Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K ₀ = (3.9 ± 0.4) × 10⁻⁴ and (1.3 ± 0.1) × 10⁻⁵ M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K _h = (4.3 ± 0.4) × 10⁻³ and (1.2 ± 0.1) × 10⁻⁴ M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H ₀ = (1.9 ± 0.2) × 10⁻⁸ M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H _d = (2.1 ± 0.2) × 10⁻⁷ M]. In the absence and presence of CO₂, the values of the second-order rate constant (l _on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10⁵ and 4.3 × 10⁵ M⁻¹ s⁻¹, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO₂. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.     

Interaction of VO ion with human serum transferrin and albumin

The complexation of VO²⁺ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO²⁺ ion occupies three different binding sites, A, B₁ and B₂, distinguishable in the X-band anisotropic spectrum recorded in D₂O. With albumin the results show that a dinuclear complex (VO)₂^dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO²⁺, the multinuclear complex (VO)_x^mHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the ⁵¹V A_z values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO²⁺/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO²⁺ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.     

New vanadium-based magnetic resonance imaging probes: clinical potential for early detection of cancer

We have developed a magnetic resonance imaging (MRI) method for improved detection of cancer with a new class of cancer-specific contrast agents, containing vanadyl (VO²⁺)-chelated organic ligands, specifically bis(acetylacetonato)oxovanadium(IV) [VO(acac)₂]. Vanadyl compounds have been found to accumulate within cells, where they interact with intracellular glycolytic enzymes. Aggressive cancers are metabolically active and highly glycolytic; an MRI contrast agent that enters cells with high glycolytic activity could provide high-resolution functional images of tumor boundaries and internal structure, which cannot be achieved by conventional contrast agents. The present work demonstrates properties of VO(acac)₂ that may give it excellent specificity for cancer detection. A high dose of VO(acac)₂ did not cause any acute or short-term adverse reactions in murine subjects. Calorimetry and spectrofluorometric methods demonstrate that VO(acac)₂ is a blood pool agent that binds to serum albumin with a dissociation constant K _d ~ 2.5 ± 0.7 × 10⁻⁷ M and a binding stoichiometry n = 1.03 ± 0.04. Owing to its prolonged blood half-life and selective leakage from hyperpermeable tumor vasculature, a low dose of VO(acac)₂ (0.15 mmol/kg) selectively enhanced in vivo magnetic resonance images of tumors, providing high-resolution images of their interior structure. The kinetics of uptake and washout are consistent with the hypothesis that VO(acac)₂ preferentially accumulates in cancer cells. Although VO(acac)₂ has a lower relaxivity than gadolinium-based MRI contrast agents, its specificity for highly glycolytic cells may lead to an innovative approach to cancer detection since it has the potential to produce MRI contrast agents that are nontoxic and highly sensitive to cancer metabolism.     

Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study

Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme–HSA has been investigated by ¹H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme–albumin), 1.0 M guanidinium chloride induces some unfolding of heme–HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K _d = (1.2 ± 0.1) × 10⁻⁶ M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K _d = (3.4 ± 0.1) × 10⁻⁵ M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme–HSA in the presence of myristate.     

Hydrolysis of the amide bond in methionine-containing peptides catalyzed by various palladium(II) complexes: Dependence of the hydrolysis rate on the steric bulk of the catalyst

