RNA-diethylstilbestrol interaction studied by Fourier transform infrared difference spectroscopy

Diethylstilbestrol (DES), a synthetic estrogen, is known to be a carcinogen in human and in animals. This study was designed to examine the interaction of DES with yeast RNA in aqueous solution at physiological pH with drug/RNA-phosphate (P) molar ratios of 1/80, 1/40, 1/20, 1/10, 1/4, and 1/2. Fourier transform infrared (FTIR) difference spectroscopy was used to determine the drug binding mode, the binding constant, the sequence selectivity, and RNA secondary structure in the RNA.DES complexes. Spectroscopic evidence showed that at low drug concentration (1/80 and 1/40), DES is intercalating through both Gua-Cyt and Ade-Urd base pairs with minor interaction with the backbone PO2 group (external binding). The calculated binding constant of K approximately 8.5 x 10(4) M-1 at a drug concentration of 3.12 x 10(-4) M shows that DES is a weaker intercalator than those of the methylene blue, acridine orange, and ethidium bromide. At high drug content (r > 1/40, where r represents the DES/RNA-phosphate molar ratio), a partial helix destabilization occurs with no alteration of RNA conformation upon drug complexation. However, a comparison with DNA.DES complexes showed that drug intercalation causes major reduction of the B-DNA structure in favor of A-DNA with no participation of the backbone PO2 group in the DES. DNA complexation.     

Effect of synthetic polymers on polymer–protein interaction

Synthetic polymers are often used for delivery of therapeutic drugs and proteins. We report the binding of milk β-lactoglobulin (β-LG) with poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) polyamidoamine (mPEG-PAMAM-G-3) and polyamidoamine (PAMAM-G4) nanoparticles in aqueous solution at pH 7.4, using Fourier Transform infrared (FTIR), circular dichroism (CD), fluorescence spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling. Structural analysis showed that polymers bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants K_{PEG-8000-β-LG} = 4.8 (±0.4) × 10⁴ M⁻¹ and K_{mPEG-PAMAM-G3-β-LG} = 5.8 (±0.6) × 10⁵ M⁻¹ and K_{PAMAM-G4-β-LG} = 6.7 (±0.9) × 10⁴ M⁻¹. The number of binding sites were occupied by polymers on protein (n) was 0.3 for PEG-8000, 0.4 for mPEG–PAMAM-G3 and 0.4 for PAMAM-G4. The order of binding is mPEG-PAMAM-G3 > PAMAM-G4 > PEG-8000. Transmission electron microscopy showed significant changes in protein morphology as polymer–protein complexation progressed with major increase in the diameter of the protein aggregate (180%). Furthermore, modeling showed several H-bonding systems between PEG and different amino acids stabilize polymer–β-LG complexes. mPEG-PAMAM-G3 is a stronger protein binder than PAMAM-G4 and PEG-8000.     

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) × 10⁵ M⁻¹ and K_{retinol-β-caseins} = 1.11 (±0.5) × 10⁵ M⁻¹ and K_{retinoic acid-α-caseins} = 6.2 (±0.6) × 10⁴ M⁻¹ and K_{retinoic acid-β-caseins} = 6.3 (±0.6) × 10⁴ 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.     

