Microcalorimetry Experiments Research Articles

A cell-based immunobiosensor with engineered molecular recognition—Part I: design feasibility

A novel bioelectronic sensor is described in which living immune cells are transformed into unique biotransducer couples by engineering their molecular recognition for preselected antigens of clinical interest. This ‘hybrid’ biosensor, constructed with mast cells interfaced to a microfabricated thermoelectric device with the use of biomolecular linkages, is capable of detecting antigens in real time by transducing minute heat changes arising from antigen-induced mast cell activation processes. The thermoelectric approach was selected based upon preliminary bioenergetic calculations which indicated that metabolic changes arising from mast cell antigen recognition result in a significant increase in exothermic heat relative to basal metabolic conditions. Experimental studies confirmed that mast cell activation and degranulation can be discriminated thermally from basal metabolic activity. Results obtained from microcalorimetry experiments using cultured mast cells (MC/9 mucosal-like mast cell line), and harvested mast cells (rat peritoneal mast cells) indicated that detectable increases in heat output (∼3 ± 0.5 pW/cell, mean peak output) immediately followed cell activation. The construction of a miniature hybrid immunobiosensor device was made possible by bioelectronic coupling achieved with the use of cellular adhesive proteins that immobilized non-adherent (MC/9) cells as well as adherent (RBL-2H3 rat basophilic leukemia) cells to the thermopile. Results from preliminary tests conducted on a hybrid biosensor prototype validated the design feasibility of a miniature, living cell immunodiagnostic biosensor. Such cell-based hybrid biosensor approaches may greatly extend the capability for selective, rapid, on-site, antigen detection for a wide range of clinically relevant antigens and offer new approaches to in vitro diagnostics.

Micellar Growth Study by Microcalorimetry in CTAB Solutions

Microcalorimetry experiments were carried out on aqueous micellar solutions of hexadecyltrimethylammonium bromide (CTAB) in the presence of potassium bromide (KBr). We have investigated the effect of surfactant concentration and of the salt content upon the specific heat. The curves of the specific heat versus temperature exhibit a maximum resulting from the transition between spherical and elongated micelles. The temperature Tmax of these maxima depends on surfactant and salt concentration as expected from simple theoretical arguments. Static and dynamic light scattering experiments were performed on the same systems. The results show that light scattering and microcalorimetry experiments yield a different kind of information: blob size and variation of the micellar length. Microcalorimetry provides an interesting tool for determining the micellar growth.

Hydration—dehydration properties of methylcellulose and hydroxypropylmethylcellulose

The degree and rates of hydration and dehydration of methylcellulose (MC) and hydroxypropylmethylcellulose (HPMC) play important roles in many commercial applications involving the use of these polymers. MC and HPMC are known to exhibit phase separation and associated gelation of aqueous solution which is caused by a decrease in stability in the hydrated polymer above the lower critical solution temperature. Hydration-dehydration phenomena of these solutions were studied using turbidimetric and microcalorimetric methods. The precipitation characteristics were observed by monitoring changes in percent light transmission with temperature. These measurements indicate a functional dependence of type and degree of substitution, molecular weight and uniformity of substitution. Scanning microcalorimetry experiments indicate that MC precipitates out of solution in a simple gelation process, while HPMC solutions exhibit broad peaks, which suggests molecular weight or branching effects. Heats of dehydration follow a linear relationship with total substitution. Hydration and rate of hydration can also be altered by controlled surface crosslinking and particle size. The importance of these hydration and rate of hydration phenomena in ceramic applications, as an example, will be illustrated.

Association of the catalytic subunit of aspartate transcarbamoylase with a zinc‐containing polypeptide fragment of the regulatory chain leads to increases in thermal stability

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamic Analysis of the Activation of Glycogen Phosphorylase b Over a Range of Temperatures

Equilibrium dialysis and isothermal microcalorimetry experiments have been carried out to characterize the thermodynamics of the binding of AMP to glycogen phosphorylase b (EC 2.4.1.1) at pH 6.9 over the temperature range of 25-35 degrees C. Thermal titrations were performed at each temperature in various buffer systems, which have afforded the calculation of the number of protons exchanged when the AMP binds to each site in the protein. Thermodynamic parameters were obtained for the binding of AMP to the two nucleotide and the two inhibitor sites of the dimeric enzyme. The former show positive cooperativity while the latter behave as independent binding sites. A positive delta Cp value was obtained for the AMP binding to the two N sites (1.3 and 1.4 kJ K-1 mol-1), while the delta Cp was negative for the binding to the I sites (-1.9 kJ K-1 mol-1). The application of Sturtevant's method to our data (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240) and their comparison with a similar analysis undertaken with phosphorylase a (Mateo, P. L., González, J. F., Barón, C., Lopez-Mayorga, O., and Cortijo, M. (1986) J. Biol. Chem. 261, 17067-17072) has opened the way to some understanding of the thermodynamics of the allosteric transition in the protein.