¹H NMR spectroscopy was applied to study the reactions of cis-[Pd(L)(H₂O)₂]²⁺ complexes (L is en, pic and dpa) with the N-acetylated tripeptides L-methionylglycylglycine, MeCOMet–Gly–Gly, and glycyl–L-methionyl–glycine, MeCOGly–Met–Gly. All reactions were performed in the pH range 2.0–2.5 with equimolar amounts of the cis-[Pd(L)(H₂O)₂]²⁺ complex and the tripeptide at 60 °C. The hydrolytic reactions of the cis-[Pd(en)(H₂O)₂]²⁺, cis-[Pd(pic)(H₂O)₂]²⁺ and cis-[Pd(dpa)(H₂O)₂]²⁺ complexes with MeCOMet–Gly–Gly were regioselective and only the amide bond involving the carboxylic group of methionine was cleaved. However, in the reactions of these three Pd(II) complexes with MeCOGly–Met–Gly, two amide bonds, Met–Gly and MeCO–Gly, were cleaved. From UV–Vis spectrophotometry studies, it was found that the rate-determining step of these hydrolytic reactions is the monodentate coordination of the corresponding Pd(II) complex to the sulfur atom of the methionine side chain. The rate of the cleavage of these amide bonds is dependent on the nature of the bidentate coordinated diamine ligand L (en > pic > dpa). The hydrolytic reaction of cis-[Pd(L)(H₂O)₂]²⁺-type complexes with MeCOMet–Gly–Gly, containing the methionine side chain in the terminal position of the peptide, is regioselective while in the reaction of these Pd(II) complexes with MeCOGly–Met–Gly, none selective cleavage of the peptide occurs. This study contributes to a better understanding of the selective cleavage of methionine-containing peptides employing palladium(II) complexes as catalysts.     

Effects of Hypoxia,Glucose Deprivation and Recovery on the Expression of Nucleoside Transporters and Adenosine Uptake in Primary Culture of Rat Cortical Astrocytes

The aim of this study was to explore effects of hypoxia, glucose deprivation (HGD) and recovery on expression and activities of equilibrative nucleoside transporters (rENT) and concentrative nucleoside transporters (rCNT) in rat astrocytes in primary culture. Amounts of cellular ATP in the control group (CG, 5% CO₂ in air, medium containing 7 mM d-glucose, 1 mM Na⁺-pyruvate, 1 h), HGD group (2% O₂/5% CO₂ in N₂, pyruvate-free medium containing 1.5 mM d-glucose and 10 mM 2-deoxy-d-glucose, 1 h) and recovery group (RG, HGD for 1 h, followed by 1 h exposure to the same conditions as the CG) were (nmol/mg protein, n = 4) 18 ± 1.6, 4.9 ± 0.6 and 10.1 ± 0.8, respectively. Extracellular adenosine concentrations increased from (nM, n = 3) 42 ± 4 in the CG, to 99 ± 8 in the HGD group and 86 ± 3 in the RG. Real-time PCR and immunoblotting revealed that in the HGD group and RG, the amounts of rENT1 mRNA and protein were reduced to 40 and 50%, when compared to the CG, respectively. Astrocyte cultures took up [³H]adenosine by concentrative and equilibrative transport processes; however, rENT1-mediated uptake was absent in the RG and cultures from the RG took up significantly less [³H]adenosine by equilibrative mechanisms than cultures from the CG.     

The vanadyl (VO2+) chelate bis(acetylacetonato)oxovanadium(IV) potentiates tyrosine phosphorylation of the insulin receptor

We have compared the insulin-like activity of bis(acetylacetonato)oxovanadium(IV) [VO(acac)₂], bis(maltolato)oxovanadium(IV) [VO(malto)₂], and bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV) [VO(OPT)₂] in differentiated 3T3-L1 adipocytes. The insulin-like influence of VO(malto)₂ and VO(OPT)₂ was decreased compared with that of VO(acac)₂. Also, serum albumin enhanced the insulin-like activity of all three chelates more than serum transferrin. Each of the three VO²⁺ chelates increased the tyrosine phosphorylation of proteins in response to insulin, including the β-subunit of the insulin receptor (IRβ) and the insulin receptor substrate-1 (IRS1). However, VO(acac)₂ exhibited the greatest synergism with insulin and was therefore further investigated. Treatment of 3T3-L1 adipocytes with 0.25 mM VO(acac)₂ in the presence of 0.25 mM serum albumin synergistically increased glycogen accumulation stimulated by 0.1 and 1 nM insulin, and increased the phosphorylation of IRβ, IRS1, protein kinase B, and glycogen synthase kinase-3β. Wortmannin suppressed all of these classical insulin-signaling activities exerted by VO(acac)₂ or insulin, except for tyrosine phosphorylation of IRβ and IRS1. Additionally, VO(acac)₂ enhanced insulin signaling and metabolic action in insulin-resistant 3T3-L1 adipocytes. Cumulatively, these results provide evidence that VO(acac)₂ exerts its insulin-enhancing properties by directly potentiating the tyrosine phosphorylation of the insulin receptor, resulting in the initiation of insulin metabolic signaling cascades in 3T3-L1 adipocytes.     

