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All-trans retinoic acid derived from vitamin A is an essential component for the modulation of angiogenesis, the process of blood vessel formation. We have investigated the binding of all-trans retinoic acid to the carrier protein, human serum albumin (HSA) under physiological conditions. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy were used for the biophysical studies. The binding parameters were determined by a Scatchard plot and the results found to be consistent with those obtained from a modified Stern-Volmer equation. From the thermodynamic parameters calculated according to the van't Hoff equation, the enthalpy change DeltaH(0) and entropy change DeltaS(0) are found to be 106.17 and 106.14J/molK, respectively. These values suggest that apart from hydrophobic interactions electrostatic interactions are present. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. Docking studies performed substantiated our experimental findings and it was observed that all-trans retinoic acid hydrogen bonded with Trp 214 and Asp 451 residues of subdomain IIA and IIIA of HSA, respectively.

The formation of specific patterns during evaporation of biofluids e.g. blood, serum etc. can be analyzed for quick identification of several diseases. Human Serum Albumin (HSA) is frequently used as the model protein to study amyloid fibril formation that is responsible for numerous neurodegenerative diseases like Parkinson’s disease, Alzheimer’s disease etc. In the present study the fibril growth of HSA is characterized by analyzing the drying patterns of the protein solution. The fibril formation of HSA is confirmed by spectrofluorimetry and the β-sheet content is quantified by far-UV Circular Dichroism spectrometry. Examinations of the drying patterns of HSA, in presence of fluorescent nanoparticles, reveals distinctive pattern transformations with increase in the β-sheet content visualized through fluorescence microscopy and FESEM. The fluorescence images of the dried droplet are analyzed using image processing by the color thresholding method. A rapid quantification methodology of the β-sheet content of HSA and the fraction of unfolded protein has been developed by comparing the results of the image analysis with those of the far-UV CD data. [ABSTRACT FROM AUTHOR]

Steady state fluorescence and time resolved fluorescence studies at 298 K and low temperature phosphorescence (LTP) studies at 77 K of the interaction of bovine serum albumin (BSA) and human serum albumin (HSA) with ligand 4-azido-2H-chromen-2-one or 4-azidocoumarin (4-AC) have been carried out to visualize the location of the binding site and perturbation of the binding site of the tryptophan (Trp)/tyrosine (Tyr) of the protein(s) by monitoring the emission maxima of Trp residue(s) in proteins. The fluorescence quenching study of Trp estimated that the binding constant for both protein–ligand complexes is in the order of ∼106 with binding site 1. Perturbation in the secondary structures of serum albumins due to binding of 4-AC is also observed from circular dichroism (CD) studies. An energy transfer (ET) study further demonstrated that the non-radiative singlet–singlet ET that takes place from the Trp singlet states of proteins to the singlet state of ligands is greater in the case of BSA. This is supported by the distance and orientation of the donor–acceptor pair obtained from molecular docking studies. The molecular docking studies were also fruitfully exploited to understand the involvement of Trp213 in BSA and Trp214 in HSA in the ET process along with the perturbation of the residues around 5 Å from the ligand 4-AC. Phosphorescence spectra at 77 K of the Trp residues in the free proteins (BSA/HSA) and in the complexes of BSA/HSA have also been utilized to specify the role of Trp residues in ET and the binding process. [ABSTRACT FROM AUTHOR]

Abstract: Green tea polyphenols, which have the ability to inhibit angiogenesis, form complexes with Cu(II), a known potent stimulator of blood vessel proliferation. Copper complexes of (−)-epicatechin gallate and (−)-epigallocatechin gallate were found to inhibit the enzymatic activity of Ribonuclease A (RNase A) as revealed by an agarose gel based assay and urea denatured gel electrophoresis. The copper complexes were found to be non-competitive inhibitors of RNase A with inhibition constants in the micromolar range. Changes in the secondary structure of the protein are found to occur due to the interaction as revealed from Fourier transform infrared and circular dichroism studies. [Copyright &y& Elsevier]

