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PubMed1998

Differential solvation of "core" trimannoside complexes of the Dioclea grandiflora lectin and concanavalin A detected by primary solvent isotope effects in isothermal titration microcalorimetry.

The thermodynamics of binding of the Man/Glc-specific seed lectin from Dioclea grandiflora (DGL) to deoxy analogs of the "core" trimannoside, 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside was determined by isothermal titration microcalorimetry (ITC) in the first paper of this series (Dam, T. K., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 32812-32817). The data showed binding of specific hydroxyl groups on all three residues of the trimannoside, similar to that observed for ConA (Gupta, D., Dam, T. K., Oscarson, S., and Brewer, C. F. (1997) J. Biol. Chem. 272, 6388-6392). However, differences exist in the thermodynamics of binding of monodeoxy analogs of the alpha(1-6) Man residue of the trimannoside to the two lectins. The x-ray crystal structure of DGL complexed to the core trimannoside, presented in the second paper in this series (Rozwarski, D. A., Swami, B. M., Brewer, C. F., and Sacchettini, J. C. (1998) J. Biol. Chem. 273, 32818-32825), showed the overall structure of the complex to be similar to that of the ConA-trimannoside complex. Furthermore, the trimannoside is involved in nearly identical hydrogen bonding interactions in both complexes. However, differences were noted in the arrangement of ordered water molecules in the binding sites of the two lectins. The present study presents ITC measurements of DGL and ConA binding to the monodeoxy analogs of the trimannoside in hydrogen oxide (H2O) and deuterium oxide (D2O). The solvent isotope effects present in the thermodynamic binding data provide evidence for altered solvation of the parent trimannoside complexes at sites consistent with the x-ray crystal structures of both lectins. The results indicate that the differences in the thermodynamics of DGL and ConA binding to alpha(1-6) monodeoxy analogs of the trimannoside do not correlate with solvation differences of the parent trimannoside complexes.

Calorimetry↗
PubMed1998

Fatty acyl-CoA binding domain of the transcription factor FadR. Characterization by deletion, affinity labeling, and isothermal titration calorimetry.

The Escherichia coli transcription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRDelta1-167, with a palmitoyl-CoA analogue, 9-p-azidophenoxy[9-3H]nonanoic acid-CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187-195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRDelta1-167 for acyl-CoA. The binding was characterized by a large negative DeltaH0, -16 to -20 kcal mol-1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, Kd of 45 and 63 nM and 68 and 59 nM, respectively. The Kd for palmitoyl-CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding.

Acyl Coenzyme A↗
PubMed2001

Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue.

Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2' region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1' amide and P2 carbonyl groups and another between the P2' amide and Nepsilon2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nepsilon2 of His(680). Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK(a) of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pK(a) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat)/K(m), and indicated that the observed pK(a) values are apparent. The procedure presented may be applicable for other serine peptidases.

Amino Acid Substitution↗
PubMed2001

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry.

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.

Animals↗
PubMed2003

Parallel measurement of Ca2+ binding and fluorescence emission upon Ca2+ titration of recombinant skeletal muscle troponin C. Measurement of sequential calcium binding to the regulatory sites.

Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca(2+) affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca(2+) titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca(2+) binding to the two N-terminal Ca(2+) binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca(2+) binding data showed that the Asp --> Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp --> Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp --> Ala mutation at position 30 eliminates the differences in Ca(2+) affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca(2+) binding to site I is dependent on the previous binding of metal to site II.

Animals↗
PubMed2003

Apparent cooperative assembly of the bacterial cell division protein FtsZ demonstrated by isothermal titration calorimetry.

