Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ～threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.     

Synchrotron radiation based STXM analysis and micro-XRF mapping of differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on Fe and S

The differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on substrates Fe^2 + and S⁰ was investigated by using synchrotron radiation based scanning transmission X-ray microscopy (STXM) imaging and microbeam X-ray fluorescence (μ-XRF) mapping. The extracellular thiol groups (SH) were first alkylated by iodoacetic acid forming Protein-SCH₂COOH and then the P-SCH₂COOH was marked by calcium ions forming P-SCH₂COOCa. The STXM imaging and μ-XRF mapping of SH were based on analysis of SCH₂COO-bonded Ca^2 +. The results indicated that the thiol group content of A. ferrooxidans grown on S⁰ is 3.88 times to that on Fe^2 +. Combined with selective labeling of SH by Ca^2 +, the STXM imaging and μ-XRF mapping provided an in situ and rapid analysis of differential expression of extracellular thiol groups.     

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.     

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k_off) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k_off value increases from (1.4 ± 0.2) × 10⁻⁴ s⁻¹, in the absence of the drug, to (9.5 ± 1.2) × 10⁻³ s⁻¹, in the presence of 1.0 × 10⁻² M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K₁ = (3.1 ± 0.4) × 10⁻⁷ M, K₂ = (1.7 ± 0.2) × 10⁻⁴ M, and K₃ = (2.2 ± 0.2) × 10⁻³ M) were determined. The K₃ value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10⁻³ M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.     

Effect of Pullulan Nanoparticle Surface Charges on HSA Complexation and Drug Release Behavior of HSA-Bound Nanoparticles

Nanoparticle (NP) compositions such as hydrophobicity and surface charge are vital to determine the presence and amount of human serum albumin (HSA) binding. The HSA binding influences drug release, biocompatibility, biodistribution, and intercellular trafficking of nanoparticles (NPs). Here, we prepared 2 kinds of nanomaterials to investigate HSA binding and evaluated drug release of HSA-bound NPs. Polysaccharides (pullulan) carboxyethylated to provide ionic derivatives were then conjugated to cholesterol groups to obtain cholesterol-modified carboxyethyl pullulan (CHCP). Cholesterol-modified pullulan (CHP) conjugate was synthesized with a similar degree of substitution of cholesterol moiety to CHCP. CHCP formed self-aggregated NPs in aqueous solution with a spherical structure and zeta potential of −19.9±0.23 mV, in contrast to −1.21±0.12 mV of CHP NPs. NPs could quench albumin fluorescence intensity with maximum emission intensity gradually decreasing up to a plateau at 9 to 12 h. Binding constants were 1.12×10⁵ M⁻¹ and 0.70×10⁵ M⁻¹ to CHP and CHCP, respectively, as determined by Stern-Volmer analysis. The complexation between HSA and NPs was a gradual process driven by hydrophobic force and inhibited by NP surface charge and shell-core structure. HSA conformation was altered by NPs with reduction of α-helical content, depending on interaction time and particle surface charges. These NPs could represent a sustained release carrier for mitoxantrone in vitro, and the bound HSA assisted in enhancing sustained drug release.     

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.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Bacterial cellulose nanofibers for albumin depletion from human serum

Cibacron Blue F3GA (CB) was covalently attached onto the bacterial cellulose (BC) nanofibers for human serum albumin (HSA) depletion from human serum. The BC nanofibers were produced by Acetobacter xylinum in the Hestrin–Schramm medium in a static condition for 14 days. The CB content of the BC nanofibers was 178 μmol/g. The specific surface area of the BC nanofibers was determined to be 914 m²/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA on the BC nanofibers was very low (1.4 mg/g polymer). CB attachment onto the BC nanofibers significantly increased the HSA adsorption (1800 mg/g). The maximum HSA adsorption was observed at pH 5.0. The HSA adsorption capacity decreased drastically with an increase of the aqueous phase concentration of sodium chloride. The elution studies were performed by adding 1 M NaCl to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the binding of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 96.5% for all studied concentrations. Proteins in the serum and eluted portion were analyzed by SDS-PAGE for testing the efficiency of HSA depletion from human serum. Eluted proteins include mainly HSA.     

Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal

Methylglyoxal (MG), a reactive α-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by ¹H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys > GSH > NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 ± 0.9%; 38.2 ± 0.7% and 39.0 ± 0.8% for Cys; 26.5 ± 0.6%; 26.6 ± 2.6% and 27.4 ± 2.5% for GSH; 10.8 ± 0.9%; and 11.2 ± 0.7% and 12.2 ± 0.9% for NAcCys, respectively. Our results explain why substances containing α-amino-β-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.     

