Reverse Titration Research Articles

Microwave Plasma Sintered Nanocrystalline Bi2O3−HfO2−Y2O3 Composite Solid Electrolyte

Processing and characterization of nanocrystalline Bi2O3−HfO2-Y2O3 composite solid electrolyte having high density and conductivity has been investigated and reported in this paper. Nanopowders of mixed bismuth oxide, hafnia, and yttrium oxide in Bi2O3:HfO2:Y2O3 = 3:2:1 molar ratio have been prepared by a reverse titration chemical coprecipitation from Bi3+, Hf4+, and Y3+ containing aqueous solution. The high density, nanocrystalline Bi2O3-HfO2−Y2O3 solid electrolyte has been synthesized by microwave plasma and pressureless sintering. From the XRD results, the growth behavior indicates that growth of both δ-Bi2O3 and c-HfO2 crystallites obeys the parabolic rate law, expressed as (D − D0)2 = kt, during the sintering process. After the samples were sintered in microwave plasma at 700 °C for 30 min, the relative density was found to be greater than 96%. Moreover, the sintered specimens exhibit considerably finer microstructure and greater densification compared to that of samples sintered by conventional pressureless sintering. Results of AC impedance spectroscopy of the solid electrolyte indicates that the conductivity of nanocrystalline Bi2O3−HfO2−Y2O3 electrolyte was more than 10-6 S cm-1 at 350 °C and 10-2 S cm-1 at 550 °C, which is significantly higher than that of microcrystalline HfO2-based solid electrolyte.

Preparation of dense nanocrystalline Bi 2O 3-HfO 2-Y 2O 3 ceramic by microwave plasma sintering

Processing of nanocrystalline Bi 2O 3-HfO 2-Y 2O 3 ceramic having high density has been investigated and reported in this paper. Nanopowders of mixed bismuth oxide, hafnia and yttrium oxide have been prepared by a reverse titration chemical coprecipitation from Bi 3+, Hf 4+ and Y 3+ containing aqueous solution. The high density, nanocrystalline Bi 2O 3-HfO 2-Y 2O 3 ceramic has been synthesized by microwave plasma sintering. The XRD results of grain growth behavior indicates that growth of both δ-Bi 2O 3 and c-HfO 2 crystallites obeys the parabolic rate law, expressed as ( D– D 0) 2 = Kt, during sintering process. After sintering at 700 °C for 60 min, the relative density of the samples sintered by microwave plasma has been found to be greater than 97%, and the samples exhibit considerably finer microstructure with an average size between 60 and 70 nm and equiaxed morphology and better density comparing with that of samples sintered by conventional pressureless sintering. In addition, mechanical properties of nanocrystalline Bi 2O 3-HfO 2-Y 2O 3 ceramic has been improved greatly compareing with nanocrystalline Bi 2O 3-Y 2O 3 ceramic.

Molecular Analysis of the Interaction between the Hematopoietic Master Transcription Factors GATA-1 and PU.1

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Dynamics of Bacteriophage T4 Presynaptic Filament Assembly from Extrinsic Fluorescence Measurements of Gp32-Single-stranded DNA Interactions

In the bacteriophage T4 homologous recombination system, presynaptic filament assembly on single-stranded (ssDNA) DNA requires UvsX recombinase, UvsY mediator, and Gp32 ssDNA-binding proteins. Gp32 exerts both positive and negative effects on filament assembly: positive by denaturing ssDNA secondary structure, and negative by competing with UvsX for ssDNA binding sites. UvsY is believed to help UvsX displace Gp32 from the ssDNA. To test this model we developed a real-time fluorescence assay for Gp32-ssDNA interactions during presynapsis, based on changes in the fluorescence of a 6-iodoacetamidofluorescein-Gp32 conjugate. Results demonstrate that the formation of UvsX presynaptic filaments progressively disrupts Gp32-ssDNA interactions. Under stringent salt conditions the disruption of Gp32-ssDNA by UvsX is both ATP- and UvsY-dependent. The displacement of Gp32 from ssDNA during presynapsis requires ATP binding, but not ATP hydrolysis, by UvsX protein. Likewise, UvsY-mediated presynapsis strongly requires UvsY-ssDNA interactions, and is optimal at a 1:1 stoichiometry of UvsY to UvsX and/or ssDNA binding sites. Presynaptic filaments formed in the presence of UvsY undergo assembly/collapse that is tightly coupled to the ATP hydrolytic cycle and to stringent competition for ssDNA binding sites between Gp32 and various nucleotide-liganded forms of UvsX. The data directly support the Gp32 displacement model of UvsY-mediated presynaptic filament assembly, and demonstrate that the transient induction of high affinity UvsX-ssDNA interactions by ATP are essential, although not sufficient, for Gp32 displacement. The underlying dynamics of protein-ssDNA interactions within presynaptic filaments suggests that rearrangements of UvsX, UvsY, and Gp32 proteins on ssDNA may be coupled to central processes in T4 recombination metabolism.

