Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM

DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, K_v4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca^2 +DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS–DREAM complexes increases upon Ca^2 + association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca^2 +DREAM were determined to be 195 ± 20 μM and 62 ± 4 μM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of K_v4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins.     

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins. Implication of the C-H...O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of 4-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha- and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H.O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.     

Lactose Binding to Galectin-1 Modulates Structural Dynamics, Increases Conformational Entropy, and Occurs with Apparent Negative Cooperativity

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K₁ = 21 ± 6 × 10³ M^− 1) than the second (K₂ = 4 ± 2 × 10³ M^− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K₁ = 20 ± 10 × 10³ M^− 1 and K₂ = 1.67 ± 0.07 × 10³ M^− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general.     

Immobilization of carbohydrate epitopes for surface plasmon resonance using the Staudinger ligation

The detection of carbohydrate-protein interactions is often performed using techniques that require surface immobilization of the lectin or the glycan. A commonly used assay for lectin binding is surface plasmon resonance (SPR). We describe an implementation of the Staudinger ligation as a method to immobilize carbohydrate epitopes to a biosensor surface. This was accomplished by first introducing an azide functionality to a carboxymethyldextran surface, followed by reaction with a phosphane-modified carbohydrate ligand. The chemistry employed is extremely mild and was easily adapted to a commercial biosensor system. Using this approach, we investigated the binding of jacalin and wheat germ agglutinin (WGA) to galactose, lactose, and N-acetyl-lactosamine. We observed that WGA binding shows evidence of multivalent interaction with the surface. Additionally, we found that jacalin binding was influenced by the presence of a flexible and hydrophobic galactosyl aglycone.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Stability studies of hydrazide and hydroxylamine-based glycoconjugates in aqueous solution

Glycoconjugates can be readily formed by the condensation of a free-reducing terminus and a strong α-effect nucleophile, such as a hydrazide or a hydroxylamine. Further characterization of a series of glycoconjugates formed from xylose, glucose and N-acetylglucosamine, and either p-toluenesulfonyl hydrazide or an N-methylhydroxylamine, was carried out to gain insight into the optimal conditions for the formation of these useful conjugates, and their stability. Their apparent association constants (9-74 M⁻¹) at pH 4.5; as well, as rate constants for hydrolysis, at pH 4.0, 5.0 and 6.0 (37 °C), were determined. The half-lives of the conjugates varied between 3 h and 300 days. All the compounds were increasingly stable as the pH approached neutrality. Conjugate hydrolysis rates mirrored those found for O-glycoside hydrolysis where conjugates formed from electron-rich monosaccharides hydrolyzed more rapidly.     

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca²⁺ binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln¹²²-Pro¹²³-Asn¹²⁴) in LvLectin-1, but QPD (Gln¹²⁸-Pro¹²⁹-Asp¹³⁰) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P < 0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P < 0.01) and 12 h (P < 0.05), but the expression level of LvLectin-1 down-regulated significantly (P < 0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.     

Immobilization of lipase on cotton cloth using the layer-by-layer self-assembly technique

Lipase from Thermomyces lanuginosus was assembled into multiple layers on polyethylenimine treated cotton flannel cloth, utilising the enzymes property of forming bimolecular aggregates via layer-by-layer (LBL) immobilization technique. An increase in lipase activity with increasing enzyme layers confirmed lipase aggregation. A study to compare the activity of enzyme bound by classical LBL technique, containing alternate layers of polyethylenimine and lipase and the modified approach indicated above, showed that more enzyme was bound to cloth in the modified approach. A total of 13 U/cm² of enzyme were bound to cloth till the fifth layer whereas only 10.2 U/cm² were bound till the fifth bilayer in the classical approach. The successful assembly of lipase molecules has shown that this modified technique is a promising approach to immobilize enzymes that aggregate through hydrophobic interactions as nano-films on cloth.     

Synthesis and spectroscopic characterization of 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide as a new fluorescent probe for the determination of proteins

A novel 4-butoxyethoxy-N-octadecyl-1,8-naphthalimide (BON) was synthesized as a fluorescent probe for the determination of proteins. The interactions between BON and bovine serum albumin (BSA) were studied by fluorescence spectroscopy and UV-vis absorption spectroscopy. Fluorescence data revealed that the fluorescence quenching of BSA by BON was likely the result of the formation of the BON-BSA complex. According to the modified Stern-Volmer equation, the binding constants of BON with BSA at four different temperatures were obtained. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be −23.27 kJ mol⁻¹ and 10.40 J mol⁻¹ K⁻¹ according to van’t Hoff equation, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding of BON to BSA. Furthermore, displacement experiments using warfarin indicated that BON could bind to site I of BSA. The effect of BON on the conformation of BSA was also analyzed by synchronous fluorescence and three-dimensional fluorescence spectra. A new fluorescence quenching assay of the proteins BSA using BON in the HCl-Tris (pH 7.4) buffer solution was developed with maximum excitation and emission wavelengths of 373 and 489 nm, respectively. The linear range was 0.1-10.0 × 10⁻⁵ mol L⁻¹ with detection limits were determined to be 1.76 × 10⁻⁸ mol L⁻¹. The effect of metal cations on the fluorescence spectra of BON in ethanol was also investigated. Determination of protein in human serum by this method gave results which were very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry.     

