Multivalent Lactose-Ferrocene Conjugates Based on Poly (Amido Amine) Dendrimers and Gold Nanoparticles as Electrochemical Probes for Sensing Galectin-3.

Galectin-3 is considered a cancer biomarker and bioindicator of fibrosis and cardiac remodeling and, therefore, it is desirable to develop convenient methods for its detection. Herein, an approach based on the development of multivalent electrochemical probes with high galectin-3 sensing abilities is reported. The probes consist of multivalent presentations of lactose-ferrocene conjugates scaffolded on poly (amido amine) (PAMAM) dendrimers and gold nanoparticles. Such multivalent lactose-ferrocene conjugates are synthesized by coupling of azidomethyl ferrocene-lactose building blocks on alkyne-functionalized PAMAM, for the case of the glycodendrimers, and to disulfide-functionalized linkers that are then used for the surface modification of citrate-stabilized gold nanoparticles. The binding and sensing abilities toward galectin-3 of both ferrocene-containing lactose dendrimers and gold nanoparticles have been evaluated by means of isothermal titration calorimetry, UV-vis spectroscopy, and differential pulse voltammetry. The highest sensitivity by electrochemical methods to galectin-3 was shown by lactosylferrocenylated gold nanoparticles, which are able to detect the lectin in nanomolar concentrations.

Design of Engineered Cyclodextrin Derivatives for Spontaneous Coating of Highly Porous Metal-Organic Framework Nanoparticles in Aqueous Media.

Nanosized metal-organic frameworks (nanoMOFs) MIL-100(Fe) are highly porous and biodegradable materials that have emerged as promising drug nanocarriers. A challenging issue concerns their surface functionalization in order to evade the immune system and to provide molecular recognition ability, so that they can be used for specific targeting. A convenient method for their coating with tetraethylene glycol, polyethylene glycol, and mannose residues is reported herein. The method consists of the organic solvent-free self-assembly on the nanoMOFs of building blocks based on ß-cyclodextrin facially derivatized with the referred functional moieties, and multiple phosphate groups to anchor to the nanoparticles' surface. The coating of nanoMOFs with cyclodextrin phosphate without further functional groups led to a significant decrease of macrophage uptake, slightly improved by polyethylene glycol or mannose-containing cyclodextrin phosphate coating. More notably, nanoMOFs modified with tetraethylene glycol-containing cyclodextrin phosphate displayed the most efficient "stealth" effect. Mannose-coated nanoMOFs displayed a remarkably enhanced binding affinity towards a specific mannose receptor, such as Concanavalin A, due to the multivalent display of the monosaccharide, as well as reduced macrophage internalization. Coating with tetraethylente glycol of nanoMOFs after loading with doxorubicin is also described. Therefore, phosphorylated cyclodextrins offer a versatile platform to coat nanoMOFs in an organic solvent-free, one step manner, providing them with new biorecognition and/or "stealth" properties.

Mannoside and 1,2-mannobioside ß-cyclodextrin-scaffolded NO-photodonors for targeting antibiotic resistant bacteria.

Two ß-cyclodextrin derivatives randomly appended on the primary face with both the nitric oxide (NO) photodonor 4-nitro-3-(trifluoromethyl)aniline and a mannose or α(1→2)mannobioside residue are reported to construct targeted NO photoreleasing nanocarriers. 2D ROESY and PGSE NMR suggested supramolecular homodimerization in water by inclusion of the nitroaniline group into the facing macrocycle cavities. Isothermal titration calorimetry on their concanavalin A lectin binding showed an exothermic binding event to the lectin and an endothermic process during the dilution of the conjugates. Both α(1→2)mannobioside and the nitroaniline moieties significantly enhanced the binding to the lectin. These effects might arise from a better fit within the carbohydrate-recognition site in the former case and a multivalent effect caused by homodimerization in the latter. Direct detection of NO by amperometric technique shows that both ß-cyclodextrin derivatives release this radical upon excitation with visible light with higher efficiency than the unfunctionalized NO photodonor.