Enthalpy and free energy of solubilization for 1-pentanol in anionic + cationic and anionic + nonionic surfactant mixtures

The standard enthalpy of solubilization as well as the partition coefficient of 1-Pentanol between micelles and water have been determined in the whole composition range of two binary surfactant systems from microcalorimetry experiments. The free energy results compare favorably with partition coefficients deduced from a head-space gas-chromatographic technique. It is shown that the surfactant mixing of sodium decylsulfate+decyltrimethylammonium bromide (system I) or sodium dodecylsulfate+dodecylpolyoxyethylene(23) (system II) is unfavorable to micellar solubilization. The solubilization of 1-pentanol is a highly endothermic process for system (I) and only slightly endothermic for system (II). The regular solution approximation predicts qualitatively the standard free energy as well as the standard enthalpy data. These results illustrate a discussion on the limits as well as the usefulness of the regular solution approach as applied to these three-component systems using available solubilization results from the literature for hydrophobic solutes in binary micellar surfactant solutions.

Specific-heat study of first-order smectic-A to smectic-B (hexatic) transitions in n-alkyl-4'-n-pentanoyloxy-biphenyl-4-carboxylates.

A recently synthesized and characterized homologous series of liquid-crystal compounds (n4COOBC) is shown by high-resolution ac microcalorimetry experiments (on the n=3,6 homologs) to most likely exhibit smectic-A to smectic-B (hexatic) (Sm${B}_{H}$) phase transitions exclusively. This is in partial contrast to earlier tentative assignments, based on low-resolution calorimetry, x-ray, and microscopy studies, which had crystal smectic-B (Sm${B}_{x}$) for n=4--8 and Sm${B}_{H}$ for n=3 and 9. The SmA-Sm${B}_{H}$ thermal anomalies reported here exhibit strong fluctuation contributions but are clearly first order. Experimental results are compared with the extensively characterized n-alkyl-4'-alkoxy-biphenyl-4-carboxylates (nmOBC's) and discussed in the context of Goodby's empirical molecular structure rules for the existence of the Sm${B}_{H}$, and in the context of a generic phase diagram (SmA, Sm${B}_{x}$, Sm${B}_{H}$) recently predicted by Aharony et al. The latter illustrates the physical content of our earlier suggestion that short-range hexagonal positional order is responsible for ubiquitous tricriticality of the SmA-Sm${B}_{H}$ phase transition.

The interaction of ubiquinone-10 and ubiquinol-10 with phospholipid bilayers. A study using differential scanning calorimetry and turbidity measurements

The interaction between ubiquinone-10 and ubiquinol-10 with dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine has been examined by differential scanning microcalorimetry and turbidity measurements. Microcalorimetry experiments showed that the phase transition of the phospholipids was largely unperturbed by the presence of ubiquinone-10, up to a phospholipid/CoQ molar ratio of 4:1. However, ubiquinol-10 markedly altered the calorimetric profile of both the main transition and the pretransition at molar ratios as high as 66:1, giving rise to a shoulder of the main transition peak at lower temperatures. This perturbation was larger for distearoylphosphatidylcholine than for dipalmitoylphosphatidylcholine samples. Turbidity measurements also showed that ubiquinol-10 perturbed both the pretransition and the main transition of both phospholipids. These results indicate that when the phospholipids are in the gel state, at temperatures below T c , and at phospholipid/CoQ molar ratios higher than 4:1, ubiquinone-10 is segregated to a region of the bilayer where it does not perturb the thermotropic phase transition. However, in the same circumstances ubiquinol-10, possibly due to its more hydrophilic character, remains in a region of the phospholipid bilayer where it perturbs the phospholipid phase transition.

ChemInform Abstract: ALKALI CATION COMPLEXATION AND TRANSPORT PROPERTIES OF SYNTHETIC BICYCLIC DECAPEPTIDES: STRUCTURAL, THERMODYNAMIC, AND KINETIC ANALYSIS

AbstractMit Hilfe spektroskopischer Methoden (NMR und UV) sowie durch Mikrocalorimetrie wurde die Komplexierung von Alkalimetallkationen durch die bicyclischen Decapeptide S,S′‐ Bis‐[cyclo‐glycyl‐L‐hemicystyll‐glycyl‐glycyl‐L‐prolyl], S,S′‐Bis‐[cyclo‐glycyl‐L‐ hemicystyl‐glycyl‐glycyl‐D‐prc)lyl], S,S′‐Bis‐[cyclo‐glycyl‐L‐hemicystyl‐sarcosyl‐sarcosyl‐ ‐L‐prolyl‐] und S,S′‐Bis‐[cyclo‐glycyl‐L‐hemicystyl‐sarcosyl‐sarcosyl‐D‐prolyl] unter strukturellen, thermodynamischen und kinetischen Gesichtspunkten sowie die Eigenschaften der Peptide in bezug auf den Ionentransport in einem künstlichen Lipoid‐ Zweischichten‐System untersucht.