Cutoff variation induces different topological properties: A new discovery of amino acid network within protein

An increasing attention has been dedicated to the characterization of complex networks within the protein world. Before now most investigations about protein structures were only considered where the interactive cutoff distance R_c=5 or 7 Å. It is noteworthy that the length of peptide bond is about 1.5 Å, the length of hydrogen bond is about 3 Å, the range of London-van der Waals force is about 5 Å and the range of hydrophobic effect can reach to 12 Å in protein molecule. Present work reports a study on the topological properties of the amino acid network constructed by different interactions above. The results indicate that the small-world property of amino acid network constructed by the peptide and hydrogen bond, London-van der Waals force and the hydrophobic effect is strong, very strong and relatively weak, respectively. Besides, there exists a precise exponential relation C∝k^−0.5 at R_c=12 Å. It means that the amino acid network constructed by the hydrophobic effect tend to be hierarchical. Functional modules could be the cause for hierarchical modularity architecture in protein structures. This study on amino acid interactive network for different interactions facilitates the identification of binding sites which is strongly linked with protein function, and furthermore provides reasonable understanding of the underlying laws of evolution in genomics and proteomics.     

Biophysical characterization and mutational analysis of the antibiotic resistance protein NimA from Deinococcus radiodurans

Metronidazole (MTZ) is an antibiotic commonly used to treat anaerobic bacterial infections in humans and animals. Antibiotic resistance toward this class of 5-nitroimidazole (5-Ni) drug derivatives has been related to the Nim genes thought to encode a reductase. Here we report the biophysical characteristics of the NimA protein from Deinococcus radiodurans (DrNimA) binding to MTZ and three other 5-Ni drugs. The interaction energies of the protein and antibiotic are studied by isothermal titration calorimetry (ITC) and with free energy and linear interaction energy (LIE) calculations, where the latter method revealed that the antibiotic binding is mainly of hydrophobic character. ITC measurements further found that one DrNimA dimer has two antibiotic binding sites which were not affected by mutation of the reactive His71. The observed association constants (K_a) were in the range of 5.1–49 ? 10⁴ M^− 1 and the enthalpy release upon binding to DrNimA for the four drugs studied was relatively low (∼ − 1 kJ/mol) but still measurable. The drug binding is mainly entropy driven and along with the hydrophobic drug binding site found by crystallography, this possibly explains the low observed enthalpy values. The effect of the His71 mutation and the presence of MTZ were studied by differential scanning calorimetry (DSC). Native DrNimA is a yellow colored protein where the interaction from His71 to the cofactor is thought to be responsible for the coloring. Mutations of His71 to Ala, Ser, Leu or Asp all gave transparent, colorless protein solutions, and the two mutant crystal structures of DrNimA-H71A and DrNimA-H71S presented revealed no cofactor binding.     

Actin as a potential target for decavanadate

ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate-protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1 × 10^− 3 s^− 1), and an apparent dissociation constant (k_dapp) of 227.4 ± 25.7 μM and 112.3 ± 8.7 μM was obtained in absence or presence of 20 μM V₁₀, respectively. Moreover, concentrations as low as 50 μM of decameric vanadate species (V₁₀) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V₁) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V₁ (0-200 μM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.     

New advances in the study on the interaction of [Cr(phen)2(dppz)]3+ complex with biological models; association to transporting proteins

The present study reports a detailed investigation with the interaction of [Cr(phen)₂(dppz)]³⁺ with serum albumins, the key protein for the transport of drugs in the blood plasma, which allows us to understand further the role of [Cr(phen)₂(dppz)]³⁺ as sensitizer in Photodynamic Therapy (PDT).Chromium(III) complex [Cr(phen)₂(dppz)]³⁺, (dppz = dipyridophenazine and phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended π system, has been found to bind strongly with bovine and human serum albumins (BSA and HSA) with an intrinsic binding constants, K_b, of (1.7 ± 0.3) × 10⁵ M^− 1 and (2.2 ± 0.3) × 10⁵ M^− 1 at 295 K, respectively. The interactions of serum albumins with [Cr(phen)₂(dppz)]³⁺ were assessed employing fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy. The serum albumins-[Cr(phen)₂(dppz)]³⁺ interactions caused conformational changes with the loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the albumin (BSA or HSA) bound to the Cr(III) complex decreased, suggesting that perturbation around the Trp 214 residue took place. The analysis of the thermodynamic parameters ΔG, ΔH, ΔS indicated that the hydrophobic interactions played a major role in both BSA-Cr(III) and HSA-Cr(III) association processes. The binding distances and transfer efficiencies for BSA-Cr(III) and HSA-Cr(III) binding reactions were calculated according to the Föster theory of non-radiation energy transfer. All these experimental results suggests that [Cr(phen)₂(dppz)]³⁺ binds to serum albumins, by which these proteins could act as carriers of this complex for further applications in PDT.     

