A dodecameric self-assembled calix[4]arene aggregate with two types of cavities.

Twelve molecules of ß-carbonyl-para-octyl-calix[4]arene assemble in an aggregate containing two types of cavities filled by water molecules and they pack in a cubic structure. Both the aggregates and the packing resemble that observed for inverse micelles.

Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin.

Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB)>multi-metal binding site (MBS)>N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9+/-5, 1.1+/-0.5, and 0.9+/-0.3x10(4)M(-1), respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.

A diadenosine 5',5''-P1P4 tetraphosphate (Ap4A) hydrolase from Arabidopsis thaliana that is activated preferentially by Mn2+ ions.

Asymmetrical diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) hydrolases are key enzymes controlling the in vivo concentration of Ap(4)A--an important signaling molecule involved in regulation of DNA replication and repair, signaling in stress response and apoptosis. Sequence homologies indicate that the genome of the model plant Arabidopsis thaliana contains at least three open reading frames encoding presumptive Ap(4)A hydrolases: At1g30110, At3g10620, and At5g06340. In this work we present efficient overexpression and detailed biochemical characteristics of the AtNUDX25 protein encoded by the At1g30110 gene. Aided by the determination of the binding constants of Mn(Ap(4)A) and Mg(Ap(4)A) complexes using isothermal titration calorimetry (ITC) we show that AtNUDX25 preferentially hydrolyzes Ap(4)A in the form of a Mn(2+) complex.

Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study.

Diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.

HEW lysozyme salting by high-concentration NaCl solutions followed by titration calorimetry.

Concentration dependence of NaCl salting of 0-1.5 mM lysozyme solution in 0.1 M sodium acetate buffer, pH 4.25, was investigated for NaCl concentration varying up to 0.9 M. Calorimetric experiments demonstrated that depending on the salt concentration the estimated number of the binding sites on the lysozyme surface varied in the range of 5 up to 13, and the increase of salt concentration caused the decrease of the number of accessible sites. The small, but significant, local maximum centered at 0.63 M NaCl concentration indicated the specific salting-out of the lysozyme accompanied by binding of approximately 2-3 chloride anions. Generalized McMillan and Mayer's approach reduced to the third-order virial coefficients demonstrates the domination of lysozyme aggregation upon salt addition (a(21)-h(xxy)) and salt organization on the lysozyme surface (a(12)-h(xyy)) processes.

Interaction between yeast eukaryotic initiation factor eIF4E and mRNA 5' cap analogues differs from that for murine eIF4E.

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (Kas) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5' cap than those of the murine and human proteins. The observed differences in the Kas values and the enthalpy changes of the association (deltaH(o)) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.