The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent–tryptophan interaction

Background

The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding.

Methods

Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy.

Results

Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations < 10 nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations.

General significance

Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1–10 nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.     

Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 to 460-470nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements.     

Formation of a wrapped DNA-protein interface: experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H' DNA

To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34-base pair (bp) H′ DNA sequence to the Escherichia coli DNA-remodeling protein integration host factor (IHF). Isothermal titration calorimetry and fluorescence resonance energy transfer are applied to determine effects of salt concentration [KCl, KF, K glutamate (KGlu)] and of the excluded solute glycine betaine (GB) on the binding thermodynamics at 20 °C. Both the binding constant K_obs and enthalpy ΔH°_obs depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., ΔH^o_obs = − 20.2 kcal·mol^− 1 in 0.04 M KCl). ΔH°_obs increases linearly with [salt] with a slope (dΔH°_obs/d[salt]), which is much larger in KCl (38 ± 3 kcal·mol^− 1 M^− 1) than in KF or KGlu (11 ± 2 kcal·mol^− 1 M^− 1). At 0.33 M [salt], K_obs is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SK_obs = dlnK_obs/dln[salt] is almost twice as large in magnitude in KCl (− 8.8 ± 0.7) as in KF or KGlu (− 4.7 ± 0.6).A novel analysis of the large effects of anion identity on K_obs, SK_obs and on ΔH°_obs dissects coulombic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K_obs, SK_obs, and ΔH°_obs to (i) displacement of a large number of water molecules of hydration [estimated to be 1.0(± 0.2) × 10³] from the 5340 Å² of IHF and H′ DNA surface buried in complex formation, and (ii) significant local exclusion of F⁻ and Glu⁻ from this hydration water, relative to the situation with Cl⁻, which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of GB on K_obs: dlnK_obs/d[GB]  = 2.7 ± 0.4 at constant KCl activity, indicating the net release of ca. 150 H₂O molecules from anionic surface.     

Effect of the phosphate substrate on drug-inhibitor binding to human purine nucleoside phosphorylase

The thermodynamics of the drug-inhibitors acyclovir, ganciclovir, and 9-benzylguanine binding to human purine nucleoside phosphorylase (hsPNP) were determined from isothermal titration calorimetry as a function of the substrate phosphate ion (Pi) concentration from 0 to 0.125 M and temperature from 15 °C to 35 °C. At 25 °C and with an increase in the Pi concentration from 0 to 50 mM, acyclovir binding becomes more entropically-driven and ganciclovir binding becomes more enthalpically-driven. At 25 °C, the tighter 9-benzylguanine binding reaction goes from an enthalpically-driven reaction in the absence of Pi to an entropically-driven reaction at 10 mM Pi, and the enthalpically-driven nature of the binding reaction is restored at 75 mM Pi. Since the dependencies of the driving-nature of the binding reactions on Pi concentration can be simulated by Pi binding to its catalytic site, it is believed that bound Pi affects the interactions of the side-chains with the ribose catalytic site. However, the binding constants are unaffected by change in the bound Pi concentration because of enthalpy-entropy compensation. The enzymatic activity of hsPNP was determined by an ITC-based assay employing 7-methylguanosine and Pi as the substrates. The heat of reaction determined from the assay increased by 7.5 kJ mol⁻¹ with increase in Pi concentration from 50 to 100 mM and is attributed to weak binding of the Pi to a secondary regulatory site. Although the binding constants of acyclovir and ganciclovir at 20 μM hsPNP were in agreement with the inverse inhibition constants determined from the ITC enzyme inhibition assays at 60 nM, the binding constant of 9-benzylguanine, which interacts with Phe159 from an adjacent subunit, decreased from 5.62 × 10⁵ M⁻¹ to 1.14 × 10⁵ M⁻¹. This reduction in the 9-benzylguanine binding affinity along with a 7-fold increase in the specific activity of hsPNP at 14.5 nM results from partial dissociation of the hsPNP trimer into monomers below the 60 nM level.     