Evaluation of PDTC for Removing Ni(II) from Human Serum Transferrin in Vitro

Pyrrolidine dithiocarbamate (PDTC)can be an oral chelator with pKa=3.300+/-0.002. It behaves as a bidentate ligand at serum pH. The effect of pH on Ni2+-Tf indicated that the maximum adsorption was at pH=7.4. The effective Ni-PDTC binding constant was determined (logk=11.1+/-0.1) for the 1:2 Ni(PDTC)2 complex using UV-vis spectra. The isosbestic point at 298 indicated that the complexation reaction was done directly (without side reaction). Removal of Ni from transferrin (Tf) was investigated by reverse titration of PDTC at 25 masculineC and pH=7.4 using UV-vis spectra. PDTC is able to remove 25% of Ni from human serum transferrin.

Fusion Peptide of Influenza Hemagglutinin Requires a Fixed Angle Boomerang Structure for Activity

The fusion peptide of influenza hemagglutinin is crucial for cell entry of this virus. Previous studies showed that this peptide adopts a boomerang-shaped structure in lipid model membranes at the pH of membrane fusion. To examine the role of the boomerang in fusion, we changed several residues proposed to stabilize the kink in this structure and measured fusion. Among these, mutants E11A and W14A expressed hemagglutinins with hemifusion and no fusion activities, and F9A and N12A had no effect on fusion, respectively. Binding enthalpies and free energies of mutant peptides to model membranes and their ability to perturb lipid bilayer structures correlated well with the fusion activities of the parent full-length molecules. The structure of W14A determined by NMR and site-directed spin labeling features a flexible kink that points out of the membrane, in sharp contrast to the more ordered boomerang of the wild-type, which points into the membrane. A specific fixed angle boomerang structure is thus required to support membrane fusion.

Ruthenium(ii) complex of 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline: synthesis, spectrophotometric pH titrations and DNA interaction

A Ru(II) complex of [(bpy)2Ru(aip)](ClO4)2·H2O (bpy = 2,2′-bipyridine, aip = 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline) was newly synthesized. The ground- and excited-state acid–base properties of the complex were studied by UV–visible and emission spectrophotometric pH titrations. The excited-state ionization constants of pKa1* = 2.57, pKa2* = 8.47 are comparable to and 1.3 orders of magnitude greater than the ground-state ones, respectively. The interaction of the complex with calf thymus DNA was investigated by UV–visible and emission spectroscopy, steady-state emission quenching by [Fe(CN)6]4–, competitive binding with ethidium bromide, reverse salt titrations, DNA melting experiments, as well as viscosity measurements. The results indicated that the complex bound to the DNA by interaction of the anthryl moiety of the complex with an intrinsic binding constant of the order of 104 M−1 in buffered 50 mM NaCl at room temperature, and non-electrostatic binding free energy made a predominant contribution to the binding free energy.

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.

Thrombin Aptamer HD1 Inhibits Prothrombin Activation by Binding Proexosite 1 on Prothrombin.