Reflex bradycardia does not influence oxygen consumption during hypoxia in the European eel (Anguilla anguilla)

Most teleost fish reduce heart rate when exposed to acute hypoxia. This hypoxic bradycardia has been characterised for many fish species, but it remains uncertain whether this reflex contributes to the maintenance of oxygen uptake in hypoxia. Here we describe the effects of inhibiting the bradycardia on oxygen consumption (MO₂), standard metabolic rate (SMR) and the critical oxygen partial pressure for regulation of SMR in hypoxia (Pcrit) in European eels Anguilla anguilla (mean ± SEM mass 528 ± 36 g; n = 14). Eels were instrumented with a Transonic flow probe around the ventral aorta to measure cardiac output (Q) and heart rate (f _H). MO₂ was then measured by intermittent closed respirometry during sequential exposure to various levels of increasing hypoxia, to determine Pcrit. Each fish was studied before and after abolition of reflex bradycardia by intraperitoneal injection of the muscarinic antagonist atropine (5 mg kg⁻¹). In the untreated eels, f _H fell from 39.0 ± 4.3 min⁻¹ in normoxia to 14.8 ± 5.2 min⁻¹ at the deepest level of hypoxia (2 kPa), and this was associated with a decline in Q, from 7.5 ± 0.8 mL min⁻¹ kg⁻¹ to 3.3 ± 0.7 mL min⁻¹ kg⁻¹ in normoxia versus deepest hypoxia, respectively. Atropine had no effect on SMR, which was 16.0 ± 1.8 μmol O₂ kg⁻¹ min⁻¹ in control versus 16.8 ± 0.8 μmol O₂ kg⁻¹ min⁻¹ following treatment with atropine. Atropine also had no significant effect on normoxic f _H or Q in the eel, but completely abolished the bradycardia and associated decline in Q during progressive hypoxia. This pharmacological inhibition of the cardiac responses to hypoxia was, however, without affect on Pcrit, which was 11.7 ± 1.3 versus 12.5 ± 1.5 kPa in control versus atropinised eels, respectively. These results indicate, therefore, that reflex bradycardia does not contribute to maintenance of MO₂ and regulation of SMR by the European eel in hypoxia.     

Novel fluorine‐containing chiral hydrazide‐hydrazones: Design,synthesis, structural elucidation,antioxidant and anticholinesterase activity,and in silico studies

In this study, a series of fluorine‐containing chiral hydrazide‐hydrazone derivatives [III‐XII] from ?‐cysteine ethyl ester hydrochloride was synthesized as new antioxidant and anticholinesterase agents. The antioxidant activity of these derivatives was evaluated by ABTS^+· and DPPH^· scavenging and CUPRAC assays and the anticholinesterase activity by the Ellman method spectrophotometrically. The results of the antioxidant assay showed that compounds V , IX , and X exhibited higher activity than BHT and α‐tocopherol used as positive standards. Among the synthesized derivatives, compound IX (IC₅₀: 2.3 ± 1.6 μM) exhibited higher acetylcholinesterase inhibitory activity than galantamine (IC₅₀: 4.5 ± 0.8 μM). Compounds XI (IC₅₀: 9.6 ± 1.0 μM), IX (IC₅₀: 12.5 ± 1.6 μM), III (IC₅₀: 16.0 ± 1.6 μM), X (IC₅₀: 17.2 ± 1.8 μM), VI (IC₅₀: 20.2 ± 0.8 μM), XII (IC₅₀: 21.5 ± 1.0 μM), and VII (IC₅₀: 24.6 ± 0.6 μM) displayed better butyrylcholinesterase inhibitory activity than galantamine (IC₅₀: 46.03 ± 0.14 μM). ADME‐Tox analysis was used to probe the drug‐like properties of the compounds. Molecular docking studies were also applied to understand the interactions between compounds and targets. The docking calculations were supported by the experimental data. In particular, compound IX , having better activity than galantamine against acetylcholinesterase and butyrylcholinesterase enzymes, was visualized using molecular docking.     