Comparative serum albumin interactions and antitumor effects of Au(III) and Ga(III) ions

In the present study, interactions of Au(III) and Ga(III) ions on human serum albumin (HSA) were studied comparatively via spectroscopic and thermal analysis methods: UV–vis absorbance spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and isothermal titration calorimetry (ITC). The potential antitumor effects of these ions were studied on MCF-7 cells via Alamar blue assay. It was found that both Au(III) and Ga(III) ions can interact with HSA, however; Au(III) ions interact with HSA more favorably and with a higher affinity. FT-IR second derivative analysis results demonstrated that, high concentrations of both metal ions led to a considerable decrease in the α-helix content of HSA; while Au(III) led to around 5% of decrease in the α-helix content at 200 μM, it was around 1% for Ga(III) at the same concentration. Calorimetric analysis gave the binding kinetics of metal–HSA interactions; while the binding affinity (K_a) of Au(III)–HSA binding was around 3.87 × 10⁵ M⁻¹, it was around 9.68 × 10³ M⁻¹ for Ga(III)–HSA binding. Spectroscopy studies overall suggest that both metal ions have significant effects on the chemical structure of HSA, including the secondary structure alterations. Antitumor activity studies on MCF7 tumor cell line with both metal ions revealed that, Au(III) ions have a higher antiproliferative activity compared to Ga(III) ions.     

Effect of cadmium on fatty acid composition of lipids in the shoots of Karelian birch cultured in vitro

The effects of cadmium (10^?6, 10^?5, 10^?4, and 10^?3 M) on the growth and development of stem apical meristem and fatty acid (FA) composition of particular lipid fractions (neutral lipids, glyco-, and phospholipids) were investigated in the shoots of Karelian birch (Betula pendula var. carelica (Mercklin) Hämet-Ahti) produced in vitro. Low cadmium concentration (10^?6 M) slightly stimulated shoot morphogenesis. However, at the concentration of 10^?4 M, shoot growth and development was terminated, and the concentration of 10^?3 M turned out to be critical (the meristem died within the first 5 days). The presence of Cd in the medium considerably affected FA composition in the shoots. The most pronounced changes occurred in the fraction of glycolipids, and the extent of adverse influence significantly depended on metal concentration in the medium. At the Cd concentration of 10^?4 M, the content of di-and trienoic FAs decreased by 3–4 times, whereas the level of monoenoic FAs rose by 5 times. The revealed differences in the FA composition of lipids in birch were considered as an indicator of adverse effect of cadmium on the structure and functions of chloroplasts and therefore on photosynthesis.     

Binding of antitumor tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen to human serum albumin

Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. The aim of this study was to examine the interaction of human serum albumin (HSA) with tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen at physiological conditions, using constant protein concentration and various drug contents. FTIR, UV-Visible, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding mode, the binding constant and the effects of drug complexation on HSA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bound HSA via both hydrophobic and hydrophilic interactions with overall binding constants of K_tam = 1.8 (±0.2) × 10⁴ M⁻¹, K_4-hydroxytam = 1.8 (±0.4) × 10⁴ M⁻¹ and K_endox = 2.0 (±0.5) × 10⁴ M⁻¹. The number of bound drugs per protein is 1.2 (tamoxifen), 1.7 (4-hydroxitamoxifen) and 1.0 (endoxifen). Structural modeling showed the participation of several amino acid residues in drug-HSA complexation, with extended H-bonding network. HSA conformation was altered by tamoxifen and its metabolites with a major reduction of α-helix and an increase in β-sheet, random coil and turn structures, indicating a partial protein unfolding. Our results suggest that serum albumins can act as carrier proteins for tamoxifen and its metabolites in delivering them to target tissues.     

Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.     

Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K _s, k ₊₂, and k ₊₂/K _s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k ₊₃ << k ₊₂). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK _a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>_a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.     

Study on the interactions of mapenterol with serum albumins using multi‐spectroscopy and molecular docking

The interactions of mapenterol with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated systematically using fluorescence spectroscopy, absorption spectroscopy, circular dichroism (CD) and molecular docking techniques. Mapenterol has a strong ability to quench the intrinsic fluorescence of BSA and HSA through static quenching procedures. At 291 K, the binding constants, K_a, were 1.93 × 10³ and 2.73 × 10³ L/mol for mapenterol–BSA and mapenterol–HAS, respectively. Electrostatic forces and hydrophobic interactions played important roles in stabilizing the mapenterol–BSA/has complex. Using site marker competitive studies, mapenterol was found to bind at Sudlow site I on BSA/HSA. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺ and Fe³⁺ on the binding. The conformation of BSA/HSA was changed by mapenterol, as seen from the synchronous fluorescence spectra. The CD spectra showed that the binding of mapenterol to BSA/HSA changed the secondary structure of BSA/HSA. Molecular docking further confirmed that mapenterol could bind to Sudlow site I of BSA/HSA. According to Förster non‐radiative energy transfer theory (FRET), the distances r₀ between the donor and acceptor were calculated as 3.18 and 2.75 nm for mapenterol–BSA and mapenterol–HAS, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Content and Composition of Branched-Chain Fatty Acids in Bovine Milk Are Affected by Lactation Stage and Breed of Dairy Cow