Functional maturation of exosites 1 and 2 during prothrombin activation endows thrombin with its physiological activities. Thrombin exosite 1 binds ligands such as fibrinogen, the thrombin receptor, and hirudin, and orients them to the active site. In contrast, thrombin exosite 2 binds ligands such as heparin, platelet glycoprotein Iba, and the chondroitin sulfate moiety of thrombomodulin molecules; ligands that do not directly interact with the active site. Neither exosite is fully functional in prothrombin. Exosite 2 only becomes functional when fragment 2 is released. In contrast, there is evidence for existence of a proexosite 1 in prothrombin which contributes to prothrombin activation by prothrombinase. This domain in prothrombin lacks fibrin-binding capacity, but has been implicated in the interaction with factor Va (fVa), a necessary step for enhanced prothrombin activation by prothrombinase. In the present study, we used fluorescent (F) HD1 and HD22, DNA aptamers that bind to exosites 1 and 2, respectively, to study the exosites in prothrombin, prothrombin intermediates and thrombin. F-HD22 does not bind to prothrombin and only binds prothrombin intermediates lacking fragment 2. In contrast, F-HD1 binds to prothrombin and all of the prothrombin intermediates with Kd values ranging from 80 to 25 nM for prothrombin and thrombin, respectively. These findings are different from what has been reported with the hirudin fragment, Hir54–65, which exhibited a 60-fold lower affinity for prothrombin than for thrombin. To better explore the involvement of proexosite 1 in the activation of prothrombin by prothrombinase, we examined the effect of HD1 on prothrombin activation by intact prothrombinase, or by various components of the prothrombinase complex. HD1 inhibits prothrombin activation by prothrombinase with an IC50 of 140 nM. Omitting fVa from the prothrombinase complex abolishes the inhibitory effect of HD1, supporting the concept that the inhibitory effect of HD1 reflects disruption of the interaction between prothrombin and fVa. In similar experiments, Hir54–65 inhibits prothrombin activation by prothrombinase with an IC50 of 225 nM, in agreement with previous studies. These data show that, like hirugen, HD1 binds to the fVa-binding domain on proexosite 1 of prothrombin. In order to refine the location of HD1 and Hir54–65 binding sites on thrombin and prothrombin, competitive binding experiments were performed. Hir54–65 displaces 80% of F-HD1 from thrombin and 70% from prothrombin. In reverse titrations, HD1 displaces 50% of F-Hir54–65 from thrombin, but does not displace F-Hir54–65 from prothrombin. Taken together, these data suggest that the HD1 binding domain is distinct, at least in part, from the Hir54–65 binding domain on prothrombin, an observation supported by examination of the crystal structures of complexes of HD1 or Hir54–65 with thrombin. In conclusion, our data demonstrate that the prsuggests that the HD1 binding site on prothrombin more closely coincides with the fVa-binding site than does the hirugen-binding site. These findings provide new avenues for investigations into the oexosite 1 domain on prothrombin is fully capacitated for binding HD1. This role of exosite 1, not only in thrombin, but also in prothrombin.

Solution Structure, Stability, and Nucleic Acid Binding of the Hyperthermophile Protein Sso10b2,

The Sso10b (or Alba) family of proteins is a conserved group of archaeal and eukaryotic proteins which are thought to play a role in both chromatin organization and RNA metabolism. We describe here the solution structure and properties of Sso10b2 from Sulfolobus solfataricus. NMR data including residual dipolar couplings and (15)N relaxation data demonstrated that the protein adopts a beta(1)alpha(1)beta(2)alpha(2)beta(3)beta(4) topology with an IF-3-like fold. The protein dimerizes in solution at 30 degrees C via a hydrophobic surface defined by the C-terminal alpha(2)beta(3)beta(4) elements with a structure similar to one of the putative dimers indicated by previous crystal structures. DSC and circular dichroism data demonstrated an unusual two-state structural transition near the growth temperature which led to an increase in beta-sheet content without dissociation of the dimer. The cooperativity of the transition exceeded that of a dimer at pH 7, demonstrating the presence of higher order oligomers near the growth temperature at pH 7. Reverse titrations of Sso10b2 with nucleic acid showed that the protein binds single-stranded DNA (K(d) of 3 x 10(-)(7) M) with higher affinity than RNA (1.3 x 10(-)(6) M) or double-stranded DNA (1.5 x 10(-)(5) M) in 10 mM KH(2)PO(4) (pH 7.0, 20 degrees C). NMR chemical shift perturbation data indicated that single-stranded DNA and RNA binding occurred across the same dimer interface and encompassed a surface defined by the C-terminal ends of the beta(1), beta(2), and beta(3) strands of each monomer.

