Philippe Bourassa

Resveratrol, Genistein, and Curcumin Bind Bovine Serum Albumin†

We report the complexation of bovine serum albumin (BSA) with resveratrol, genistein, and curcumin, at physiological conditions, using constant protein concentration and various polyphenol contents. FTIR, CD, and fluorescence spectroscopic methods were used to analyze the ligand binding mode, the binding constant, and the effects of complexation on BSA stability and conformation. Structural analysis showed that polyphenols bind BSA via hydrophilic and hydrophobic interactions with the number of bound polyphenol (n) being 1.30 for resveratrol-BSA, 1.30 for genistein-BSA, and 1.0 for curcumin-BSA. The polyphenol-BSA binding constants were K(Res-BSA) = 2.52(+/-0.5) x 10(4) M(-1), K(Gen-BSA) = 1.26(+/-0.3) x 10(4) M(-1), and K(Cur-BSA) = 3.33(+/-0.8) x 10(4) M(-1). Polyphenol binding altered BSA conformation with a major reduction of alpha-helix and an increase in beta-sheet and turn structures, indicating a partial protein unfolding.

Milk β-lactoglobulin complexes with tea polyphenols.

The effect of milk on the antioxidant capacity of tea polyphenols is not fully understood. The complexation of tea polyphenols with milk proteins can alter the antioxidant activity of tea compounds and the protein secondary structure. This study was designed to examine the interaction of β-lactogolobulin (β-LG) with tea polyphenols (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) at molecular level, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on β-LG stability and secondary structure were determined. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of KC-β-LG=2.2 (±0.8)×10(3)M(-1), KEC-β-LG=3.2 (±1)×10(3)M(-1), KECG-β-LG=1.1 (±0.6)×10(4)M(-1) and KEGCG-β-LG=1.3 (±0.8)×10(4)M(-1). The number of polyphenols bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 0.9 (ECG) and 1.3 (EGCG). Molecular modelling showed the participation of several amino acid residues in polyphenol-protein complexation with extended H-bonding network. The β-LG conformation was altered in the presence of polyphenols with an increase in β-sheet and α-helix suggesting protein structural stabilisation. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.

Folic acid complexes with human and bovine serum albumins

The interaction of folic acid with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various folic acid contents was investigated. FTIR, UV-visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse folic acid binding sites, the binding constant and the effect on HSA and BSA stability and conformations. Structural analysis showed that folic acid binds HSA and BSA via both hydrophilic and hydrophobic contacts with overall binding constants of Kfolic acid-HSA=8.1 (±0.5)×10(4)M(-1) and Kfolic acid-BSA=1.0 (±0.3)×10(5)M(-1). The number of bound acid molecules per protein was 1.7 (±0.4) for HSA and 1.5 (±0.3) for BSA complexes. Molecular modelling showed participation of several amino acids in folic acid-protein complexes stabilised by hydrogen bonding network. Folic acid complexation altered protein secondary structure by major reduction of α-helix from 59% (free HSA) to 35% (acid-complex) and 62% (free BSA) to 25% (acid-complex) with an increase in random coil, turn and β-sheet structures indicating protein unfolding. The results suggest that serum albumins might act as carrier proteins for folic acid in delivering it to target molecules.

Probing the Binding Sites of Antibiotic Drugs Doxorubicin and N-(trifluoroacetyl) Doxorubicin with Human and Bovine Serum Albumins

We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K DOX-BSA = 7.8 (±0.7)×103 M−1, K FDOX-BSA = 4.8 (±0.5)×103 M−1 and K DOX-HSA = 1.1 (±0.3)×104 M−1, K FDOX-HSA = 8.3 (±0.6)×103 M−1. The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47–44% (drug-complex) and 57% (free HSA) to 51–40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.