Cis-diammineplatinum(II) forms a macrochelate with 2′-deoxycytidine 5′-monophosphate (dCMP2–)! Reactivity and acid-base properties of cis-Pt(NH3)2(dCMP)

 The synthesis of cis-Pt(NH₃)₂(dCMP) is reported and by various physico-chemical methods it is demonstrated that it is a macrochelate in which Pt(II) is bound simultaneously to the N3 site of cytosine in dCMP^2– and to a phosphate-oxygen atom. According to the NOESY spectra (cross-peaks between cytosine H6 and H2′ and H3′) the cytosine ring adopts an anti orientation. Highly unusual is the significant (1 ppm) downfield shift of the sugar proton H5″ in the ¹H-NMR spectrum and the sensitivity of the cytosine H6 resonance on the protonation state of the phosphate group. Based on these three features a geometry for the macrochelate is proposed. The compound is a major product of the reaction of cis-[Pt(NH₃)₂(H₂O)₂]²⁺ with dCMP^2– at neutral pH, but it even forms at pH 5. By applying pD-dependent NMR spectroscopy (¹H, ³¹P) and potentiometric pH titration, it is demonstrated that the Pt-coordinated phosphate group can be protonated (pK _a/1=3.21±0.10 and 3.31±0.05, respectively), and ¹H- and ³¹P-NMR spectra also indicate deprotonation (pK _a/2=13.35±0.25) of the exocyclic amino group of the cytosine moiety. The metal ion binding affinity of cis-Pt(NH₃)₂(dCMP) is very small, as shown for Cu²⁺ (log K<0.6). The cis-Pt(NH₃)₂(dCMP) complex reacts with nucleosides and nucleotides (L′) by losing its chelate structure and forming mixed ligand complexes, cis-Pt(NH₃)₂(dCMP)(L′); this means that the phosphate group is released from the coordination sphere of Pt(II), indicating that the Pt(II)-O(phosphate) bond is not very strong. Received: 23 October 1997 / Accepted: 17 February 1998     

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [³H]bestim was found to bind with high affinity to mouse peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [³H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [³H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.     

Antioxidant reactivity toward nitroxide probes anchored into human serum albumin. A new model for studying antioxidant repairing capacity of protein radicals

A new strategy to evaluate accessibility of antioxidants to radical proteins has been developed using nitroxide prefluorescent probes anchored into human serum albumin (HSA). Binding association constants for the nitroxide probes C₃₄₃T and QT with HSA were 5 × 10⁴ and 9 × 10⁴ M⁻¹, respectively. Rate constants for the nitroxide reduction by antioxidants in HSA were determined finding k_HSA/k_buffer ratio of 0.8, 1.9, and 0.075 for ascorbic acid, Trolox, and caffeic acid, respectively, for the nitroxide C₃₄₃T reduction.     

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn²⁺, suppressed by Cu²⁺ and Zn²⁺ but inhibited by Fe³⁺. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg⁻¹ protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg⁻¹ was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC₅₀ = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC₅₀ = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD₅₀ = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm⁻²). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT₅₀ = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn²⁺-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.     

Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay

A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum–acridine agents represented by the formula [Pt(am₂)LCl](NO₃)₂, where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5′-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am₂ is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k _obs = 1.4 ± 0.37 × 10⁻⁴ s⁻¹ (t _1/2 = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k _obs = 5.7 ± 0.58 × 10⁻⁴ s⁻¹, t _1/2 = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k _obs = 2.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k _obs = 1.1 ± 0.40 × 10⁻⁴ s⁻¹, t _1/2 = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine–purine cross-links are discussed.     

