Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time T_M for HSA labeled with OX063 is 6.3 μs at 50 K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.     

In‐Situ Spin Labeling of His‐Tagged Proteins: Distance Measurements under In‐Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin‐labeled chemical recognition unit for switchable and concomitantly high affinity binding to His‐tagged proteins was synthesized. In combination with an orthogonal site‐directed spin label, this novel spin probe, Proxyl‐trisNTA (P‐trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate‐binding protein, 2) its substrate‐dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site‐specific spin labeling in cell lysates under in‐cell conditions. This approach will open new avenues towards in‐cell EPR.     

Association of Valdecoxib,a Nonsteroidal Anti‐Inflammatory Drug,with Human Serum Albumin

Valdecoxib addition quenches the intrinsic human serum albumin (HSA) fluorescence. This allows an evaluation of the drug–protein association. However, both the number of binding sites and their affinity for the drug depend upon the methodology employed for their evaluation and the employed protein concentration. In this work, we measured the effect of valdecoxib on HSA fluorescence yield over a wide range of experimental conditions and discuss the validity of the binding parameters derived from the different data treatments: Stern–Volmer, Scatchard, double logarithmic, quadratic equation, Benesi–Hilderand, and Encinas–Lissi. It is proposed that a combination of Encinas–Lissi and Scatchard treatments of the data renders the most reliable results. From these data, it is concluded that HSA presents three high‐affinity binding sites for valdecoxib (K_as = 4.5 × 10⁴ m ^?1) and several secondary sites of smaller activity.     

Identification and Characterization of a Single High‐Affinity Fatty Acid Binding Site in Human Serum Albumin

A single high‐affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐C₁₂ fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site‐specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA‐binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow‐sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X‐ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug‐protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.     

Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with Cu^II spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent Cu^II coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (K_D) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm Cu^II K_Ds under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled Cu^II–nitroxide systems using a commercial Q‐band EPR instrument.     

Ibuprofen induces an allosteric conformational transition in the heme complex of human serum albumin with significant effects on heme ligation

Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.     

Binding of Organic Dyes with Human Serum Albumin: A Single‐Molecule Study

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single‐molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 ( C153 , neutral) and rhodamine 6G ( R6G , cationic) were chosen. For both the dyes, a major (ca. 96‐98 %) and minor (ca. 3 %) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ_off and τ_on) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst‐integrated lifetime analysis. Distributions of lifetime histograms for both the probes ( C153 and R6G ) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153 . However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.     

Phase Separation of Poly(N‐isopropylacrylamide) in Mixtures of Water and Methanol: A Spectroscopic Study of the Phase‐Transition Process with a Polymer Tagged with a Fluorescent Dye and a Spin Label

The thermoreversible phase transition of poly(N‐isopropylacrylamide) randomly labeled with a spin label, 4‐amino‐2,2′,6,6′‐tetramethylpiperidine 1‐oxide (TEMPO), and a fluorescent dye, 4‐(pyren‐1‐yl)butyl (PNIPAM‐Py‐T), in different H₂O/MeOH mixtures was studied by turbidimetry, continuous‐wave electron paramagnetic resonance spectroscopy (CW‐EPR), and fluorescence spectroscopy. The macroscopic phase diagram of PNIPAM‐Py‐T in H₂O/MeOH measured by turbidimetry was identical to those of poly(N‐isopropylacrylamide) (PNIPAM) and of TEMPO‐labeled PNIPAM (PNIPAM‐T) in H₂O/MeOH mixtures. However, distinct differences among the three polymers were detected in their solvent‐dependent EPR and fluorescence‐spectroscopic properties. The EPR spectra were analyzed in terms of the isotropic hyperfine coupling constants, which monitor the variation in environmental polarity of the radical labels occurring for the conformational transitions of the polymer as a function of temperature, as well as the correlation time for reorientation motion, the increase of which is indicative of the increased viscosity of the radical environment and interactions occurring between the radical and other surface groups of the precipitated polymer, if compared to the soluble polymer. The fluorescence of Py in PNIPAM‐Py‐T displayed contributions from isolated excited pyrenes (monomer emission) and from preformed pyrene ground‐state aggregates (excimer emission). The quantum efficiencies of monomer and excimer emission were monitored as a function of solvent composition. By the two experimental approaches, we demonstrate the profound influence of the PNIPAM‐attached pyrene units in increasing the hydrophobicity of the nanodomains formed upon heat‐induced precipitation of PNIPAM‐Py‐T.     

Studies of Interaction between Ergosta‐4,6,8(14),22‐tetraen‐3‐one (Ergone) and Human Serum Albumin by Molecular Spectroscopy and Modeling

Our previous experimental results have shown that ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) is one of the main bioactive components of Polyporus umbellatus. The efficacy of ergone binding to human serum albumin (HSA) is critical for pharmacokinetic behavior of ergone. The interactions between ergone and HSA under simulative physiological conditions were investigated by the methods of fluorescence spectroscopy, absorption and circular dichroism spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by ergone was the result of the formation of the ergone‐HSA complex. According to the modified Stern‐Volmer equation, the binding constants (K_a) between ergone and HSA were determined. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be 0.989 kJ mol^‐1 and 11.214 J mol^‐1 K^‐1, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding of ergone to HSA. The conformational investigation showed that the presence of ergone decreased the α‐helical content of HSA and induced the slight unfolding of the polypeptides of protein. Furthermore, displacement experiments using warfarin and ibuprofen indicated that ergone could bind to site I of HSA, which was also in agreement with the results of the molecular modeling.     