Binding Sites of Resveratrol, Genistein, and Curcumin with Milk α- and β-Caseins

The binding sites of antioxidant polyphenols resveratrol, genistein, and curcumin are located with milk α- and β-caseins in aqueous solution. FTIR, CD, and fluorescence spectroscopic methods and molecular modeling were used to analyze polyphenol binding sites, the binding constant, and the effects of complexation on casein stability and conformation. Structural analysis showed that polyphenols bind casein via hydrophilic and hydrophobic interactions with the number of bound polyphenol molecules (n) 1.20 for resveratrol, 1.42 for genistein, and 1.43 for curcumin with α-casein and 1.14 for resveratrol, 1.27 for genistein, and 1.27 for curcumin with β-casein. The overall binding constants of the complexes formed are K(res-α-casein) = 1.9 (±0.6) × 10(4) M(-1), K(gen-α-casein) = 1.8 (±0.4) × 10(4) M(-1), and K(cur-α-casein) = 2.8 (±0.8) × 10(4) M(-1) with α-casein and K(res-β-casein) = 2.3 (±0.3) × 10(4) M(-1), K(gen-β-casein) = 3.0 (±0.5) × 10(4) M(-1), and K(cur-β-casein) = 3.1 (±0.5) × 10(4) M(-1) for β-casein. Molecular modeling showed the participation of several amino acids in polyphenol-protein complexes, which were stabilized by the hydrogen bonding network with the free binding energy of -11.56 (resveratrol-α-casein), -12.35 (resveratrol-β-casein), -9.68 (genistein-α-casein), -9.97 (genistein-β-casein), -8.89 (curcumin-α-casein), and -10.70 kcal/mol (curcumin-β-casein). The binding sites of polyphenols are different with α- and β-caseins. Polyphenol binding altered casein conformation with reduction of α-helix, indicating a partial protein destabilization. Caseins might act as carriers to transport polyphenol in vitro.

Dendrimers Bind Antioxidant Polyphenols and cisPlatin Drug

Synthetic polymers of a specific shape and size play major role in drug delivery systems. Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape with potential applications in gene and drug delivery. We examine the interaction of several dendrimers of different compositions mPEG-PAMAM (G3), mPEG-PAMAM (G4) and PAMAM (G4) with hydrophilic and hydrophobic drugs cisplatin, resveratrol, genistein and curcumin at physiological conditions. FTIR and UV-visible spectroscopic methods as well as molecular modeling were used to analyse drug binding mode, the binding constant and the effects of drug complexation on dendrimer stability and conformation. Structural analysis showed that cisplatin binds dendrimers in hydrophilic mode via Pt cation and polymer terminal NH2 groups, while curcumin, genistein and resveratrol are located mainly in the cavities binding through both hydrophobic and hydrophilic contacts. The overall binding constants of durg-dendrimers are ranging from 102 M−1 to 103 M−1. The affinity of dendrimer binding was PAMAM-G4>mPEG-PAMAM-G4>mPEG-PAMAM-G3, while the order of drug-polymer stability was curcumin>cisplatin>genistein>resveratrol. Molecular modeling showed larger stability for genisten-PAMAM-G4 (ΔG = −4.75 kcal/mol) than curcumin-PAMAM-G4 ((ΔG = −4.53 kcal/mol) and resveratrol-PAMAM-G4 ((ΔG = −4.39 kcal/mol). Dendrimers might act as carriers to transport hydrophobic and hydrophilic drugs.

Binding of cationic lipids to milk β-lactoglobulin.

We determined the bindings of several lipids such as cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) to β-lactoglobulin (β-LG) at physiological conditions. FTIR, CD, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of lipid-protein complexes. Structural analysis showed that lipids bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K(CHOL-β-LG) = 6.0 (±0.6) × 10(3) M(-1), K(DOPE-β-LG) = 6.5 (±0.7) × 10(3) M(-1), K(DDAB-β-LG) = 1.6 (±0.3) × 10(4) M(-1), and K(DOTAP-β-LG) = 2.2 (±0.67) × 10(4) M(-1). The number of lipid bound per protein molecule (n) was 0.8 (CHOL), 0.7 (DOPE), 1.0 (DDAB), and 1.3 (DOTAP). Molecular modeling showed the participation of several amino acid residues in lipid-protein complexation with the order of binding DOTAP > DDAB > DOPE > CHOL. Alterations of the protein conformation were observed in the presence of lipids with a minor decrease in β-sheet and an increase in turn structure.