Electrochemical detection of glutathione S-transferase: an important enzyme in the cell protective mechanism against oxidative stress.

Oxidative stress arises when the antioxidant capacity of cells to clean the excess production of reactive oxygen species (ROS) decreases. Several human diseases seem to be related with an increment in the oxidative stress. In this regard, GSH present in the cells works by neutralizing ROS and other xenobiotics through the glutathione S-transferase (GST) enzyme. Thus, the level of expression of GST is an important factor in determining the sensitivity of cells to toxic chemicals or xenobiotic compounds. Therefore, the detection of GST levels is fundamental in the clinical diagnosis of ROS-related diseases. Here, we describe a methodology, based on the voltammetric properties of the ferrocene group (used as electrochemical probe), which can be applied for selective detection of GST levels in human cells. The electrochemical signal measured is associated to the specific interaction of a ferrocenyl-GSH derivate with the G- and H-sites of this enzyme.

Assessing solvent derivatization and carbon dioxide supercritical fluid simultaneous extraction/derivatization of cyprodinil.

Derivatization of cyprodinil with different reagents and solvents has been evaluated to improve the GC/MS characterization of this fungicide. After assessing some preliminary acylation and silylation reactions, derivatization with anhydrous heptafluorobutyric anhydride (HFBA) was selected as the best derivatization option for cyprodinil. The HFBA-cyprodinil derivative was clearly identified and characterized by GC/MS (ion-trap). The spectrum of the HFBA derivative of cyprodinil was characterized by the base peak, 252 m/z ion, and two other ions with relative abundances of 5% (224 m/z ion) and 4% (420 m/z molecular ion). Conversion rates in the range of 83-92% were obtained when 0.1-1 µg cyprodinil were derivatized in vial without solvent at 25ºC temperature for 120 min, with 5 µL HFBA and 5 µL pyridine. Simultaneous extraction-derivatization of cyprodinil in supercritical carbon dioxide was only achieved when no modifier was present, but conversion/recovery rates obtained in the replicate experiments carried out with 15 mL supercritical carbon dioxide at 50°C and 200 atm (n = 5), 300 atm (n = 7), and 400 atm (n = 5) were no reproducible (RSD > 50%) and ranged between 10% and 45% (related to the signal obtained for derivatization in vial).

Asn112 in Plasmodium falciparum glutathione S-transferase is essential for induced reversible tetramerization by phosphate or pyrophosphate.

The glutathione S-transferase from Plasmodium falciparum presents distinct features which are absent from mammalian GST isoenzyme counterparts. Most apparent among these are the ability to tetramerize and the presence of a flexible loop. The loop, situated between the 113-119 residues, has been reported necessary for the tetramerization process. In this article, we report that a residue outside of this loop, Asn112, is a key to the process - to the point where the single Asn112Leu mutation prevents tetramerization altogether. We propose that a structural pattern involving the interaction of the Asn112 and Lys117 residues from two neighboring subunits plays a role in keeping the tetramer structure stable. We also report that, for the tetramerization of the wild-type PfGST to occur, phosphate or pyrophosphate anions must be present. In other words, tetramerization is a phosphate- or pyrophosphate-induced process. Furthermore, the presence of magnesium reinforces this induction. We present experimental evidence for these claims as well as a preliminary calorimetric and kinetic study of the dimeric Asn112Leu PfGST mutant. We also propose a putative binding site for phosphate or pyrophosphate anions through a comparative structural analysis of PfGST and pyrophosphatases from several organisms. Our results highlight the differences between PfGST and the human isoenzymes, which make the parasite enzyme a suitable antimalarial target.

ß-Cyclodextrin-bearing gold glyconanoparticles for the development of site specific drug delivery systems.