Spin‐Labeled Heparins as Polarizing Agents for Dynamic Nuclear Polarization

A potentially biocompatible class of spin‐labeled macromolecules, spin‐labeled (SL) heparins, and their use as nuclear magnetic resonance (NMR) signal enhancers are introduced. The signal enhancement is achieved through Overhauser‐type dynamic nuclear polarization (DNP). All presented SL‐heparins show high ¹H DNP enhancement factors up to E=?110, which validates that effectively more than one hyperfine line can be saturated even for spin‐labeled polarizing agents. The parameters for the Overhauser‐type DNP are determined and discussed. A striking result is that for spin‐labeled heparins, the off‐resonant electron paramagnetic resonance (EPR) hyperfine lines contribute a non‐negligible part to the total saturation, even in the absence of Heisenberg spin exchange (HSE) and electron spin‐nuclear spin relaxation (T_1ne). As a result, we conclude that one can optimize the use of, for example, biomacromolecules for DNP, for which only small sample amounts are available, by using heterogeneously distributed radicals attached to the molecule.     

Kinetic and thermodynamic analysis of 9‐nitrocamptothecin hydrolysis at physiological pH in the presence and absence of human serum albumin

Kinetic and thermodynamic analysis of the 9‐nitrocamptothecin (9NC) hydrolysis reaction in the presence and absence of human serum albumin (HSA) in phosphate‐buffered saline (PBS) of pH 7.4 was carried out by first derivative absorption spectroscopy. The thermodynamic parameters determined in these studies provided a mechanistic explanation toward the endothermic but yet thermodynamically favorable hydrolysis of 9NC at physiological temperature and pH. In the presence of HSA, the apparent rate constant of 9NC hydrolysis was 3–3.5 times higher than in 9NC solutions alone, whereas the apparent equilibrium constant of 9NC hydrolysis was found to increase at a higher extent in the presence of HSA than in PBS with increasing temperature, reaching almost complete hydrolysis of the 9NC to the 9NC‐carboxylate at 315.15 K. Importantly, the E_a of the 9NC hydrolysis reaction in the presence of HSA was determined to be on average 17 kJ mol^?1 lower than the E_a determined in plain PBS. Moreover, analysis of binding isotherms constructed for the HSA interaction with 9NC, using infinitely cooperative and independent binding models, demonstrated an incredibly higher binding constant for the 9NC‐carboxylate form as compared to the very weak and concentration‐dependent binding for the 9NC‐lactone species at 310.15 K. Taken together, the preferential association of the carboxylate form with HSA and the lower E_a of 9NC hydrolysis in the presence of HSA provide a mechanistic explanation for the shift of lactone–carboxylate equilibria toward the carboxylate product under physiological conditions of pH and ionic strength. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 693–703, 2010     

Degree of Complexation of Anticoagulant Rodenticides with Human Serum Albumin: Effect of Saccharose and Sodium

A theoretical treatment has been developed to describe association between rodenticides and human serum albumin (HSA). The degree of complexation n_c (the amount (%) of complexed guest) and the association constant (K) for rodenticide–HSA binding were determined. Enthalpy–entropy compensation was also investigated in relation to this mathematical model to confirm rodenticide binding behavior with HSA. The role of the sodium cation and saccharose in this association were also analyzed. As expected for a salting-out agent, addition of Na⁺ cation to the mobile phase led to enhancement of n_c and K values by increasing the hydrophobic effect. When saccharose was increased above a critical x_c value equal to 4 mM, a decrease of the association constant and the degree of complexation was observed, owing to reduction of the radius of curvature of the HSA cavity.     

High-performance affinity chromatography and the analysis of drug interactions with modified proteins: binding of gliclazide with glycated human serum albumin

This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (K _a) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high-affinity sites (average K _a, 7.1–10 × 10⁴ M⁻¹) and a group of lower-affinity sites (average K _a, 5.7–8.9 × 10³ M⁻¹) at pH 7.4 and 37 °C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the K _a values for gliclazide at these sites being 1.9 × 10⁴ and 6.0 × 10⁴ M⁻¹, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification.     