Discovery of 1,2,4-thiadiazolidine-3,5-dione analogs that exhibit unusual and selective rapid cell death kinetics against acute myelogenous leukemia cells in culture

4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) was previously identified as an antileukemic agent exhibiting no evident toxicity toward normal hematopoietic cells. An SAR study has been carried out to examine the effect of varying the C-2 and C-4- substituents on the thiadiazolidinone ring of TDZD-8 on antileukemic activity. These studies resulted in the identification of more druglike analogs that exhibited comparable potency to TDZD-8 in killing acute myelogenous leukemia (AML) cells in culture. Surprisingly, the cell death kinetics induced by several of these novel analogs on MV-411 cells were extremely fast, with commitment to death occurring within 30 min. At a concentration of 10 μM, 3f (LD₅₀ = 3.5 μM) completely eradicated cell viability of MV-411 cells within 2 h, while analog 3e (LD₅₀ = 2.0 μM) decimated cell viability within 30 min at a concentration of 10 μM and effectively abolished cell viability at 5 μM within 1-2 h.     

Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii

The thermotolerant yeast strain isolated from sugarcane juice through enrichment technique was identified as a strain of Pichiakudriavzevii (Issatchenkiaorientalis) through molecular characterization. The P. kudriavzevii cells adapted to galactose medium produced about 30% more ethanol from sugarcane juice than the non-adapted cells. The recycled cells could be used for four successive cycles without a significant drop in ethanol production. Fermentation in a laboratory fermenter with galactose adapted P. kudriavzevii cells at 40 °C resulted in an ethanol concentration and productivity of 71.9 g L⁻¹ and 4.0 g L⁻¹ h⁻¹, respectively from sugarcane juice composed of about 14% (w/v) sucrose, 2% (w/v) glucose and 1% (w/v) fructose. In addition to ethanol, 3.30 g L⁻¹ arabitol and 4.19 g L⁻¹ glycerol were also produced, whereas sorbitol and xylitol were not formed during fermentation. Use of galactose adapted P. kudriavzevii cells for ethanol production from sugarcane juice holds potential for scale-up studies.     

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

DNA binding and cleavage activity of [Ru(NH3)4(diimine)]Cl2 complexes

The interaction of a series of mixed ligand complexes of the type [Ru(NH₃)₄(diimine)]Cl₂, where diimine=2,2^′-bipyridine (bipy), 1,10-phenanthroline (phen), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp), 4,7-dimethyl-1,10-phenanthroline (4,7-dmp), 2,9-dimethyl-1,10-phenanthroline (2,9-dmp), 3,4,7,8-tetra-methyl-1,10-phenanthroline (Me₄phen), with calf thymus DNA has been studied using absorption, emission and circular dichroic spectral measurements and viscometry and electrochemical techniques. On interaction with DNA the complexes show hypochromism and red-shift in their MLCT band suggesting that the complexes bind to DNA. The magnitude of the binding constant (K_b) obtained from absorption spectral titration varies depending upon the nature of the diimine ligand: Me₄phen > 5,6-dmp > 4,7-dmp > phen suggesting the use of diimine ‘face’ of the octahedral complexes in binding to DNA. The interaction of phen complex possibly involves phen ring partially inserted into the DNA base pairs. In contrast, the methyl-substituted phen complexes would involve hydrophobic interaction of the phen ring in the grooves of DNA, which is supported by hydrogen bonding interactions of the ammonia ligands with the intrastrand nucleobases. Also the shape and size of the phen ligand as modified by the methyl substituents determine the DNA binding site sizes (0.12-0.45 base pairs). The relative emission intensities (I/I₀) of the DNA-bound complexes parallel the variation in K_b values. Almost all the metal complexes exhibit induced CD bands on binding to B DNA, with the 4,7-dmp and Me₄phen complexes inducing certain structural modifications on the biopolymer. DNA melting curves obtained in the presence of metal complexes reveal a monophasic melting of the DNA strands, the Me₄phen complex exhibiting a slightly enhanced tendency to stabilize the double-stranded DNA. There were slight to appreciable changes in the relative viscosities of DNA, which are consistent with enhanced hydrophobic interaction of the methyl-substituted phen rings. Upon interaction with CT DNA, the Me₄phen, 4,7-dmp and 5,6-dmp complexes, in contrast to bipy, phen and 2,9-dmp complexes, show a decrease in anodic peak current in their cyclic voltammograms suggesting that they exhibit enhanced DNA binding. DNA cleavage experiments show that all the complexes induce cleavage of pBR322 plasmid DNA, the Me₄phen and 5,6-dmp complexes being remarkably more efficient than other complexes.     

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.