Processing of dense nanocrystalline Bi 2O 3-Y 2O 3 solid electrolyte

Processing of nanocrystalline Bi 2O 3-Y 2O 3 solid electrolyte having high density has been investigated and reported in this paper. Nanopowders of mixed bismuth oxide and yttrium oxide (75 mol% Bi 2O 3 + 25 mol% Y 2O 3) have been prepared by reverse titration chemical coprecipitation from Bi 3+ and Y 3+ containing aqueous solution. The high density, nanocrystalline Bi 2O 3-Y 2O 3 solid electrolyte has been synthesized by pressureless reactive sintering. The XRD results of the formation of nano δ-Bi 2O 3 and its grain growth indicates that the solid solution reaction between Y 2O 3 and β-Bi 2O 3 occurs essentially in the initial stage of sintering and the growth of nano δ-Bi 2O 3 crystallites obeys the parabolic rate law, expressed as ( D − D 0) 2 = K· t, during sintering process. After sintering at 600 °C for 2 h, the relative density of the samples has been found to be greater than 96%. The microstructure reveals that the nanocrystalline Bi 2O 3-Y 2O 3 solid electrolyte samples have dense equiaxed microstructure with few isolated pores and δ-Bi 2O 3 grains are less than 100 nm in diameter.

Role of Regulatory F-domain in Hepatocyte Nuclear Factor-4α Ligand Specificity

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.

Determination of Metal Speciation by Reverse Titrations

A new method is proposed to determine metal speciation by varying the concentration of a competing ligand at a constant metal concentration, with detection by cathodic stripping voltammetry. The free metal ion concentration is gradually lowered from its natural level while the method probes progressively deeper into the already complexed metal fraction: it is therefore a reverse titration rather than the forward titration which is used for conventional complexing ligand titrations. The sensitivity is greatest at the lowest free metal ion concentration, where it matters most, and the method can be carried out in a single sample aliquot in the voltammetric cell. The method is applied here to copper speciation, but in principle, it can also be used for other metals. Modeling shows that this method has good sensitivity at ligand concentrations near the metal concentration (lower as well as greater). Comparative measurements of copper speciation using reverse and forward titrations of representative water samples of oceanic and coastal origin show good agreement. The data showed that these samples did not contain low levels of strong ligands in addition to the ligands detected by the forward titrations.

Overexpression of Cyclin D1 in Accelerated-Phase Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a bi- or triphasic disease. Molecular markers distinct for the phase evolution would be clinically helpful. For signaling transformation and proliferation activities in CML, Bcr-Abl is the pivotal protein. As downstream signals of Bcr-Abl, RAS, PI3-K, and Stat 5 may lead to cell cycle progression mediated by increased expression of cyclin Ds. We analyzed copy numbers of bcr-abl and cyclin D1 transcripts by reverse transcription (RT) and competitive PCR titration in bone marrow cells of 20 patients with CML, 10 in chronic phase (CP) and the other 10 in accelerated phase (AP). The level of bcr-abl transcripts in the AP was not significantly higher than that in the CP; in contrast, the level of cyclin D1 transcripts in the AP was significantly higher than that in the CP (p < 0.001). Cyclin D1 RNA expression in the CP of CML was also found to have clinical relevance to time to AP transformation. The median time to AP transformation for the CP patients with cyclin D1 transcripts of ⩾̸ 1.50 × 104/μg RNA was significantly shorter than that for those with cyclin D1 transcripts < 1.50 × 104/μg RNA (15 vs. 67 months, p = 0.0354) although confirmation to conduct in a larger patient group is required. These results suggest that the expression level of cyclin D1 RNA in bone marrow cells is predictive of the phase evolution in CML and may be helpful in treatment decision-making.

Interaction of erythroid spectrin with hemoglobin variants: implications in β-thalassemia

Among the few studies, producing contradictory results, done on the interaction of erythroid membrane skeletal spectrin with hemoglobin (Hb), none has been able to provide a quantitative estimate of the association of spectrin with Hb. In this work, studies on the interactions of erythroid spectrin with Hb have been elaborated upon using a novel fluorescence technique. The concentration-dependent change in the fluorescence intensity of fluorescein-conjugated spectrin (F-spectrin) in presence of oxy-Hb indicated binding with a dissociation constant of ∼20 μM that has been directly evaluated from the increase in the extent of quenching of the fluorescein fluorescence of F-spectrin by reverse titration with the increasing concentrations of different Hb samples isolated from both normal and β-thalassemic patients. The Hb compositions, with major components of the normal HbA, the fetal HbF, and the variant HbA2, of each individual were estimated using the Variant HPLC device of Bio-Rad. Results of the present study indicated that the dissociation constant, K d, of spectrin binding to Hb decreased from 19.5 ± 2 μM in normal individuals to of 6.5 ± 0.5 μM in the presence of 73% HbA2 along with coeluted variants in the blood samples of patients suffering from β-thalassemia, indicating differential interactions of the Hb variants with spectrin.

Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge.

Responses in three chimpanzees were compared following challenge with a clonal hepatitis C virus (HCV) contained in plasma from an animal that had received infectious RNA transcripts. Two of the chimpanzees (Ch1552 and ChX0186) had recovered from a previous infection with HCV, while the third (Ch1605) was a naïve animal. All animals were challenged by reverse titration with decreasing dilutions of plasma and became serum RNA positive following challenge. Ch1605 displayed a typical disease profile for a chimpanzee. We observed increasing levels of serum RNA from week 1 postinoculation (p.i.), reaching a peak of 10(6) copies/ml at week 9 p.i., and alanine aminotransferase (ALT) elevations and seroconversion to HCV antibodies at week 10 p.i. In contrast, both Ch1552 and ChX0186 exhibited much shorter periods of viremia (4 weeks), low serum RNA levels (peak, 10(3) copies/ml), and minimal ALT elevations. A comparison of intrahepatic cytokine levels in Ch1552 and Ch1605 showed greater and earlier gamma interferon (IFN-gamma) and tumor necrosis factor alpha responses in the previously infected animal, responses that were 30-fold greater than baseline responses at week 4 p.i. for IFN-gamma in Ch1552 compared to 12-fold in Ch1605 at week 10 p.i. These data indicate (i) that clonal HCV generated from an infectious RNA transcript will lead to a typical HCV infection in naïve chimpanzees, (ii) that there are memory immune responses in recovered chimpanzees that control HCV infection upon rechallenge, and (iii) that these responses seem to be T-cell mediated, as none of the animals had detectable antibody against the HCV envelope glycoproteins. These observations have encouraging implications for the development of a vaccine for HCV.

Control of the Pump Cycle in Bacteriorhodopsin: Mechanisms Elucidated by Solid-State NMR of the D85N Mutant

By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of [ζ- 15N]lys-, [12- 13C]retinal-, and [14,15- 13C]retinal-labeled D85N samples indicate a deprotonated, 13- cis,15- anti chromophore. On the other hand, at neutral pH, the NMR spectra of D85N show a mixture of protonated Schiff base species similar to that seen in the wild-type protein at low pH, and more complex than the two-state mixture of 13- cis,15- syn, and all- trans isomers found in the dark-adapted wild-type protein. These results lead to several conclusions. First, the reversible titration of order in the D85N chromophore indicates that electrostatic interactions have a major influence on events in the active site. More specifically, whereas a straight chromophore is preferred when the Schiff base and residue 85 are oppositely charged, a bent chromophore is found when both the Schiff base and residue 85 are electrically neutral, even in the dark. Thus a “bent” binding pocket is formed without photoisomerization of the chromophore. On the other hand, when photoisomerization from the straight all- trans,15- anti configuration to the bent 13- cis,15- anti does occur, reciprocal thermodynamic linkage dictates that neutralization of the SB and D85 (by proton transfer from the former to the latter) will result. Second, the similarity between the chromophore chemical shifts in D85N at alkaline pH and those found previously in the M n intermediate of the wild-type protein indicate that the latter has a thoroughly relaxed chromophore like the subsequent N intermediate. By comparison, indications of L-like distortion are found for the chromophore of the M o state. Thus, chromophore strain is released in the M o→M n transition, probably coincident with, and perhaps instrumental to, the change in the connectivity of the Schiff base from the extracellular side of the membrane to the cytoplasmic side. Because the nitrogen chemical shifts of the Schiff base indicate interaction with a hydrogen-bond donor in both M states, it is possible that a water molecule travels with the Schiff base as it switches connectivity. If so, the protein is acting as an inward-driven hydroxyl pump (analogous to halorhodopsin) rather than an outward-driven proton pump. Third, the presence of a significant C N syn component in D85N at neutral pH suggests that rapid deprotonation of D85 is necessary at the end of the wild-type photocycle to avoid the generation of nonfunctional C N syn species.