Binding of caffeic acid to human serum albumin by the retention data and frontal analysis

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high‐performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 10⁴/m . The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm . The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug–protein interaction. The proposed model also has the advantages of ligand saving and rapid operation. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis

In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA–HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated β‐cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K₁), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K₁ values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction of Human Serum Albumin with Indomethacin: Spectroscopic and Molecular Modeling Studies

In this work, the interaction between indomethacin (IM) and human serum albumin (HSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling. The experiment results showed that the fluorescence quenching of HSA by IM was a result of the formation of an IM–HSA complex and the corresponding association constants (K _a) between IM and HSA at four different temperatures were determined according to the modified Stern–Volmer equation. The resulting thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicate that the hydrophobic force plays a major role for IM–HSA association, but hydrogen bonds also could not be excluded. A molecular modeling study further confirmed the binding mode and indicated that the binding of IM to HSA primarily takes place in sub-domain IIA (site I). The conformational investigation showed that the presence of IM decreased the α-helical content of HSA and induced slight unfolding of the polypeptides of protein, which confirmed that some microenvironmental and conformational changes occur for HSA molecules.     

Binding equilibrium study between Mn( Ⅱ ) and HSA or BSA

The binding of Mn( Ⅱ ) to human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by equilibrium dialysis at physiological pH (7. 43). The Scatchard analysis indicates that there are 1.8 and 1.9 strong binding sites of Mn( Ⅱ ) in HSA and BSA, respectively. The successive stability constants which are reported for the first time are obtained by non-linear least-squares methods fitting Bjerrum formula. For both Mn( Ⅱ )-HSA and Mn( Ⅱ )-BSA systems, the order of magnitude of K1 was found to be 104. The analyses of Hill plots and free energy coupling show that the positive cooperative effect was found in both Mn( Ⅱ )-HSA and Mn( Ⅱ )-BSA systems . The results of Mn ( Ⅱ ) competing with Cu ( Ⅱ ) 、 Zn(Ⅱ)、Cd( Ⅱ) or Ca( Ⅱ ) to bind to HSA or BSA further support the conjecture that there are two strong binding sites of Mn( Ⅱ) in both HSA and BSA. One is most probably located at the tripeptide segment of N- terminal sequence of HSA and BSA molecules involving four groups composed of n     

Tuning Human Serum Albumin (HSA) Hydrogels through Albumin Glycation

The changes of technological properties of albumin-based hydrogels induced by increasing degrees of post-translational modification of the protein are reported. Maillard-type modification of amino acids arginine and lysine of albumin is achieved through glyoxal as an α-dicarbonyl compound. The degrees of modification are fine-tuned using different molar ratios of glyoxal. Hydrogels are thermally induced by heating highly concentrated precursor solutions above the protein's denaturation temperature. While the post-translational modifications are determined and quantified with mass spectrometry, continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy shed light on the protein fatty acid binding capacity and changes thereof in solution and in the gel state. The viscoelastic behavior is characterized as a measure of the physical strength of the hydrogels. On the nanoscopic level, the modified albumins in low concentration solution reveal lower binding capacities with increasing degrees of modification. On the contrary, in the gel state, the binding capacity remains constant at all degrees of modifications. This indicates that the loss of fatty acid binding capacity for individual albumin molecules is partially compensated by new binding sites in the gel state, potentially formed by modified amino acids. Such, albumin glycation offers a fine-tuning method of technological and nanoscopic properties of these gels.     

Fluorescence spectroscopic studies on binding of a flavonoid antioxidant quercetin to serum albumins

Binding of quercetin to human serum albumin (HSA) was studied and the binding constant measured by following the red-shifted absorption spectrum of quercetin in the presence of HSA and the quenching of the intrinsic protein fluorescence in the presence of different concentrations of quercetin. Fluorescence lifetime measurements of HSA showed decrease in the average lifetimes indicating binding at a location, near the tryptophan moiety, and the possibility of fluorescence energy transfer between excited tryptophan and quercetin. Critical transfer distance (R _o ) was determined, from which the mean distance between tryptophan-214 in HSA and quercetin was calculated. The above studies were also carried out with bovine serum albumin (BSA).     

Characterization of Interactions Between Fluoroquinolones and Human Serum Albumin by CE–Frontal Analysis

The binding of fluoroquinolones to the transport protein, human serum albumin (HSA), under simulated physiological conditions has been studied by capillary electrophoresis–frontal analysis (CE–FA). The binding of these drugs to human plasma was evaluated by using ultrafiltration and capillary electrophoresis. The free drug concentration [D]_f at each HSA concentration was determined by the plateau height in the electropherograms and the calibration lines. The binding constants of fluoroquinolones and HSA were estimated using nonlinear regression with origin 7.5 software. The fluoroquinolones were found to show low affinity toward HSA, with binding constants ranging from 1.73 × 10² to 5.40 × 10² M⁻¹. The percentages of protein binding (PB) for fluoroquinolones to HSA were between 8.6 and 22.2%, while the PB percentages for fluoroquinolones to human plasma were between 10.2 and 33.1%. It can be found that the PB percentages for fluoroquinolones to HSA are mostly lower than those for fluoroquinolones to human plasma. It suggests that HSA is the primary protein responsible for the binding of fluoroquinolones in human plasma. The thermodynamic parameters were obtained by CE–FA. The positive ∆H and ∆S values obtained by CE–FA showed that the binding reaction was an endothermic process, and the entropy drive the binding and hydrophobic interaction played major roles in the binding of fluoroquinolones to HSA.