Understanding the interaction of proteins with nanoparticles has become an important area of research in biomedical and pharmaceutical fields. Morin is a flavonol which shows several properties including antioxidant, anticancer, and anti-inflammatory activities. However, the major limitation is its poor aqueous solubility. Therefore, morin-loaded polylactic-co-glycolic acid (PLGA) nanoparticles (MPNPs) were prepared to improve the solubility of morin. The resulting MPNPs were characterized by spectroscopic and microscopic techniques. The nanoparticles were spherical with an average size of 237 ± 17 nm. UV-visible, fluorescence, and circular dichroism (CD) spectroscopy were employed to study the interaction of the MPNPs with human serum albumin (HSA). Our study revealed that a static fluorescence quenching mechanism was involved in the interaction between HSA and MPNPs. Hydrophobic interactions also play an important role in stabilizing the HSA-MPNP complex. CD results suggest that there is an alteration of the secondary structure of HSA in the presence of MPNPs. MPNPs exhibit antioxidant properties which are supported by the DPPH assay. We have further checked the effect of HSA on the antioxidant property of morin and MPNPs. HSA binding with MPNPs was also found to influence the in vitro release property of morin from MPNPs wherein a delayed release response is observed. [ABSTRACT FROM AUTHOR]

Abstract: Green tea polyphenols (GTPs) are found to be potent inhibitors of amyloid fibril formation. We report the effective inhibitory property of (−)-epicatechin gallate (ECG) during the alkali-salt induced fibrillogenesis of hen egg white lysozyme (HEWL) at 37°C. Spectroscopic techniques such as fluorescence, circular dichroism and microscopic images show that (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin gallate (EGCG) show moderate inhibition of fibrillation with ECG as the most potent polyphenol. Aromatic interactions, hydrophobic interactions, the radical scavenging activity and autoxidation of polyphenols are likely to be the major reasons for ECG being the most effective inhibitor. [Copyright &y& Elsevier]

The binding interaction of Rutin, a flavonoid, with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), were investigated using different spectroscopic techniques, such as fluorescence, time-resolved single photon counting (TCSPC) and circular dichroism (CD) spectroscopy as well as molecular docking method. The emission studies revealed that the fluorescence quenching of BSA/HSA by Rutin occurred through a simultaneous static and dynamic quenching process, and we have evaluated both the quenching constants individually. The binding constants of Rutin-BSA and Rutin-HSA system were found to be 2.14 × 10 6  M −1 and 2.36 × 10 6  M −1 at 298 K respectively, which were quite high. Further, influence of some biologically significant metal ions (Ca 2+ , Zn 2+ and Mg 2+ ) on binding of Rutin to BSA and HSA were also investigated. Thermodynamic parameters justified the involvement of hydrogen bonding and weak van der Waals forces in the interaction of Rutin with both BSA and HSA. Further a site-marker competitive experiment was performed to evaluate Rutin binding site in the albumins. Additionally, the CD spectra of BSA and HSA revealed that the secondary structure of the proteins was perturbed in the presence of Rutin. Finally protein–ligand docking studies have also been performed to determine the probable location of the ligand molecule. [ABSTRACT FROM AUTHOR]