Three novel gold nanoparticles containing multiple long, flexible linkers decorated with lactose, ß-cyclodextrin, and both simultaneously have been prepared. The interaction of such nanoparticles with ß-d-galactose-recognizing lectins peanut agglutinin (PNA) and human galectin-3 (Gal-3) was demonstrated by UV-vis studies. Gal-3 is well-known to be overexpressed in several human tumors and can act as a biorecognizable target. This technique also allowed us to estimate their loading capability toward the anticancer drug methotrexate (MTX). Both results make these glyconanoparticles potential site-specific delivery systems for anticancer drugs.

Calorimetric studies of ligands binding to glutathione S-transferase from the malarial parasite Plasmodium falciparum.

Glutathione S-transferase, from the malarial parasite Plasmodium falciparum (PfGST), exerts a protective role in the organism and is thus considered an interesting target for antimalarial drug development. In contrast to other GSTs, it is present in solution as a tetramer and a dimer in equilibrium, which is induced by glutathione (GSH). These properties prevent a calorimetric titration from being conducted upon binding of ligands to this protein's G-site. Thermodynamic characterization can be an optimal strategy for antimalarial drug development, and isothermal titration calorimetry (ITC) is the only technique that allows the separation of the binding energy into both enthalpic and entropic contributions. This information facilitates an understanding of the changes in the drugs' substituents, improving their affinity and specificity. In this study, we have applied a nontypical ITC procedure, based on the dissociation of the ligand-protein complex, to calorimetrically study the binding of the GSH substrate, and the glutathione sulfonate competitive inhibitor, to dimeric PfGST over a temperature range of 15-37 °C. The optimal experimental conditions for applying this procedure have been optimized by studying the dimer to tetramer conversion using size exclusion chromatography. The binding of these ligands to dimeric PfGST is noncooperative, the affinity of glutathione sulfonate being approximately 2 orders of magnitude higher than that of its natural substrate GSH. The binding of both ligands is enthalpically favorable and entropically unfavorable at all the studied temperatures. These results demonstrate that, although PfGST presents differences when compared to other known GSTs, these ligands bind to its dimeric form with a similar affinity and energetic balance. However, in contrast to that of other GSTs, the binding of GSH to protein, in the absence of the ligand, is slow.

Poly(amido amine)-based mannose-glycodendrimers as multielectron redox probes for improving lectin sensing.

An easy-to-prepare series of electroactive poly(amido amine) (PAMAM)-based dendrimers of generations G0 to G2 having mannopyranosylferrocenyl moieties in the periphery to detect carbohydrate-protein interactions is reported. The synthesis involved the functionalization of the PAMAM surface with azidomethylferrocenyl groups and subsequent coupling of mannoside units by the Cu(I)-catalyzed Huisgen reaction. The binding affinity of the series of electroactive glycodendrimers was studied by isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Upon complexation of the glycodendrimers conjugates with prototypical concanavalin A (Con A), voltammograms showed a decrease of the peak current. Such dendrimers showed a notable improvement of redox sensing abilities toward Con A when compared with mono- and divalent analogues, based on both the glycoside multivalent and ferrocene dendritic effects.

Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1.

The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene-glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1.

Identifying and characterizing binding sites on the irreversible inhibition of human glutathione S-transferase P1-1 by S-thiocarbamoylation.

