Thermodynamics of aminoglycoside binding to aminoglycoside-3'-phosphotransferase IIIa studied by isothermal titration calorimetry

The aminoglycoside-3'-phosphotransferase IIIa [APH(3')-IIIa] phosphorylates aminoglycoside antibiotics and renders them ineffective against bacteria. APH(3')-IIIa is the most promiscuous aminoglycoside phosphotransferase enzyme, and it modifies more than 10 different aminoglycoside antibiotics. A wealth of information exists about the enzyme; however, thermodynamic properties of enzyme-aminoglycoside complexes are still not known. This study describes the determination of the thermodynamic parameters of the binary enzyme-aminoglycoside and the ternary enzyme-metal-ATP-aminoglycoside complexes of structurally related aminoglycosides using isothermal titration calorimetry. Formation of the binary enzyme-aminoglycoside complexes is enthalpically driven and exhibits a strongly disfavored entropic contribution. Formation of the ternary enzyme-metal-ATP-aminoglycoside complexes yields much smaller negative DeltaH values and more favorable entropic contributions. The presence of metal-ATP generally increases the affinity of aminoglycosides to the enzyme. This is consistent with the kinetic mechanism of the enzyme in which ordered binding of substrates occurs. However, the observed DeltaH values neither correlate with kinetic parameters k(cat), K(m), and k(cat)/K(m) nor correlate with the molecular size of the substrates. Comparison of the thermodynamic properties of the complexes formed by structurally similar aminoglycosides indicated that the 2'- and the 6'-amino groups of the substrates are involved in binding to the enzyme. Thermodynamic properties of the complexes formed by aminoglycosides differing only at the 3'-hydroxyl group suggested that the absence of this group does not alter the thermodynamic parameters of the ternary APH(3')-IIIa-metal-ATP-aminoglycoside complex. Our results also indicate that protonation of ligand and protein ionizable groups is coupled to the complex formation between aminoglycosides and APH(3')-IIIa. Comparison of DeltaH values for different aminoglycoside-enzyme complexes indicates that enzyme and substrates undergo significant conformational changes in complex formation.     

An automated differential thermal and potentiometric titration apparatus for binding studies

A differential pH-termal titration apparatus is described which can detect pH differences with a sensitivity of ±0.0001 pH units and a thermal sensitivity of ±0.00002°C at a time constant of 0.1 s. With a reaction which yields 1 kcal mol⁻¹, the current system can detect concentrations as low as 4×10⁻⁶ M or, in a 2 ml volume, a total amount of 40 nmol. With a time constant of 0.1 s, the sensitivity is 20±4 μ°C. The experimental protocol is specified by a microprocessor and three modes of operation are possible: titration at constant rate of reagent addition, titration at variable rates of addition so that the contents of both cells are at either constant pH or at a constant temperature and variable rate when a rate of change is specified. Experimental data are collected in files, corrected for heat loss, initial baseline drift, and changes in volume. The final corrected from the standardized run of 0.01338 M HCl in 0.2 M KCl at 25°C calibrate the pH scale yielded the calorimetric conversion constants and pK_w which are calculated and stored for subsequent corrections for the titration of an unknown acid or the measurement of bindin constants and heats.     

Screening of metal complex–amino acid side chain interactions by potentiometric titration

Reversible coordination of amino acid side chains to metal complexes is widely used in protein purification (IMAC technique), but available data on affinity and selectivity of such binding processes are limited. We use potentiometric titration of a series of metal complexes with vacant coordination sites in the presence of molecules resembling amino acid side chain functionalities to screen for new affinities. The investigation confirms documented affinities of imidazole to nickel(II) and copper(II) IDA and NTA complexes, and discovers a hitherto unknown binding of zinc(II)- and cadmium(II) cyclen complexes to imidazole.     

Determination of binding constants of cyclodextrin inclusion complexes with amino acids and dipeptides by potentiometric titration

Cyclodextrins are well known for their ability to separate enantiomers of drugs, natural products, and other chiral substances using HPLC, GC, or CE. The resolution of the enantiomers is due to the formation of diastereomeric complexes between the cyclodextrin and the pairs of enantiomers. The aim of this study was to determine the binding constants of the complexes between alpha- and beta-cyclodextrin and the enantiomers of a series of aliphatic and aromatic amino acids, and dipeptides, using a potentiometric titration method. The results of this method are compared to other methods, and correlated to findings in cyclodextrin-modified capillary electrophoresis and possible complex structures. Potentiometric titration was found to be an appropriate tool to determine the binding constants of cyclodextrin inclusion complexes.     