The assembly dynamics of FtsZ, a prokaryotic homolog of tubulin, are important for their role in bacterial cytokinesis. Here we used isothermal titration calorimetry (ITC) to measure the heat of FtsZ self-association under various conditions. The measurements were designed to test whether FtsZ protofilaments are assembled by an isodesmic (linear aggregates in which each bond has an identical equilibrium constant) or a cooperative (aggregates only become stable after forming a oligomeric nucleus) assembly process. The isodesmic model can fit the assembly in GDP closely but cannot fit the assembly in GTP. FtsZ-GTP without Mg(2+) exhibits an apparent critical concentration, which is indicative of cooperative assembly, near 2.9 microm. With 2.5 mm Mg(2+) (which allows FtsZ to hydrolyze GTP) the critical concentration is reduced 10-fold to approximately 0.31 microm. Both with and without Mg(2+) there is no evidence for assembly below the critical concentration, but there is an abrupt transition to full assembly above. The ITC data are highly suggestive of a cooperative assembly, although this is difficult to reconcile with the 1-subunit-thick protofilaments observed by electron microscopy.

Bacterial Proteins↗
PubMed2004

Direct measurement of the thermodynamic parameters of amyloid formation by isothermal titration calorimetry.

Amyloid fibril deposition is associated with over 20 degenerative diseases, including Alzheimer's, Parkinson's, and prion diseases. Although research over the last few years has revealed the morphology and structural features of the amyloid form, knowledge about the thermodynamics of amyloid formation is limited. Here, we report for the first time a direct thermodynamic study of amyloid formation using isothermal titration calorimetry. Beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, was used for extending amyloid fibrils in a seed-controlled reaction in the cell of the calorimeter. We investigated the enthalpy and heat capacity changes of the reaction, where the monomeric, acid-denatured molecules adopt an ordered, cross-beta-sheet structure in the rigid amyloid fibrils. Despite the dramatic difference in morphology, beta(2)-microglobulin exhibited a similar heat capacity change upon amyloid formation to that of the folding to the native globular state, whereas the enthalpy change of the reaction proved to be markedly lower. In comparison with the native state, the results outline the important structural features of the amyloid fibrils: a similar extent of surface burial even with the supramolecular architecture of amyloid fibrils, a lower level of internal packing, and the possible presence of unfavorable side chain contributions.

Amyloid↗
PubMed2005

Insights into hydrophobicity and the chaperone-like function of alphaA- and alphaB-crystallins: an isothermal titration calorimetric study.

Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.

Anilino Naphthalenesulfonates↗
PubMed1984

Theophylline therapy. Titrating dosage requirements.

Theophylline has long been a cornerstone of bronchodilator therapy for asthma and chronic obstructive lung disease, but until recently its use carried the risk of serious, even fatal, toxicity. With recent advances in understanding of this drug's pharmacologic properties and pharmacokinetic characteristics, serious toxicity now can usually be avoided. The dosage requirements for theophylline demonstrate wide interpatient and intrapatient variation. Several factors responsible for this variation, most notably age, have been identified and used in definition of dosage guidelines for different patient groups. However, substantial variation still exists within these groups, necessitating measurement of serum concentration in selected patients. The measurements obtained are used to titrate the patient's dosage requirement in such a way as to maximize therapeutic response while minimizing risk of toxicity.

Administration, Oral↗
PubMed1991

Immunosorbent binding bioassay: a solid phase biological immunoassay for the titration of antisera to alpha interferons.

An Immuno Sorbent Binding Bio-Assay (ISBBA) for the detection and the titration of antisera to the highly biologically active proteins, alpha interferons, is described. This method is similar to a classical solid phase immunoassay, except for the last step which uses the biological activity of the captured antigen. With specific serum antibodies the bound antigen prevents further virus induced cell lysis. On the contrary, with negative serum or preserum the antigen is washed out and virus induced cell lysis occurs, therefore no labelling is required. ISBBA exhibits three main differences when compared to the reference method i.e. the neutralization assay (NA): i) ISBBA is 10 to 1000 fold more sensitive than NA; ii) in contrast to NA, ISBBA is based on the production of an antiviral effect; iii) ISBBA makes it possible to use unpurified antigen. The applications of ISBBA to subtypes study in natural alpha interferon samples are discussed.

Animals↗
PubMed1995

DNA interaction of the imidazole-containing lexitropsin ImPy: titration viscometric study in comparison to netropsin.