Human glutathione S-transferase P1-1 (hGST P1-1) is involved in cell detoxification processes through the conjugation of its natural substrate, reduced glutathione (GSH), with xenobiotics. GSTs are known to be overexpressed in tumors, and naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are effective cancer chemopreventive compounds. To identify and characterize the potential inhibitory mechanisms of GST P1-1 induced by isothiocyanate conjugates, we studied the binding of GST P1-1 and some cysteine mutants to the BITC-SG conjugate as well as to the synthetic S-(N-benzylcarbamoylmethyl)glutathione conjugate (BC-SG). We report here the inactivation of GST P1-1 through the covalent modification of two Cys47 residues per dimer and one Cys101. The evidence has been compiled by isothermal titration calorimetry (ITC) and electrospray ionization mass spectrometry (ESI-MS). ITC experiments suggest that the BITC-SG conjugate generates adducts with Cys47 and Cys101 at physiological temperatures through a corresponding kinetic process, in which the BITC moiety is covalently bound to these enzyme cysteines through an S-thiocarbamoylation reaction. ESI-MS analysis of the BITC-SG incubated enzymes indicates that although the Cys47 in each subunit is covalently attached to the BITC ligand moiety, only one of the Cys101 residues in the dimer is so attached. A plausible mechanism is given for the emergence of inactivation through the kinetic processes with both cysteines. Likewise, our molecular docking simulations suggest that steric hindrance is the reason why only one Cys101 per dimer is covalently modified by BITC-SG. No covalent inactivation of GST P1-1 with the BC-SG inhibitor has been observed. The affinities and inhibitory potencies for both conjugates are high and very similar, but slightly lower for BC-SG. Thus, we conclude that the presence of the sulfur atom from the isothiocyanate moiety in BITC-SG is crucial for its irreversible inhibition of GST P1-1.

Binding properties of ferrocene-glutathione conjugates as inhibitors and sensors for glutathione S-transferases.

The binding properties of two electroactive glutathione-ferrocene conjugates that consist in glutathione attached to one or both of the cyclopentadienyl rings of ferrocene (GSFc and GSFcSG), to Schistosoma japonica glutathione S-transferase (SjGST) were studied by spectroscopy fluorescence, isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Such ferrocene conjugates resulted to be competitive inhibitors of glutathione S-transferase with an increased binding affinity relative to the natural substrate glutathione (GSH). We found that the conjugate having two glutathione units (GSFcSG) exhibits an affinity for SjGST approximately two orders of magnitude higher than GSH. Furthermore, it shows negative cooperativity with the affinity for the second binding site two orders of magnitude lower than that for the first one. We propose that the reason for such negative cooperativity is steric since, i) the obtained thermodynamic parameters do not indicate profound conformational changes upon GSFcSG binding and ii) docking studies have shown that, when bound, part of the first bound ligand invades the second site due to its large size. In addition, voltammetric measurements show a strong decrease of the peak current upon binding of ferrocene-glutathione conjugates to SjGST and provide very similar K values than those obtained by ITC. Moreover, the sensing ability, expressed by the sensitivity parameter shows that GSFcSG is much more sensitive than GSFc, for the detection of SjGST.

ß-Cyclodextrin dimers linked through their secondary faces with rigid spacer arms as hosts for bile salts.

A convenient synthesis of ß-cyclodextrin dimers in which the two cyclodextrin units are linked by rigid tethers of relatively short length through their secondary sides is reported. Compounds hexa-2,4-diynediyl- and 1,4-phenylenediethyne-briged ß-cyclodextrin dimers are obtained in good yields from mono-2-O-propargyl-ß-cyclodextrin through Pd-mediated oxidative homo- and heterocoupling reactions. Isothermal titration calorimetry and NMR spectroscopy (PGSE and 2D-ROESY) are used to determine the thermodynamic parameters (K, ΔH, and TΔS°) for the complexation of such ß-cyclodextrin dimers with sodium cholate, deoxycholate, and chenodeoxycholate as well as to estimate the size of the supramolecular structures. The binding of bile salts is enhanced relative to that of native ß-cyclodextrin. Although chenodeoxycholate salt binds in a 1:1 fashion, cholate and deoxycholate salts bind in a 1:2 sequential mode.

Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V.