Erythrocyte permeability to urea and water: comparative study in rodents,ruminants, carnivores,humans, and birds

Mammalian erythrocytes exhibit high urea permeability (P _urea) due to UT-B expression in their cytoplasmic membrane. This high P _urea allows fast equilibration of urea in erythrocytes during their transit in the hyperosmotic renal medulla. It also allows more urea (in addition to that in plasma) to participate in counter-current exchange between ascending and descending vasa recta, thus improving the trapping of urea in the medulla and improving urine concentrating ability. To determine if P _urea in erythrocytes is related to diet and urine concentrating ability, we measured P _urea in erythrocytes from 11 different mammals and 5 birds using stopped-flow light scattering. Carnivores (dog, fox, cat) exhibited high P _urea (in ×10⁻⁵ cm/s, 5.3 ± 0.6, 3.8 ± 0.5 and 2.8 ± 0.7, respectively). In contrast, herbivores (cow, donkey, sheep) showed much lower P _urea (0.8 ± 0.2, 0.7 ± 0.2, 1.0 ± 0.1, respectively). Erythrocyte P _urea in human (1.1 ± 0.2), and pig (1.5 ± 0.1), the two omnivores, was intermediate. Rodents and lagomorphs (mouse, rat, rabbit) had P _urea intermediate between carnivores and omnivores (3.3 ± 0.4, 2.5 ± 0.3 and 2.4 ± 0.3, respectively). Birds that do not excrete urea and do not express UT-B in their erythrocytes had very low values (<0.1 × 10⁻⁵ cm/s). In contrast to P _urea, water permeability, measured simultaneously, was relatively similar in all mammals. The species differences in erythrocytes P _urea most probably reflect adaptation to the different types of diet and resulting different needs for concentrating urea in the urine.     

Calorimetric studies of the interaction between the insulin-enhancing drug candidate bis(maltolato)oxovanadium(IV) (BMOV) and human serum apo-transferrin

Bis(maltolato)oxovanadium(IV) (BMOV), and its ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), are candidate insulin-enhancing agents for the treatment of type 2 diabetes mellitus; in mid-2008, BEOV advanced to phase II clinical testing. The interactions of BMOV and its inorganic congener, vanadyl sulfate (VOSO₄), with human serum apo-transferrin (hTf) were investigated using differential scanning calorimetry (DSC). Addition of BMOV or VOSO₄ to apo-hTf resulted in an increase in thermal stability of both the C- and N-lobes of transferrin as a result of binding to either vanadyl compound. A series of DSC thermograms of hTf solutions containing different molar ratios of BMOV and VOSO₄ were used to determine binding constants; at 25 °C the binding constants of BMOV to the C- and N-lobes of apo-hTf were found to be 3 (±1) × 10⁵ and 1.8 (±0.7) × 10⁵ M⁻¹, respectively. The corresponding values for VOSO₄ were 1.7 (±0.3) × 10⁵ and 7 (±2) × 10⁴ M⁻¹. The results show that the vanadium species initially presented as either BMOV or VOSO₄ had similar affinities for human serum transferrin due to oxidation of solvated vanadyl(IV) prior to complexation to transferrin. Binding of metavanadate () was confirmed by DSC and isothermal titration calorimetry (ITC) experiments of the interaction between sodium metavanadate (NaVO₃) and hTf.     

Hydrogen Peroxide Inhibits Chloride Channels of the Sarcoplasmic Reticulum of Skeletal Muscle