Several DNA binding proteins exist in dimeric form when bound with DNA to be able to exhibit various biological processes such as DNA repair, DNA replication and gene expression. Various dimeric forms of Ribonuclease A (RNase A) and other members of the ribonuclease A superfamily are endowed with a multitude of biological activities such as antitumor and antiviral activity. In the present study, we have compared the DNA binding properties between the RNase A monomer and the dityrosine (DT) cross-linked RNase A dimer, and checked the inhibitory effect of DNA on the ribonucleolytic activity of the dimeric protein. An agarose gel based assay shows that like the monomer, the dimer also binds with DNA. The number of nucleotides bound per monomer unit of the dimer is higher than the number of nucleotides that bind with the each monomer. From fluorescence measurements, the association constant ( K a ) values for complexation of the monomer and the dimer with ct-DNA are (4.95 ± 0.45) × 10 4 M − 1 and (1.29 ± 0.05) × 10 6 M − 1 respectively. Binding constant ( K b ) values for the binding of the monomer and the dimer with ct-DNA were determined using UV–vis spectroscopy and were found to be (4.96 ± 1.67) × 10 4 M − 1 and (4.32 ± 0.31) × 10 5 M − 1 respectively. Circular dichroism studies shows that the dimer possesses significant effect on DNA conformation. The melting profile for the ct-DNA–dimer indicated that the melting temperature ( T m ) for the ct-DNA–dimer complex is lower compared to the ct-DNA–monomer complex. The ribonucleolytic activity of the dimer, like the monomer, diminishes upon binding with DNA. [ABSTRACT FROM AUTHOR]

Abstract: In consideration of the various medicinal aspects of the flavonoid polyphenols, the interaction of morin with bovine serum albumin (BSA) has been investigated using multi-spectroscopic approaches. The pKa 1 of morin being 5.09, which is below physiological pH, binding studies provide important insights into its potential use as a biotherapeutic. The binding was performed under different pH (5, 7 and 9) conditions and in absence and presence of Cu(II) and Fe(III) ions. It is observed that the presence of metal ions affect the binding of morin towards BSA. The binding with BSA results in a motional restriction of morin in solution that causes an increase in anisotropy (r), rotational correlation time (t r ) and steady-state lifetime (t av ) of the ligand. Urea causes denaturation of BSA resulting in the release of morin from the protein core as determined from both the steady-state fluorescence and anisotropy (r) measurements. The possibility of non-radiative energy transfer from the donor tryptophan to the acceptor morin is detected following the Förster's theory. The site marker displacement studies along with the molecular docking results indicated that morin binds to the hydrophobic pocket of site 1 (subdomain IIA) near Trp 213 of BSA. [Copyright &y& Elsevier]

Abstract: The interaction of genistein with bovine serum albumin (BSA) has been characterized via UV–vis, fluorescence spectroscopy and Circular Dichroism (CD) measurements under physiological conditions. In this study, we have investigated the effect of some common metal ions on the binding of genistein with BSA using fluorescence studies. The fluorescence data reveal that the binding affinity of genistein to BSA increases in presence of certain metal ions. The possibility of non-radiative energy transition from the donor tryptophan to the acceptor genistein has been observed in absence and presence of metal ions. The observed similarities in the values of efficiency of energy transfer (E) and the separation between the donor and acceptor (r) in both the cases may be correlated with the complexation between the genistein and metal ions, which is also observed from the UV–vis studies. The changes in enthalpy (ΔH°) and entropy (ΔS°) of the interaction were found to be −14.64kJmol−1 and +42.75Jmol−1 K−1 respectively. These values indicate the involvement of electrostatic interactions along with a hydrophobic association that results in a positive entropy change. CD analysis shows that there is a slight increase in the% α-helical content of BSA on binding with genistein at lower molar ratios. Warfarin and ibuprofen displacement studies in accordance with the molecular docking show that genistein binds to site I (subdomain IIA) of BSA. [Copyright &y& Elsevier]

Abstract: Polyphenols find wide use as antioxidants, cancer chemopreventive agents and metal chelators. The latter activity has proved interesting in many aspects. We have probed the binding characteristics of the polyphenol quercetin–Cu(II) complex with human serum albumin (HSA) and bovine serum albumin (BSA). Fluorescence studies reveal that the quercetin–Cu(II) complex can quench the fluorescence of the serum albumins. The binding constant (K b ) values are of the order of 105 M−1 which increased with rise in temperature in case of HSA and BSA interacting with the quercetin–Cu(II) complex. Displacement studies reveal that both the ligands bind to site 1 (subdomain IIA) of the serum albumins. However, thermodynamic parameters calculated from temperature dependent studies indicated that the mode of interaction of the complexes with the proteins differs. Both ΔH° and ΔS° were positive for the interaction of the quercetin–Cu(II) complex with both proteins but the value of ΔH° was negative in case of the interaction of quercetin with the proteins. This implies that after chelation with metal ions, the polyphenol alters its mode of interaction which could have varying implications on its other physicochemical activities. [Copyright &y& Elsevier]