Alkali Cation Complexation and Transport Properties of Synthetic Bicyclic Decapeptides: Structural, Thermodynamic, and Kinetic Analysis

AbstractAlkali cation complexation and bilayer transport by the bicyclic decapeptides S,S′‐bis‐cyclo‐glycyl‐L‐hemicystyl‐glycyl‐glycyl‐L‐prolyl (1), S, S′‐bis‐cyclo‐glycyl‐L‐hemicystyl‐glycyl‐glycyl‐D‐prolyl (2), S, S′‐bis‐cyclo‐glycyl‐L‐ hemicystyl‐sarcosyl‐sarcosyl‐L‐prolyl (3), and S, S′‐bis‐cyclo‐glycyl‐L‐hemicystyl‐sarcosyl‐sarcosyl‐D‐prolyl (4) were analyzed according to structural, thermodynamic and kinetic criteria; valinomycin was used as a reference ionophoretic system.Structural analysis of peptide 3 with spectroscopic methods showed different conformational arrangements in the bicyclic system depending on its state of complexation. Circular dichroism indicated the presence of a multitude of conformations with differing helicities around the disulfide bridge in both free and complexed states.Thermodynamic analysis by microcalorimetry demonstrated a far lower cation selectivity among the synthetic peptides than displayed by valinomycin. Peptide 3 shows cation affinities of about two orders of magnitude higher than peptide 4, but still much lower than found for the complexes of valinomycin with K+ and Rb+. In contrast to the latter case, the complexation reactions of peptides 3 and 4 are driven by both enthalpy and entropy contributions. Neither peptide 1 and 2 nor the cyclic partial structures of all four peptides displayed significant cation complexation.A kinetic analysis of the K+‐complexation by peptide 3 based on the microcalorimetry experiments showed far lower rates of cation exchange for the synthetic peptide than those reported for valinomycin. Transport studies with peptide 3 using artificial lipid bilayer membranes gave negative results. The apparent lack of ionophoretic properties of these synthetic peptides despite their considerable ability to form complexes with cations is discussed in terms of structural parameters.

Thermodynamics of nucleotides binding to phosphorylase b

The effects of several chemical modifications in the AMP molecule on its interaction with phosphorylase b are examined by microcalorimetry, equilibrium dialysis, light scattering and ultracentrifuge experiments. In this work we report the results obtained for eight AMP analogues corresponding to different substituents in the puric base or in the ribose, or to different positions of the phosphate. The thermodynamic properties of the interaction between the phosphorylase b and the above mentioned nucleotides are also reported. The following conclusions were obtained: a) Except for IMP and 2'dIMP all the nucleotides studied clearly show two types of binding sites in the enzyme. b) The interaction of the nucleotide with its weaker affinity binding site is highly dependent upon chemical alterations in the puric base. c) Both the amino group in C(6) and the N(1) of the adenine in the AMP seem to play an important role in the conformational transitions induced by the nucleotide on the enzyme. d) The tetramerization of phosphorylase b in the presence of 10(-2) M AMP and in the conditions of the ultracentrifuge experiments is drastically affected by modifications in the ribose-phosphate part of the AMP molecule.

Infra-red spectroscopic studies of transformations of isomeric cresols over a [formula omitted] catalyst

As the scale and intensity of mining increase, the application of mineral agents raises concerns about its environmental impact, but little information is available on the combined toxic effect of cresol frothing agent and Cu2+ on the soil microbial activity. In this work, microcalorimetric method, enzyme activity and Illumina HiSeq 2500 high throughput sequencing technology were combined to determine the effect of binary toxicants on soil microorganisms. The dose-toxicity relationship obtained from microcalorimetry experiments indicated that an increase in dosage was associated with a decrease in metabolic rate constant k (or an increase in the inhibitory ratio). The ratio of 400/0.08 (mg·L−1/mg·g−1) of Cu2+/cresols stimulated the growth of microorganisms in soil. Urease and fluorescein diacetate esterase activities upon the exposure of binary toxic mixture showed a negative impact and the toxicity order followed: Cu2+/p-cresol > Cu2+/o-cresol > Cu2+/m-cresol. By Illumina HiSeq 2500 high throughput sequencing technology, the results of microbial communities in 3 soil samples showed no significant differences, and Proteobacteria, Acidobacteria, Gemmatimonadetes and Bacteroidetes phyla were the dominant species as Gram-negative bacteria.