The imidazole (Im) containing lexitropsin ImPy related to netropsin (Nt) is a sequence reading DNA ligand which, in contrast to Nt, permits binding to a GC base pair. The ImPy induced DNA conformational changes differ significantly from those induced by Nt as monitored by titration viscometry, although interaction modes have also been resolved with boundaries at the same ligand to DNA phosphate ratio, r. Evidently ImPy covers similar binding sites (in the same sequence) as Nt for natural calf thymus DNA at r < 0.023. This result suggests that the preferred binding sites of ImPy are A tracts (cf. K.E.R. JBSD 9(1993) 973), in agreement with previous data. The respective DNA coil expansion, most probably caused by unbending (l.c.), is similar but smaller compared to the Nt-DNA interaction. These results again suggest that, at low r values, the van der Waals interaction in the narrowed minor groove of AT clusters provides a dominating energy contribution to ImPy binding. At r > 0.03 the DNA coil expansion increases to extremely high values in that r range where Nt binding (to mixed AT/GC sequences) induces no effect at all owing to steric hindrance with the amino group of guanine. On the basis of many quantitative results for the Nt-DNA systems these effects can be understood in terms of an unbending of intrinsic helix bends (l.c.). They are of considerable interest in connection with the ability of such compounds to influence the direction of the local regulatory relevant DNA curvature.

Animals↗
PubMed2006

Association constants of Pb2+ with binding sites of fungal biomass using metal-based titrations.

Biosorption is perceived as an alternative method for toxic heavy metal removal/recovery from aqueous effluents. This work focused on derivation of some key quantitative physico-chemical characteristics of a representative biosorbent material required for its further effective exploitation. The newly developed acid-base and metal-based titrator allowed the characterization of the chemisorption active sites of Rhizopus arrhizus biomass and the study of their metal affinity. This experimental approach, combined with an analytical method consisting of transforming the initial data enabled the calculation of the number and capacity of the reactive sites (Qads) and the metal affinity constants (Km) for lead sorption by R. arrhizus biomass. The pKm values for Rhizopus biomass varied between -3 and -6 for sites releasing no protons, -1 and 1 for sites releasing one proton, and > 8 for sites releasing two protons - combined with the Pb precipitation phenomenon. At low temperatures, the active binding site number was lower at lower lead concentrations whereas the precipitation was promoted at higher lead concentration values. Lead adsorption contributed modestly (11%) to its overall uptake and occurred at low lead concentrations onto strong and medium affinity binding sites. Micro-precipitation quickly commenced around active binding sites distinguished by their weak affinity whenever the solution lead concentrations reached 10(-6) or 10(-5) M and represented more than 85% of the total sorbed metal quantity. The work also demonstrated the usefulnes of the methodology reported here for characterizing complex biosorbent materials.

Binding Sites↗
PubMedDate not supplied

Differences in the adsorption behaviour of poly(ethylene oxide) copolymers onto model polystyrene nanoparticles assessed by isothermal titration microcalorimetry correspond to the biological differences.

The adsorption behaviour of a tetrafunctional copolymer of poly (ethylene oxide)-poly (propylene oxide) ethylene diamine (commercially available as Poloxamine 908) and a diblock copolymer of poly (lactic acid)-poly (ethylene oxide) (PLA/PEG 2:5) onto a model colloidal drug carrier (156 nm sized polystyrene latex) is described. The adsorption isotherm, hydrodynamic thickness of the adsorbed layers and enthalpy of the adsorption were assessed. The close similarity in the conformation of the poly (ethylene oxide) (PEO) chains (molecular weight 5,000 Da) in the adsorbed layers of these two copolymers was demonstrated by combining the adsorption data with the adsorbed layer thickness data. In contrast, the results from isothermal titration microcalorimetry indicated a distinct difference in the interaction of the copolymers with the polystyrene colloid surface. Poloxamine 908 adsorption to polystyrene nanoparticles is dominated by an endothermic heat effect, whereas, PLA/PEG 2:5 adsorption is entirely an exothermic process. This difference in adsorption behaviour could provide an explanation for differences in the biodistribution of Poloxamine 908 and PLA/PEG 2:5 coated polystyrene nanoparticles observed in previous studies. A comparison with the interaction enthalpy for several other PEO-containing copolymers onto the same polystyrene colloid was made. The results demonstrate the importance of the nature of the anchoring moiety on the interaction of the adsorbing copolymer with the colloid surface. An endothermic contribution is found when an adsorbing molecule contains a poly (propylene oxide) (PPO) moiety (e.g. Poloxamine 908), whilst the adsorption is exothermic (i.e. enthalpy driven) for PEO copolymers with polylactide (PLA/PEG 2:5) or alkyl moieties.