Effect of the distal C162S mutation on the energetics of drug binding to p38alpha MAP kinase

The binding reactions of the inhibitor drugs, SB 203580, SKF 86002, and p38 INH.1 to the isoforms 1 and 2 splice variants of p38α MAP kinase and their C162S mutants, as determined from ITC measurements from 25 to 35 °C, are totally enthalpically driven with binding constants ranging from 10⁷ M⁻¹ for SKF 86002 and SB 203580 to 10⁹ M⁻¹ for p38 INH.1. Interactions of p38 INH.1 with an additional hydrophobic pocket of the kinase would account for its large increase in K_b. DSC scans exhibited single unfolding transitions for the isoforms, their mutants, and the mutants bound to the drug inhibitors. Two transitions, however, were observed for the isoform-drug complexes of SB 203580 and p38 INH.1 and were attributed to decoupled unfolding of the N- and C-terminal domains of the kinase. The C-terminal domain of isoform 1 is estimated to be less stable than of isoform 2 by 15 kJ mol⁻¹.     

The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is a 2-Cys peroxiredoxin extremely abundant in the erythrocyte. The peroxidase activity was studied in a steady-state approach yielding an apparent K_M of 2.4 μM for human thioredoxin and a very low K_M for H₂O₂ (?0.7 μM). Rate constants for the reaction of peroxidatic cysteine with the peroxide substrate, H₂O₂ or peroxynitrite, were determined by competition kinetics, k₂ = 1.0 × 10⁸ and 1.4 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.4, respectively. Excess of both oxidants inactivated the enzyme by overoxidation and also tyrosine nitration and dityrosine were observed with peroxynitrite treatment. Prx2 associates into decamers (5 homodimers) and we estimated a dissociation constant K_d < 10⁻²³ M⁴ which confirms the enzyme exists as a decamer in vivo. Our kinetic results indicate Prx2 is a key antioxidant enzyme for the erythrocyte and reveal red blood cells as active oxidant scrubbers in the bloodstream.     

Frataxin from Psychromonas ingrahamii as a model to study stability modulation within the CyaY protein family

Adaptation of life to low temperatures influences both protein stability and flexibility. Thus, proteins from psychrophilic organisms are excellent models to study relations between these properties. Here we focused on frataxin from Psychromonas ingrahamii (pFXN), an extreme psychrophilic sea ice bacterium that can grow at temperatures as low as − 12 °C. This α/β protein is highly conserved and plays a key role in iron homeostasis as an iron chaperone. In contrast to other frataxin homologs, chemical and temperature unfolding experiments showed that the thermodynamic stability of pFXN is strongly modulated by pHs: ranging from 5.5 ± 0.9 (pH 6.0) to 0.9 ± 0.3 kcal mol^− 1 (pH 8.0). This protein was crystallized and its X-ray structure solved at 1.45 Å. Comparison of B-factor profiles between Escherichia coli and P. ingrahamii frataxin variants (51% of identity) suggests that, although both proteins share the same structural features, their flexibility distribution is different. Molecular dynamics simulations showed that protonation of His44 or His67 in pFXN lowers the mobility of regions encompassing residues 20–30 and the C-terminal end, probably through favorable electrostatic interactions with residues Asp27, Glu42 and Glu99. Since the C-terminal end of the protein is critical for the stabilization of the frataxin fold, the predictions presented may be reporting on the microscopic origin of the decrease in global stability produced near neutral pH in the psychrophilic variant. We propose that suboptimal electrostatic interactions may have been an evolutionary strategy for the adaptation of frataxin flexibility and function to cold environments.     

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308 307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from −14.0 to −17.09 kcal mol⁻¹ were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CL^pro. Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CL^pro expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC₅₀ ranged from 38.57 ± 2.41 to 101.38 ± 3.27 μM. Two strong 3CL^pro inhibitors were further identified as competitive inhibitors of 3CL^pro with K_i values of 9.11 ± 1.6 and 9.93 ± 0.44 μM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL^pro were also identified.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

2-Aminopurine/cytosine base pair containing oligonucleotides: fluorescence spectroscopy studies on DNA-polyamide binding