Abstract: Copper is known to exert diverse effects on the self-association of proteins and has been found in amyloid deposits that are involved in neurodegenerative disease processes. The effects of the metal ion on the protein during fibrillation were investigated by fluorescence, circular dichroism spectroscopy and fluorescence microscopy. We report for the first time, the complete reduction of Cu(II)→Cu(I) in vitro during fibrillation of hen egg white lysozyme at pH 7. This was confirmed by the lack of any signal for Cu(II) in electron paramagnetic resonance spectroscopy and quantification of Cu(I) was achieved by a bathocuproine disulfonate based assay. [Copyright &y& Elsevier]

Abstract: The interactions of two stereoisomeric antioxidant flavonoids, catechin (C) and epicatechin (EC) with bovine serum albumin (BSA) and human serum albumin (HSA), have been investigated by steady state and time resolved fluorescence, phosphorescence, circular dichroism (CD), FTIR and protein–ligand docking studies. The steady-state fluorescence studies indicate a single binding site for both the ligands. FTIR spectra suggest that in both the albumins, C and EC stabilize the α-helix at the cost of a corresponding loss in the β-sheet structure. CD studies have been carried out using (±)C, and both the epimers (+)C and (−)C. The low temperature phosphorescence and protein–ligand [(+), (−) and (±) forms of C and EC] docking studies indicate that the ligands bind in the proximity of Trp 134 of BSA and Trp 214 of HSA, thereby changing their solvent accessible surface areas (ASA). Asn 158 and Glu 130 side chains are found to be within the hydrogen bonding distance from the phenolic –OH groups of C and EC in the case of BSA complex. C and EC are located within the binding pocket of sub-domain IIa of HSA. [Copyright &y& Elsevier]

Abstract: Interaction of a genotoxic fungicide methyl thiophanate (MT) has been studied in vitro with calf thymus DNA. Fluorescence quenching data revealed the binding constant (K a =3.23×104 M−1) and binding capacity (n =1.1) of MT with ctDNA. Ligand displacement studies using specific probes suggested the MT binding at DNA minor groove. The docking analysis further substantiated MT interaction with at least three AT base pairs within the DNA groove. A discernable change in E 0′ value with decreased peak currents in cyclic voltammogram, and peak shifts in CD spectra reflected the formation of MT–ctDNA and MT–ctDNA–Cu(II) complexes. The results elucidate the significance of specific MT–DNA interactions as an initiating event in MT-induced DNA damage. [Copyright &y& Elsevier]

Abstract: The interaction of copper complexes of (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) with calf thymus DNA (ct-DNA) was investigated by UV–visible (UV–Vis), fluorescence and circular dichroism along with melting studies. It was observed that both copper complexes quench the fluorescence intensity of ethidium bromide bound ct-DNA upon binding, resulting in a ground state complex formation by a static quenching process. The binding constants evaluated from fluorescence data were supported by the UV–Vis study. The values ranged from 0.84 to 1.07×105 M−1 and 1.14 to 1.04×105 M−1 for Cu(II)–ECG and Cu(II)–EGCG, respectively for the temperature range 21–42°C with two binding sites. Thermodynamic parameters obtained are suggestive of the involvement of different modes of interaction during binding for each complex although both were found to be intercalating in nature. Circular dichroism studies and variations in the melting temperature reveal unwinding of the ct-DNA helix with conformational changes due to binding. [Copyright &y& Elsevier]