Data obtained with the lipid bilayer technique indicate that cis (cytoplasmic) concentration of 4.4–22 mm hydrogen peroxide (H₂O₂), is a water-soluble oxidant. [H₂O₂] _cis (n= 26) reversibly inhibits the multisubconductance SCl channel of the sarcoplasmic reticulum vesicles from rabbit skeletal muscle. At −40 mV, the mean values of the current amplitude (I) and the probability of the SCl channel being open (P _o ) were reduced significantly (n= 8) from −6.14 ± 0.42 pA and 0.69 ± 0.06 (for all conductance levels) in control 0.0 mm [H₂O₂] _cis to −1.10 ± 0.51 pA and 0.13 ± 0.04 (for the intermediate subconductance states) in 8.8 mm [H₂O₂] _cis , respectively. The [H₂O₂] _cis -induced decrease in P _o is mainly due to a decrease in the mean open time T _o . The mechanism of [H₂O₂] _cis effects on the multiconductance SCl channel is characterized by a mode shift in the channel state from the main conductance state to the low subconductance states. The estimated concentration of the [H₂O₂] _cis for the half inhibitory constant, K _i , was 11.78 mm, higher than the estimated 8.0 and 8.1 mm for the parameters P _o and T _o , respectively, indicating that the conductance of the SCl channel is less sensitive than the gating kinetics of the channel. After a lag period of between 30 to 60 sec, the lipophilic SH-oxidizing agent 4,4′-dithiodipyridine (4,4′-DTDP) added to the cis side at 1.0 mm removed the inhibitory effects of 8.8 mm [H₂O₂] _cis . The 4,4′-DTDP-enhanced SCl channel activity was blocked after the addition of 0.5 mm ATP to the cis side of the channel. The addition of 1.0 mm 4,4′-DTDP to the cis or trans solutions facing an SCl channel already subjected to 0.5 mm [ATP] _cis or [ATP] _trans failed to activate the ATP-inhibited SCl channel. These findings suggest that 4,4′-DTDP is not preventing the binding of ATP to its binding site on the channel protein. The interaction of H₂O₂ with the SCl channel proteins is consistent with a thiol-disulfide redox state model for regulating ion transport, where SH groups can directly modify the function of the channel and/or the availability of regulatory sites on the channel proteins. The H₂O₂ effects on the Ca²⁺ countercurrent through the SCl channel are also consistent with H₂O₂-modification of the mechanisms involved in the Ca²⁺ regulation, which underlies excitation-contraction coupling in skeletal muscle. Received: 27 April 1999/Revised: 1 July 1999     

Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein

Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be K_Umb-1=3.1 ± .01 × 10⁴ M^?1, K_Umb-2 = 7 ± .01 × 10⁴ M^?1, which corresponds to ?6.1 and ?6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of K_Umb-1-AGP=3.1 ± .01 × 10³ M^?1 and K_Umb-2-AGP = 4.6 ± .01 × 10³ M^?1. Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster’s theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1–HSA and Umb-2–HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.     

Influence of stearic acids on resveratrol-HSA interaction

The interaction between the natural polyphenol resveratrol and human serum albumin (HSA), the most abundant transport protein in plasma, has been studied in the absence and in the presence of up to six molecules of stearic acids (SA) pre-complexed with the protein. The study has been carried out by using the intrinsic fluorescence of both HSA and resveratrol. Protein and polyphenol fluorescence data indicate that resveratrol binds to HSA with an association constant k _a ?=?(1.10?±?0.14)?×?10⁵?M^?1 and (1.09?±?0.02)?×?10⁵?M^?1, respectively, whereas Job plot evidences the formation of an equimolar protein/drug complex. Low SA content associated with HSA does not affect significantly the structural conformation of the protein and its interaction with resveratrol, whereas high SA content induces conformational changes in the protein, and reduces resveratrol binding affinity. The photostability of resveratrol in the different samples changes in the order: buffer <?(high [SA]/HSA)?<?HSA?<?(low [SA]/HSA). The results on (SA/HSA)-resveratrol samples highlight the ability of the protein to bind hydrophobic and amphiphilic ligands and to protect from degradation an important antioxidant molecule under biologically relevant conditions.     

d-Aspartate binding sites in rat Harderian gland

Radioligand binding of d-[³H]aspartic and l-[³H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for d-Asp and l-Glu was saturable, and Eadie–Hofstee analysis revealed interaction with a single population of binding sites (for d-Asp K _d = 860 ± 28 nM, B _max = 27.2 ± 0.5 pmol/mg protein; for l-Glu, K _d = 580 ± 15 nM and B _max = 51.3 ± 0.8 pmol/mg protein). l-[³H]glutamate had higher affinity and a greater percentage of specific binding than did d-[³H]aspartate. The pharmacological binding specificity of l-[³H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was l-Glu > d-Asp > NMDA > MK801 > d-AP5 > glycine. For d-[³H]aspartate, the data revealed an interaction of d-Asp with either NMDA-type receptors or putative specific binding sites.