Interaction of milk α- and β-caseins with tea polyphenols

The interaction of α- and β-caseins with tea polyphenols (+)-catechin (C), (−)-epicatechin (EC), (−)-epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCG) was examined at a molecular level, using FTIR, UV–visible, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on casein stability and conformation were determined. Structural analysis showed that polyphenols bind casein via both hydrophilic and hydrophobic interactions with overall binding constants of K_C–α-cas = 1.8 (±0.8) × 10³ M⁻¹, K_EC–α-cas = 1.8 (±0.6) × 10³ M⁻¹, K_EGC–α-cas = 2.4 (±1.1) × 10³ M⁻¹ and K_{EGCG–α-cas} = 7.4 (±0.4) × 10³ M⁻¹, K_C–β-cas = 2.9 (±0.3) × 10³ M⁻¹, K_EC–β-cas = 2.5 (±0.6) × 10³ M⁻¹, K_EGC–β-cas = 3.5 (±0.7) × 10³ M⁻¹ and K_{EGCG–β-cas} = 1.59 (±0.2) × 10⁴ M⁻¹. The number of polyphenol bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 1.1 (EGC), 1.5 (EGCG) for α-casien and 1.0 (C), 1.0 (EC), 1.1 (EGC) and 1.5 (EGCG) for β-casein. Structural modelling showed the participation of several amino acid residues in polyphenol–protein complexation with extended H-bonding network. Casein conformation was altered by polyphenol with a major reduction of α-helix and β-sheet and increase of random coil and turn structure suggesting further protein unfolding. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.     

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 K_C–β-LG = 2.2 (±0.8) × 10³ M⁻¹, K_EC–β-LG = 3.2 (±1) × 10³ M⁻¹, K_ECG–β-LG = 1.1 (±0.6) × 10⁴ M⁻¹ and K_EGCG–β-LG = 1.3 (±0.8) × 10⁴ 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 K_{folic acid–HSA} = 8.1 (±0.5) × 10⁴ M⁻¹ and K_{folic acid–BSA} = 1.0 (±0.3) × 10⁵ 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.     

RNA-ascorbate interaction

Ascorbic acid and divalent iron salts have been widely used to investigate the effects of reactive oxygen species in different biological targets such as nucleic acids, proteins and lipids. This study was designed to examine the interaction of yeast RNA with vitamin C in aqueous solution at physiological pH with drug/RNA(P)(P=phosphate) molar ratios of r=1/80, 1/40, 1/20, 1/10, 1/4 and 1/2. Absorption spectra and Fourier transform infrared (FTIR) difference spectroscopy were used to determine the ascorbate binding mode, binding constant, sequence selectivity and RNA secondary structure in aqueous solution. Spectroscopic evidence showed that at low drug concentration (r=1/80 and 1/40), no major ascorbate-RNA interaction occurs, while at higher drug concentrations (r>1/40), a major drug-RNA complexation was observed through both G-C and A-U base pairs and the backbone phosphate groups with k=31.80 M(-1). Evidence for this comes from large perturbations of the G-C vibrations at 1698 and 1488 cm(-1) and the A-U bands at 1654 and 1608 cm(-1) as well as the phosphate antisymmetric stretch at 1244 cm(-1). At r>1/10, minor structural changes occur for the ribose-phosphate backbone geometry with RNA remaining in the A-family structure. The drug distributions around double helix were about 55% with G-C, 33% A-U and 12% with PO2 groups. A comparison between ascorbate-RNA and ascorbate-DNA complexes showed minor differences. The ascorbate binding (H-bonding) is via anion CO and OH groups.     

Dual effect of milk on the antioxidant capacity of green,Darjeeling, and English breakfast teas

Past studies present contradictory results regarding the effect of milk on the antioxidant capacities of teas, possibly because of the different methods used. Here, we re-address the question by using three complementary assays, ABTS⁺ free radical scavenging, voltammetry, and lipid peroxidation inhibition, to estimate how milk affects the antioxidant capacities of green, Darjeeling, and English breakfast teas. We observed that milk decreased the antioxidant capacities of Darjeeling (−8.3%), green (−6.0%), and English breakfast (−19.6%) teas, estimated with the ABTS⁺ method. These inhibitions were four times larger using voltammetry. In contrast, milk enhanced the chain-breaking antioxidant capacity of teas in the lipid peroxidation method by 19%, 12%, and 10%, for green, English breakfast, and Darjeeling teas, respectively. Therefore, milk can have dual effects on the tea antioxidant capacity, an inhibitory effect for reactions occurring in solution or at a solid–liquid interface and an enhancing effect for those in oil-in-water emulsion. The mechanisms responsible for these different milk–tea interactions are discussed.