Abstract: Ongoing research on curcumin and its structural derivatives are a subject of growing interest because of their demonstrated biological properties. Diacetylcurcumin (DAC), a synthetic derivative of natural non-toxic curcumin has been shown to affect a host of activities ranging from wound healing to life threatening diseases like AIDS, cancer etc. The interaction of diacetylcurcumin (DAC) with calf thymus-DNA (ct-DNA) has been investigated by spectroscopic and viscometric techniques. The fluorescence intensity of DAC was quenched by ct-DNA. The mean binding constant obtained from the spectroscopic techniques was 3.97±0.31×105 M−1. Circular dichroism studies did not reveal any unwinding of the DNA helix on interaction with DAC, implying no conformational changes. The binding mode was analyzed by competitive binding between ethidium bromide (EB) and DAC for ct-DNA and also by viscometric studies. DAC was found to be a minor groove binder with a preference for the A-T region compared to the G-C region. This was substantiated by displacement studies with Hoechst 33258, a known minor groove binder. Docking studies were found to corroborate the experimental results. [Copyright &y& Elsevier]

Abstract: In this study, compounds with a carboxy ester in lieu of the phosphate ester at the 3′-position have been employed to inhibit the ribonucleolytic activity of ribonuclease A (RNase A). Phosphates at the 3′-position of pyrimidine bases are well-known inhibitors of the protein. We have investigated the inhibition of RNase A by 3′-O-carboxy esters of thymidine. The compounds behave as competitive inhibitors with inhibition constants ranging from 42 to 95μM. The mode of inhibition has also been confirmed by 1H NMR studies of the active site histidines of RNase A. Docking studies have further substantiated the experimental results. The compounds are also found to inhibit the ribonucleolytic activity of angiogenin, a homologous protein and potent inducer of blood vessel formation. [Copyright &y& Elsevier]

Abstract: Silibinin is a well-known naturally occurring compound used for the treatment of a wide range of hepatitic diseases. In this study, we report the binding of silibinin to the carrier protein, human serum albumin (HSA) under physiological conditions. UV–Vis and fluorescence quenching methods in combination with Fourier transformed infrared (FT-IR) and circular dichroism (CD) spectroscopy have been used to study the nature and mode of binding. The binding parameters were determined from tryptophan fluorescence quenching by a Scatchard plot and the results were found to be consistent with those obtained from a modified Stern–Volmer equation. The association constant is of the order of 105. The change in enthalpy (ΔH°) and entropy (ΔS°) due to the interaction were found to be −6.76kJmol−1 and 73.69Jmol−1 K−1, respectively. These values suggest that apart from an initial hydrophobic association, electrostatic interactions play a decisive role during complexation of silibinin with HSA. Observations from FT-IR and CD spectra upon ligand binding indicate changes in the secondary structure of HSA. Docking studies that corroborate our experimental results reveal that the silibinin molecule lies within hydrogen bonding distance of Trp 214 and Asp 451 residues of subdomains IIa and IIIa of HSA, respectively. [Copyright &y& Elsevier]

Abstract: The interaction of isoxazolcurcumin (IOC), a synthetic derivative of curcumin, with calf thymus-DNA (ct-DNA) has been investigated by UV–Vis, fluorescence, circular dichroism spectroscopies, viscosity measurements and docking studies. From these analyses, the binding constant, number of binding sites and mode of binding of IOC to ct-DNA has been determined. The binding constant of IOC to DNA calculated from both UV–Vis and CD spectra was found to be in the 104 M−1 range. Analyses of fluorescence spectra, viscosity measurements and molecular modeling of IOC–DNA interactions indicate that IOC is a minor groove binder of ct-DNA and preferentially binds to AT rich regions. Ethidium bromide displacement studies revealed that IOC did not have any effect on ethidium bromide bound DNA which is indicative of groove binding. To elucidate the preferred region of binding of IOC to DNA, docking studies have been performed and changes in accessible surface area (ΔASA) of nucleobases determined due to IOC–DNA complexation. [Copyright &y& Elsevier]