyflavone and chrysin) were estimated using the Föster's theory and the possibility of non-radiative energy transfer from BHb to 6-hydroxyflavone/chrysin was observed. The ligands, 6-hydroxyflavone and chrysin induced conformational change around Trp residues in BHb as confirmed by synchronous and 3D fluorescence results. CD and FT-IR studies indicated that the % α-helicity of BHb was enhanced due to 6-hydroxyflavone/chrysin binding. Both the flavonoids showed remarkable inhibitory effect towards BHb glycation. Hydrophobic probe (8-anilino-1-naphthalenesulfonic acid, ANS) displacement and molecular docking studies revealed that the ligands bind within the hydrophobic pocket of BHb., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hemoglobin serves a multitude of biological functions and its activity and stability are influenced by structural alterations. Fibrillation of this protein is one such complication that can hinder its physiological levels resulting in numerous health disorders. This study focuses on providing a comprehensive elucidation on the binding interaction of bioactive esculin and esculetin with bovine hemoglobin (BHb) and ultimately determining the anti-fibrillation properties of these compounds. UV/vis spectroscopy and fluorescence measurements verified the presence of a static form of quenching mechanism for the BHb-esculin complex and a combined quenching mechanism for the BHb–esculetin complex. The binding affinity (Kb) was determined to be higher for the BHb–esculetin complex than for the BHb-esculin complex. This could be due to the lower binding distance observed between a β2-Trp37 residue and esculetin, as observed through Förster's theory, molecular docking, and molecular dynamic (MD) simulation. The calculated thermodynamic parameters indicated the presence of hydrophobic forces and hydrogen bonding stabilizing the complexes. Synchronous, 3D fluorescence, REES, and anisotropy measurements revealed microenvironmental alterations around the Trp residue induced by esculin/esculetin binding. Secondary structural changes of BHb in the presence of esculin/esculetin were observed through UV/vis absorption, CD, and FTIR studies. BHb was subjected to fibrillation in the presence of fructose, and ThT fluorescence, fluorescence microscopy, FESEM, Soret absorption, ANS measurements, and Congo red assay provided evidence of the anti-fibrillation effects of esculin and esculetin. [ABSTRACT FROM AUTHOR]

Background: Iron is important for brain development and cognitive function. Iron deficiency may cause alteration of neurotransmitters and may be manifested by different central nervous system disorders including attention deficit hyperactivity disorder (ADHD). Aims: As studies are scarce in the Indian context, we had undertaken this study to find out the association between iron deficiency and ADHD. Settings and Design: Hospital-based cross-sectional study. Materials and Methods: Hematological parameters indicating iron status (hemoglobin [Hb], ferritin, Iron, total iron binding capacity [TIBC], mean corpuscular volume [MCV], and mean corpuscular Hb [MCH]) were measured among 119 ADHD patients selected by complete enumeration method and 119 controls. Statistical Analysis: Shapiro–Wilk test, Mann–Whitney U-test, Spearman's correlation, and binary logistic regression were used. P < 0.01 was taken as statistically significant. Results: Hb, iron, ferritin, MCV, and MCH were lower among cases and negatively correlated to ADHD, while reverse is true for TIBC and ADHD. Iron deficiency anemia makes one 3.82 times more prone for ADHD. Conclusion: Iron deficiency was associated with ADHD. [ABSTRACT FROM AUTHOR]

[Display omitted] • Binding of coumarin derivatives with bovine hemoglobin have been investigated using biophysical techniques. • Static quenching mechanism is involved in the binding interactions of coumarins with bovine hemoglobin. • Binding constant was found moderate in nature. • Secondary structure of the protein was perturbed during ligand binding. • Results from computational studies correlated well with the experimental findings. Biological functioning takes place through the interaction of biomacromolecules with each other or with other small molecules. The molecular recognition of a carrier protein, bovine hemoglobin (BHb), with 7-hydroxycoumarin (7-HC) and 4-methyl-7-hydroxycoumarin (4-Me-7-HC) was investigated using various biophysical and computational techniques. Fluorescence spectroscopy and molecular docking revealed the interaction of 7-HC and 4-Me-7-HC with β 2 -Trp37 fluorophore, quenching the intrinsic fluorescence of BHb. The mechanism of quenching was determined to be static. The binding constant (K b) for BHb with 7-HC and 4-Me-7-HC was found to be 6.15 × 104 M−1 and 5.73 × 104 M−1 at 298 K, respectively. This moderate form of protein–ligand association could result in reversible binding to transport and release the ligand in the target tissue. Negative Δ G for both complexes suggested spontaneous binding. Positive Δ H and Δ S for BHb-7-HC indicated hydrophobic forces played a dominant role in binding. However, hydrogen bonding and hydrophobic forces dictated the binding process for BHb-4-Me-7-HC due to obtaining negative Δ H and positive Δ S values. Changes in the microenvironment of the binding site were observed through 3D fluorescence studies. Through Förster resonance energy transfer (FRET), a binding distance of less than 7 nm was measured between BHb and 7-HC/4-Me-7-HC. The interaction of coumarin derivatives with BHb resulted in a loss of α-helical content of the protein, as proven by circular dichroism (CD) and Fourier transform infrared (FTIR) measurements. Molecular dynamic simulations showed the binding of 7-HC supplemented the stability of BHb, whereas 4-Me-7-HC binding resulted in conformational changes in the structure of BHb. [ABSTRACT FROM AUTHOR]