The diuretic drug ethacrynic acid (EA), both an inhibitor and substrate of pi class glutathione S-transferase (GST P1-1), has been tested in clinical trials as an adjuvant in chemotherapy. We recently studied the role of the active site residue Tyr-108 in binding EA to the enzyme and found that the analysis was complicated by covalent binding of this drug to the highly reactive Cys-47. Previous attempts to eliminate this binding by chemical modification yielded ambiguous results and therefore we decided here to produce a double mutant C47S/Y108V by site directed mutagenesis and further expression in Escherichia coli and the interaction of EA and its GSH conjugate (EASG) examined by calorimetric studies and X-ray diffraction. Surprisingly, in the absence of Cys-47, Cys-101 (located at the dimer interface) becomes a target for modification by EA, albeit at a lower conjugation rate than Cys-47. The Cys-47 → Ser mutation in the double mutant enzyme induces a positive cooperativity between the two subunits when ligands with affinity to G-site bind to enzyme. However, this mutation does not seem to affect the thermodynamic properties of ligand binding to the electrophilic binding site (H-site) and the thermal or chemical stability of this double mutant does not significantly affect the unfolding mechanism in either the absence or presence of ligand. Crystal structures of apo and an EASG complex are essentially identical with a few exceptions in the H-site and in the water network at the dimer interface.

Kinetic properties and specificity of trimeric Plasmodium falciparum and human dUTPases.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. Hydrolysis of other nucleotides similar in structure to dUTP would be physiologically negative and therefore high substrate specificity is essential. Binding and hydrolysis of nucleotides different to dUTP by the dUTPases from Plasmodium falciparum (PfdUTPase) and human (hdUTPase) was evaluated by applying isothermal titration calorimetry (ITC). The ribo and deoxyribonucleoside triphosphates dGTP, dATP, dCTP, dTTP, UTP, FdUTP and IdUTP have been analysed. dUTP and FdUTP were the most specific substrates for both enzymes. The specificity constants (k(cat)/K(m)) for the remaining ones, except for the IdUTP, were very similar for both enzymes, although PfdUTPase showed a slightly higher specificity for dCTP and UTP and the human enzyme for dTTP and dCTP. PfdUTPase was very efficient in using FdUTP as substrate indicating that small size substituents in the 5' position are well tolerated. In addition product inhibition was assessed by binding studies with the nucleoside monophosphate derivatives and thermodynamic parameters were established. When FdUTP hydrolysis was monitored, Plasmodium dUTPase was more sensitive to end-product inhibition by FdUMP than the human enzyme. Taken together these results highlight further significant differences between the human and Plasmodium enzymes that may be exploitable in selective inhibitor design.

Influence of the H-site residue 108 on human glutathione transferase P1-1 ligand binding: structure-thermodynamic relationships and thermal stability.

The effect of the Y108V mutation of human glutathione S-transferase P1-1 (hGST P1-1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 --> Val resulted in a 3D-structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H-site) and glutathione binding site (G-site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H-site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K(d) approximately 0.5 microM) when compared with those of the parent compounds, K(d) (EA) approximately 13 microM, K(d) (GSH) approximately 25 microM. The EA moiety of the conjugate binds in the H-site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the Delta C(p) values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.

Ferrocene-beta-cyclodextrin conjugates: synthesis, supramolecular behavior, and use as electrochemical sensors.

Ferrocene with a beta-cyclodextrin unit bound to one or both cyclopentadienyl rings through the secondary face were conveniently synthesized by regiospecific copper(I)-catalyzed cycloaddition of 2-O-propargyl-beta-cyclodextrin to azidomethyl or bis(azidomethyl)ferrocene. The supramolecular behavior of the synthesized conjugates in both the absence and presence of bile salts (sodium cholate, deoxycholate, and chenodeoxycholate) was studied by using electrochemical methods (cyclic and differential pulse voltammetry), isothermal titration calorimetry, and NMR spectroscopy (PGSE, CPMG, and 2D-ROESY). These techniques allowed the determination of stability constants, mode of inclusion, and diffusion coefficients for complexes formed with the neutral and, in some cases, the oxidized states of the ferrocenyl conjugates. It was found that the ferrocenyl conjugate with one beta-cyclodextrin unit forms a redox-controllable head-to-head homodimer in aqueous solution. The ferrocene-bis(beta-cyclodextrin) conjugate is present in two distinguishable forms in aqueous solution, each one having a different half-wave oxidation potential for the oxidation of the ferrocene. By contrast, only one distinguishable form for the oxidized state of the ferrocene-beta-cyclodextrin conjugate is detectable. The redox-sensing abilities of the synthesized conjugates towards the bile salts were evaluated based on the observed guest-induced changes in both the half-wave potential and the current peak intensity of the electroactive moiety.