Dairy products contain bioactive fatty acids (FA) and are a unique dietary source of an emerging class of bioactive FA, branched-chain fatty acids (BCFA). The objective of this study was to compare the content and profile of bioactive FA in milk, with emphasis on BCFA, among Holstein (HO), Jersey (JE), and first generation HO x JE crossbreeds (CB) across a lactation to better understand the impact of these factors on FA of interest to human health. Twenty-two primiparous cows (n = 7 HO, n = 7 CB, n = 8 JE) were followed across a lactation. All cows were fed a consistent total mixed ration (TMR) at a 70:30 forage to concentrate ratio. Time points were defined as 5 days in milk (DIM), 95 DIM, 185 DIM, and 275 DIM. HO and CB had a higher content of n-3 FA at 5 DIM than JE and a lower n-6:n-3 ratio. Time point had an effect on the n-6:n-3 ratio, with the lowest value observed at 5 DIM and the highest at 185 DIM. The content of vaccenic acid was highest at 5 DIM, yet rumenic acid was unaffected by time point or breed. Total odd and BCFA (OBCFA) were higher in JE than HO and CB at 185 and 275 DIM. Breed affected the content of individual BCFA. The content of iso-14:0 and iso-16:0 in milk was higher in JE than HO and CB from 95 to 275 DIM. Total OBCFA were affected by time point, with the highest content in milk at 275 DIM. In conclusion, HO and CB exhibited a higher content of several bioactive FA in milk than JE. Across a lactation the greatest content of bioactive FA in milk occurred at 5 DIM and OBCFA were highest at 275 DIM.     

Analysis of lidocaine interactions with serum proteins using high-performance affinity chromatography

High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α₁-acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (K_a) of 1.1–1.7 × 10⁵ M^?1 at 37 °C and pH 7.4. Lidocaine had weak to moderate binding to HSA, with a K_a in the range of 10³ to 10⁴ M^?1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes.     

Molecular Interaction Studies of Trimethoxy Flavone with Human Serum Albumin

Background

Human serum albumin (HSA) is the most abundant protein in blood plasma, having high affinity binding sites for several endogenous and exogenous compounds. Trimethoxy flavone (TMF) is a naturally occurring flavone isolated from Andrographis viscosula and used in the treatment of dyspepsia, influenza, malaria, respiratory functions and as an astringent and antidote for poisonous stings of some insects.

Methodology/Principal Findings

The main aim of the experiment was to examine the interaction between TMF and HSA at physiological conditions. Upon addition of TMF to HSA, the fluorescence emission was quenched and the binding constant of TMF with HSA was found to be K_TMF = 1.0±0.01×10³ M⁻¹, which corresponds to −5.4 kcal M⁻¹ of free energy. Micro-TOF Q mass spectrometry results showed a mass increase of from 66,513 Da (free HSA) to 66,823 Da (HAS +Drug), indicating the strong binding of TMF with HSA resulting in decrease of fluorescence. The HSA conformation was altered upon binding of TMF to HSA with decrease in α-helix and an increase in β-sheets and random coils suggesting partial unfolding of protein secondary structure. Molecular docking experiments found that TMF binds strongly with HSA at IIIA domain of hydrophobic pocket with hydrogen bond and hydrophobic interactions. Among which two hydrogen bonds are formed between O (19) of TMF to Arg 410, Tyr 411 and another one from O (7) of TMF to Asn 391, with bond distance of 2.1 Å, 3.6 Å and 2.6 Å, respectively.

Conclusions/Significance

In view of the evidence presented, it is imperative to assign a greater role of HSA''s as a carrier molecule for many drugs to understand the interactions of HSA with TMF will be pivotal in the design of new TMF-inspired drugs.     

Properties of pig heart aconitase

Comparison of pig heart aconitase (Kennedy et al., 1972) with yeast (Candida lipolytica) aconitase (Suzuki et al., 1973) reveals similarities in molecular weight and iron content but not in sulphide content. Comparison with the Mildvan & Villafranca (1971) pig heart aconitase preparation reveals differences in iron ligands, specific activity and other properties; these differences possibly arise from protein association as pig heart protein associates under a variety of conditions. The electron spin resonance spectrum, g 4.25, and the low molar relaxivity, 473m⁻¹·s⁻¹, of water H⁺ suggest the presence of high-spin Fe(III) unco-ordinated to water in the enzyme. The iron chromophore on acid titration at 320nm gives a curve with an inflexion at pH4.2. Ten of 16 expected thiol equivalents are titrated with p-hydroxymercuribenzoate suggesting the presence of cystine as well as cysteine residues. Inhibition of the activation of inactive (activatable) enzyme is sigmoidally related to the molar ratio, p-hydroxymercuribenzoate/enzyme with 10–11mol of mercurial compound causing complete inhibition. Active enzyme, free from activating reagents, requires high molar ratios of mercurial compound for rapid inhibition. In terms of p-hydroxymercuribenzoate the enzyme then lacks an essential thiol group.