Adsorption↗
PubMed2004

Inhibition of beta-amylase activity by calcium, magnesium and zinc ions determined by spectrophotometry and isothermal titration calorimetry.

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH = 4.8 (sodium acetate 16 mM) and T = 300K. The Ki and enthalpy of binding for calcium (13.4, 13.1 mM and -14.3 kJ/mol), magnesium (18.6, 17.8mM and -17.7 kJ/mol) and zinc (17.5, 17.7 mM and -20.0 kJ/mol) were found by spectrophotometric and ITC methods respectively.

Calcium↗
PubMed1981

Titration of penicilloyl antibody by enzyme-immunoassay: method and data analysis to assign titer.

Antisera to the hapten, penicilloyl, were titered in an indirect enzyme immunoassay with the marker enzyme, horseradish peroxidase. A multivalent solid-phase antigen for stable solid-phase immunocomplex adsorption and a 10 min peroxidase light-assay with photolabile ortho-phenylenediamine were features of the assay. The penicilloyl antisera titration curves formed the basis for an evaluation of 4 methods to assign titer. Titer was expressed as either the antiserum dilution-1 that produced an assigned absorbance, exhibited 50% maximal antigen-antibody binding, and equaled zero absorbance by extrapolation, or as absorbance at 10(-3) antiserum dilution. The accuracy for extrapolated titer was improved with 2 or more solid-phase antigen concentrations. Absorbance at 10(-3) antiserum dilution was most useful as a screen for relative titer and for specific titer from a standard curve generated with many test sera. The antiserum dilution may vary with a particular enzyme immunoassay.

Animals↗
PubMed2004

Simulating titration to goal clinical trials with statins.

Many countries have local guidelines on the management of subjects' lipid levels with and without pharmaceutical intervention. The statin class of drugs is the preferred class for reducing low density lipoprotein cholesterol (LDL-C). Different statins have different potencies and different dose ranges. It is of interest to simulate clinical trials in which subjects are titrated through the dose ranges of various statins in accordance with local guidelines, in order to estimate the proportion of subjects who reach treatment goal of LDL-C at any particular dose of any particular statin.

Cholesterol, LDL↗
PubMed2004

A new end-point for ELISA titrations.

This report describes a new ELISA procedure based on end-point titrations. This end-point ELISA takes advantage of the change of color intensity that occurs when peroxidase-containing wells of an ELISA plate are revealed with diaminobenzidine-nickel and further intensification with silver: as antibody concentration and, therefore, peroxidase concentration, decreased, the color became stronger in some wells and, afterwards (i.e., at lower antibody and peroxidase concentrations), the color faded toward clear background. It is proposed that the reciprocal of the sample dilution at which the color intensifies can be used as a measure of the sample antibody content. This report verifies the validity and precision of that procedure.

3,3'-Diaminobenzidine↗
PubMedDate not supplied

Isothermic titration calorimetry to study CMCs of neutral surfactants and of the liposome-forming bolaamphiphile dequalinium.

Isothermic titration calorimetry was used to measure the heat of micelle formation (molar enthalpy of transfer of surfactants monomers from water into micellar aggregates. The problems associated with the estimation of the CMC and the whole therodynamic profile of micellization of surfactants via Gibbs-Helmholtz-Equation are discussed. CMC's of octylthioglucoside and the peculiar bolaamphilphile dequalinium which concentrates in mitochondria are measured. In contrast to earlier reports, no CMC of dequalinium could be found inspite of extensive systematic measurements.

Anti-Infective Agents, Local↗