Ferrocene-carbohydrate conjugates as electrochemical probes for molecular recognition studies.

We report two methods that have allowed the attachment of glucose, mannose and lactose to one or both of the cyclopentadienyl rings of ferrocene. The resulting ferrocene-carbohydrate conjugates were synthesised by the reaction of thioglycosides with ferrocenemethanol and 1,1'-ferrocenedimethanol in acidic media. A second method based on the regiospecific copper(I)-catalysed cycloaddition of propargyl glycoside, azidomethyl and bis(azidomethyl)ferrocene as well as azidoethyl glycoside and ethynylferrocene was also used and led to the synthesis of 1,2,3-triazole-containing glycoconjugates. The electrochemical behaviour of the synthesised glycoconjugates was investigated. In addition, their binding interactions with beta-cyclodextrin were studied by means of NMR spectroscopy, isothermal titration calorimetry, and cyclic and differential pulse voltammetric experiments. These techniques allowed the determination of the thermodynamic parameters of the complexes, the stability constants for the complexes formed with both the neutral and the oxidised states of the ferrocenyl glycoconjugates, the mode of inclusion and the diffusion coefficients for both the glycoconjugates and the complexes.

Ferrocene-mannose conjugates as electrochemical molecular sensors for concanavalin A lectin.

The binding affinity of a series of electroactive glycoconjugates, based on a ferrocene core bearing alpha-mannose units on one or both of its cyclopentadienyl rings, to lectin Con A was studied by isothermal titration calorimetry (ITC) and voltammetry. Voltammetric measurements were performed by differential pulse adsorptive stripping voltammetry (DPAdSV). Upon complexation of ferrocene-mannose conjugates with Con A, voltammograms showed a decrease of the peak current. Both the monomannosylated ferrocene and the bis(mannosylated) ferrocene derivatives form more stable complexes with Con A than methyl alpha-D-mannopyranoside. Bis(mannosylated) ferrocene conjugates were found to bind to Con A with enhanced affinity due to the multivalent effect. A comparison of the thermodynamic data obtained by ITC and voltammetry is presented.

Kinetic and thermodynamic characterization of dUTP hydrolysis by Plasmodium falciparum dUTPase.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and plays an important role in nucleotide metabolism and DNA replication controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. Isothermal titration calorimetry has been applied to the determination of the kinetic and thermodynamic parameters of the trimeric Plasmodium falciparum dUTPase, a potential drug target against malaria. The role of divalent ions in binding, and inhibition by different uridine derivatives has been assessed. When dUTP hydrolysis in the presence of EDTA was evaluated, a 105-fold decrease and a 12-fold increase of the k(cat) and Km values, respectively, were observed when compared with the dUTP.Mg2+ complex. Calculation of the activation energy, E(a), and the thermodynamic activation parameters showed that the energetic barrier was approximately 4-fold higher when Mg2+ was depleted. Other divalent ions such as Co2+ or Mn2+ can substitute the physiological cofactor, however the k(cat) was significantly reduced compared to dUTP.Mg2+. Binding and inhibition by dU, dUMP, dUDP, and alpha,beta-imido-dUTP were analysed by ITC and compared with data obtained by spectrophotometric methods and binding equilibrium studies. Product inhibition (Kip dUMP: 99.34 microM) was insignificant yet Ki values for dUDP and alpha,beta-imido-dUTP were in the low micromolar range. The effect of ionic strength on protein stability was also monitored. DSC analysis evidenced a slight increase in the unfolding temperature, Tm, with increasing salt concentrations. Moreover, the thermal unfolding pathway in the presence of salt fits adequately to an irreversible two-state model (N3-->3D).