Binding of vitamin A with milk α- and β-caseins

The binding sites of retinol and retinoic acid with milk α- and β-caseins were determined, using constant protein concentration and various retinoid contents. FTIR, UV-visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse retinol and retinoic acid binding sites, the binding constant and the effect of retinoid complexation on the stability and conformation of caseins. Structural analysis showed that retinoids bind caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(retinol-)(α)(-caseins)=1.21 (±0.4)×10(5) M(-1) and K(retinol-)(β)(-caseins)=1.11 (±0.5)×10(5) M(-1) and K(retinoic acid-)(α)(-caseins)=6.2 (±0.6)×10(4) M(-1) and K(retinoic acid-)(β)(-caseins)=6.3 (±0.6)×10(4) M(-1). The number of bound retinol molecules per protein (n) was 1.5 (±0.1) for α-casein and 1.0 (±0.1) for β-casein, while 1 molecule of retinoic acid was bound in the α- and β-casein complexes. Molecular modelling showed different binding sites for retinol and retinoic acid on α- and β-caseins with more stable complexes formed with α-casein. Retinoid-casein complexation induced minor alterations of protein conformation. Caseins might act as carriers for transportation of retinoids to target molecules.

The effect of milk alpha-casein on the antioxidant activity of tea polyphenols.

In this study, we report how the antioxidant capacities of major tea polyphenols are affected by their interactions with milk alpha-casein (milk protein) using three complimentary oxidation methods: ABTS(+) radical cation scavenging, cyclic voltammetry and lipid peroxidation inhibition. We found that using the ABTS(+) assays, the antioxidant activity of all polyphenols was lowered by 11-27% in the presence of caseins. Using cyclic voltammetry, the overall current measured at the electrode was decreased by the presence of the protein, from 21% to 61%. The peak potentials were also shifted to higher values varying from 13 to 41 mV. However, using lipid peroxidation method, we noticed of the antioxidant activity of all the polyphenols changed (from 6% up to 75%) after the addition of alpha-casein. The results show using this method the larger gallate esters containing polyphenols epicatechingallate (ECG) and (epigallocatechingallate (EGCG) were less affected by the presence of casein than smaller polyphenols catechins (C), epicatechin (EC) and epicgallocatechine (EGC). Alpha-casein caused a small effect on the chain breaking antioxidant capacity of theaflavins as well. Therefore, casein has different effects on the overall antioxidant capacities of tea compounds depending on the methods used. We aim to understand those results with the types of protein-polyphenol interactions that take place in various settings and their effects on the antioxidant capacities of those compounds.

Structural modeling for DNA binding to antioxidants resveratrol, genistein and curcumin.

Several models are presented here for the bindings of the antioxidant polyphenols resveratrol, genistein and curcumin with DNA in aqueous solution at physiological conditions. Multiple spectroscopic methods and molecular modeling were used to locate the binding sites of these polyphenols with DNA duplex. Structural models showed that intercalation is more stable for resveratrol and genistein than groove bindings, while curcumin interaction is via DNA grooves. Docking showed more stable complexes formed with resveratrol and genistein than curcumin with the free binding energies of -4.62 for resveratrol-DNA (intercalation), -4.28 for resveratrol-DNA (groove binding), -4.54 for genistein-DNA (intercalation), -4.38 for genistein-DNA (groove binding) and -3.84 kcal/mol for curcumin-DNA (groove binding). The free binding energies show polyphenol-DNA complexation is spontaneous at room temperature. At high polyphenol concentration a major DNA aggregation occurred, while biopolymer remained in B-family structure.