Analytic Binding Isotherms Describing Competitive Interactions of a Protein Ligand with Specific and Nonspecific Sites on the Same DNA Oligomer

Many studies of specific protein-nucleic acid binding use short oligonucleotides or restriction fragments, in part to minimize the potential for nonspecific binding of the protein. However, when the specificity ratio is low, multiple nonspecifically bound proteins may occupy the region of DNA corresponding to one specific site; this situation was encountered in our recent calorimetric study of binding of integration host factor (IHF) protein to its specific 34-bp H′ DNA site. Here, beginning from the analytical McGhee and von Hippel infinite-lattice nonspecific binding isotherm, we derive a novel analytic isotherm for nonspecific binding of a ligand to a finite lattice. This isotherm is an excellent approximation to the exact factorial-based Epstein finite lattice isotherm even for short lattices and therefore is of great practical significance for analysis of experimental data and for analytic theory. Using this isotherm, we develop an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecific binding sites. Analysis of calorimetric data for IHF-H′ DNA binding using this treatment yields enthalpies and binding constants for both specific and nonspecific binding and the nonspecific site size. This novel analysis demonstrates the potential contribution of nonspecific binding to the observed thermodynamics of specific binding, even with very short DNA oligomers, and the need for reverse (constant protein) titrations or titrations with nonspecific DNA to resolve specific and nonspecific contributions. The competition treatment is useful in analyzing low-specificity systems, including those where specificity is weakened by mutations or the absence of specificity factors.

Natural pyrite as an electrochemical sensor for potentiometric titrations with EDTA, mercury(II) and silver(I)

The results obtained in potentiometric titrations of copper(II), mercury(II) and iron(III) with standard EDTA solutions are presented. The titration of copper(II) at pH values in the range from 8.11 to 10.99 (ammonia buffer) and the titration of mercury(II) and iron(III) at pH values from 3.59 to 5.65 (acetate buffer) were performed. The titration end-point (TEP) was detected with an indicator electrode made from natural crystalline pyrite as an electrochemical sensor. The results obtained in potentiometric titration with the pyrite electrode were compared with those obtained using a platinum electrode (Fe3+), a Cu ion selective electrode (Cu2+) and a Hg electrode (Hg2+). Accurate and reproducible results with good agreement were obtained, but higher potential changes at the TEP were obtained using the pyrite electrode. In the course of the titration the potential was established within less than 1 min, whereas at the TEP it was within about 2–3 min. The potential changes at the TEP were in the range from 60 to 200 mV per 0.1 ml EDTA, according to the stability constant of the complex formed. The highest potential changes, ranging from 160 to 200 mV, were obtained in the titration of iron(III) at pH 3.59. Reverse titration was also performed and accurate and reproducible results were obtained. Moreover, titration of halogenide and thiocyanate with standard mercury(II) solutions, as well as cyanide with silver(I) solution, were performed and accurate and reproducible results were again obtained.

Influence of HMG-1 and Adenovirus Oncoprotein E1A on Early Stages of Transcriptional Preinitiation Complex Assembly

The TATA-binding protein (TBP) in the TFIID complex binds specifically to the TATA-box to initiate the stepwise assembly of the preinitiation complex (PIC) for RNA polymerase II transcription. Transcriptional activators and repressors compete with general transcription factors at each step to influence the course of the assembly. To investigate this process, the TBP.TATA complex was titrated with HMG-1 and the interaction monitored by electrophoretic mobility shift assays. The titration produced a ternary HMG-1.TBP. TATA complex, which exhibits increased mobility relative to the TBP. TATA complex. The addition of increasing levels of TFIIB to this complex results in the formation of the TFIIB.TBP.TATA complex. However, in the reverse titration, with very high mole ratios of HMG-1 present, TFIIB is not dissociated off and a complex is formed that contains all factors. The simultaneous addition of E1A to a mixture of TBP and TATA; or HMG-1, TBP, and TATA; or TFIIB, TBP, and TATA inhibits complex formation. On the other hand, E1A added to the pre-established complexes shows a significantly reduced capability to disrupt the complex. In add-back experiments with all complexes, increased levels of TBP re-established the complexes, indicating that the primary target for E1A in all complexes is TBP.