Studies on the binding of a triamide f-IPI (1) to its cognate sequence labeled with a 2-aminopurine (2AP or G^∗) group are described. ITC studies showed that f-IPI (1) bound to the cognate site (ACG^∗CGT) with only 3.5-fold lower affinity than binding to the unlabeled DNA (ACGCGT) (K_eq = 2 × 10⁷ and 7 × 10⁷ M⁻¹, respectively). Titration of f-IPI (1) to both sequences gave strong induced bands at 330 nm via circular dichroism studies. The compound also gave comparable ΔT_m values of 5.0 and 7.8 °C, respectively. These techniques also proved that the sequence selectivity of f-IPI (1) was uncompromised, as only limited binding to the non-cognate sequence ACCG^∗GT was observed. Fluorescence studies demonstrated a 2:1 ligand:DNA binding motif as anticipated, and indicated that the limit of detection for this technique was 20 μM DNA concentration. The results demonstrate that 2-aminopurine is a sufficient substitute for guanine in a G·C base pair useful in DNA binding studies.     

Heme impairs allosterically drug binding to human serum albumin Sudlow's site I

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand (e.g., heme and drugs) binding capacity. Here, the binding of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, and tolbutamide to HSA and ferric heme-HSA is reported. Moreover, ferric heme binding to HSA in the absence and presence of drugs has been investigated. Values of the association equilibrium constant for drug binding to Sudlow’s site I of ferric heme-HSA (ranging between 1.7 × 10³ and 1.6 × 10⁵ M⁻¹) are lower by one order of magnitude than those for drug binding to ferric heme-free HSA (ranging between 1.9 × 10⁴ and 1.8 × 10⁶ M⁻¹). According to linked functions, the value of the association equilibrium constant for heme binding to HSA decreases from 7.8 × 10⁷ M⁻¹, in the absence of drugs to 7.0 × 10⁶ M⁻¹, in the presence of drugs. These findings represent a clear-cut evidence for the allosteric inhibition of drug binding to HSA Sudlow’s site I by the heme. According to linked functions, drugs impair allosterically heme binding to HSA. These results appear to be relevant in the drug therapy and management.     

Substituent effect on the preferred DNA binding mode and affinity of a homologous series of naphthalene diimides

A combination of isothermal titration calorimetry (ITC), topoisomerase I DNA unwinding assays, and ethidium bromide displacement studies were employed to investigate the binding of a homologous series of naphthalene diimides (NDI) to DNA. Our results suggest that the nature of the substituent plays a significant role in both the preferred binding mode and relative binding affinity of the compounds of this study. Only intercalative-type binding (K = 15 ± 3 × 10⁶ M⁻¹) was observed for the NDI with the smallest substituent (trimethyl-ethylamino), while larger members of the series (diethylmethyl-, dipropylmethyl- and dibutylmethyl-ethylamino substituents) adopted an additional binding mode of higher affinity (K₁ = 31 − 78 × 10⁶ M⁻¹).     

The Contribution of a Covalently Bound Cofactor to the Folding and Thermodynamic Stability of an Integral Membrane Protein

The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol^− 1 to 23.4 ± 1.5 kcal mol^− 1 at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol^− 1 towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol^− 1 to 10.9 ± 1.0 kcal mol^− 1, indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3-6 kcal mol^− 1. Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k_u^H2O, from ~1 × 10^− 16 s^− 1 to ~1 × 10^− 4 s^− 1 at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol^− 1 in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein-retinal interactions that increase the rigidity and compactness of the polypeptide chain.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.     

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant K_A = 7.0(± 0.8) × 10⁴ M^− 1. Taken together, the FAD–protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase

F₁-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F₁-ATPase over the temperature range 4-50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 10⁶ M⁻¹ s⁻¹ at 4°C to 4.3 × 10⁷ M⁻¹ s⁻¹ at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4-50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65°C. F₁-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.     

Studies on the interaction of copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate with calf thymus DNA

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.     

Characterization of the binding of sulfonylurea drugs to HSA by high-performance affinity chromatography

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (K_a) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average K_a values of 1.3 (±0.2) × 10⁵ and 3.5 (±3.0) × 10² M⁻¹ for acetohexamide and values of 8.7 (±0.6) × 10⁴ and 8.1 (±1.7) × 10³ M⁻¹ for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with K_a values of 1.3 (±0.1) × 10⁵ and 4.3 (±0.3) × 10⁴ M⁻¹, respectively, at 37 °C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with K_a values of 5.5 (±0.2) × 10⁴ and 5.3 (±0.2) × 10⁴ M⁻¹, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug–protein interactions.