Interaction of anthracyclines with nucleotides and related compounds studied by spectroscopy

Interaction of the antitumour anthracyclines with mononucleotides and related compounds can be assessed through the perturbation of the spectral properties of the drugs. Purine-derived compounds induce spectral changes more efficiently than pyrimidine derivatives. No marked differences are observed when mono-, di- or triphosphate derivatives, deoxy forms, nucleosides or free nitrogen bases are used for the experiments. Visible absorbance data indicate the existence of a drug/purine nucleotide complex in solution. Assuming a simple equilibrium, this complex would be of low affinity (Keq 100 M-1). Circular dichroism spectra of daunomycin in the presence of ATP suggest that the resulting daunomycin/ATP complexes are not comparable to those formed by intercalation of the anthracycline into DNA. 31P-NMR of ATP in the presence of daunomycin does not support the notion that anthracycline/nucleotide complex formation involves interaction through the phosphate group(s) of the nucleotide. Analysis of the quenching of the drug's intrinsic fluorescence in the presence of nucleotides indicates a predominantly collisional, dynamic quenching mechanism. Values in the 2-6 mM and 85-100 mM range, respectively, are estimated for the reciprocal of the Stern-Volmer quenching constant for a variety of purine and pyrimidine derivatives. This indicates that purine derivatives are highly efficient quenchers of the fluorescence of anthracyclines, while pyrimidine derivatives are not. The fluorescence lifetime of daunomycin in the absence of quencher and the Stern-Volmer quenching constants obtained for different nucleotides are used to calculate the apparent bimolecular rate constants for collisions between fluorophore and quencher to occur. Values of (2-3) X 10(11) and 1 X 10(10) M-1 X s-1 are obtained, respectively, for purine and pyrimidine derivatives. This suggests a combination of static and dynamic quenching processes for purine compounds, which is consistent with the drug/purine nucleotide complex formation detected by visible absorbance. Because of the high intracellular concentration of certain nucleotides, particularly ATP, the above processes are predicted to be highly significant 'in vivo'.     

Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding.

We characterized the thermodynamics of binding reactions of nucleotides ADP and ATPgammaS (a nonhydrolyzable analog of ATP) to GroEL in a temperature range of 5 degrees C to 35 degrees C by isothermal titration calorimetry. Analysis with a noncooperative binding model has shown that the bindings of nucleotides are driven enthalpically with binding constants of 7x103 M-1 and 4x104 M-1 for ADP and ATPgammaS, respectively, at 26 degrees C and that the heat capacity change DeltaCp is about 100 cal/mol.K for both the nucleotides. The stoichiometries of binding were about 8 and 9 molecules for ADP and ATPgammaS, respectively, per GroEL tetradecamer at 5 degrees C, and both increased with temperature to reach about 14 (ADP) and 12 (ATPgammaS) for both nucleotides at 35 degrees C. The absence of initial increase of binding heat as well as Hill coefficient less than 1.2, which were obtained from the fitting to the model curve by assuming positive cooperativity, showed that there was virtually no positive cooperativity in the nucleotide bindings. Incorporating a difference in affinity for the nucleotide (ADP and ATPgammaS) between the two rings of GroEL into the noncooperative binding model improved the goodness of fitting and the difference in the affinity increased with decreasing temperature.     

Oleanolic acid and related derivatives as medicinally important compounds

Oleanolic acid has been isolated from chloroform extract of Olea ferruginea Royle after removal of organic bases and free acids. The literature survey revealed it to be biologically very important. In this review the biological significance of oleanolic acid and its derivatives has been discussed. The aim of this review is to update current knowledge on oleanolic acid and its natural and semisynthetic analogs, focussing on its cytotoxic, antitumer, antioxidant, anti-inflamatory, anti-HIV, acetyl cholinesterase, alpha-glucosidase, antimicrobial, hepatoprotective, anti-inflammatory, antipruritic, spasmolytic activity, anti-angiogenic, antiallergic, antiviral and immunomodulatory activities. We present in this review, for the first time, a compilation of the most relevant scientific papers and technical reports of the chemical, pre-clinical and clinical research on the properties of oleanolic acid and its derivatives.     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.