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At least 1,063 records · Page 59Linked to original sources
PubMed1998

[The effects of midazolam on oxygen consumption and the level of vigilance. The possibility of the sedative effect of the plexiglas dome (canopy) of the Deltatrac indirect calorimetry device].

OBJECTIVE: To assess the effects of midazolam on oxygen consumption (VO2) and the level of vigilance. To search for a possible "sedative effect" of the canopy of the Deltatrac (Datex) when a placebo is administered instead of midazolam. STUDY DESIGN: Prospective, comparative, randomized, double-blinded, cross-over study. PATIENTS: Eight healthy volunteers of ASA physical class 1, not under stress, fasting and at rest. METHODS: The volunteers were administered at T0 either midazolam (0.07 mg.kg-1, IM), or a placebo at a one week interval in an order at random. The VO2 was measured with a Deltatrac. Circulatory status was monitored with non invasive methods and the level of vigilance assessed using the Ramsay scale. Statistical analysis was obtained with Anova for repeated measurements with two within factors (drug and time). RESULTS: VO2 decreased similarly and significantly between T0 and T45 min (P < 0.05). The decrease continued only after midazolam until T60 min. However the variation of VO2 between the midazolam and placebo group was not significant. The haemodynamic variables remained unchanged. Midazolam had a stronger sedative effect at T120 min.

Adult↗
PubMed1996

Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions.

Biomolecular interactions can be defined by combining thermodynamic data on the energetic properties of the interaction with high-resolution structural data. The development of high sensitivity isothermal titration calorimetric equipment provides a dramatic advance in the gathering of thermodynamic data, and the interactions between biological macromolecules can now be described with unprecedented accuracy.

Calorimetry↗
PubMed2006

Pressure-modulated differential scanning calorimetry: theoretical background.

We demonstrate in this work and in the accompanying paper that it is possible to measure simultaneously heat capacity and expansibility of biomolecules in a single DSC experiment. In this study, we provide the theoretical basis for this new method based on rigorous statistical thermodynamics. The theoretical treatment presented here demonstrates that there are two additive contributions to the heat capacity at variable pressure, viz. (1) the isobaric heat capacity and (2) an expansibility term, and that these contributions can be experimentally separated to obtain simultaneously both heat capacity and expansibility in continuous DSC temperature scans preformed under pressure modulation. Equations that describe the mixed heat capacity and expansibility signal are derived, and experimental strategies as well as data extraction procedures are discussed.

Calorimetry, Differential Scanning↗
PubMed2006

Isothermal calorimetry as a tool to investigate slow conformational changes in proteins and peptides.

A new calorimetric method has been developed to follow the time course of slow conformational changes during the refolding of denatured proteins. The method is based on the ability of isothermal titration calorimeters (ITC) to detect small amounts of heat continuously over a minute to an hour time range without being disturbed by baseline drift. We benchmarked the method on the basis of the slow kinetic phases resulting from prolyl cis/trans isomerization of oligopeptides. Using this method, the simultaneous investigation of the kinetics and thermodynamics of slow phases in the refolding of GdmCl-denatured RNase A by single jump techniques was performed. Time traces of heat production in the presence of a peptidyl prolyl cis/trans isomerase support the classical model of rate-limiting prolyl trans to cis isomerizations in the folding reactions of RNase A. However, we also observed that, unlike prolyl cis/trans isomerizations in oligopeptides, those found in RNase A refolding are highly exothermic. It appears that coupling between slow prolyl trans to cis isomerization and relocation of remote backbone segments increases the number of contacting sites during formation of the native protein. The results demonstrate that calorimetrically monitored folding kinetics will be of relevance in the detection of otherwise silent folding events.

Calorimetry↗
PubMed1995

Thermodynamic evaluation of binding interactions in the methionine repressor system of Escherichia coli using isothermal titration calorimetry.

The binding interactions of the methionine repressor protein, MetJ, from Escherichia coli with its cognate, metbox DNA sequence and corepressor S-adenosylmethionine were examined using calorimetric methods. A detailed thermodynamic characterization of this system which exhibits the recently reported (beta alpha alpha)2 binding motif provides values for delta G, delta H, and delta S for each step in the repressor binding cycle. These studies show that, in the presence of corepressor, MetJ binds to a single metbox operator site with delta G = -7.7 kcal.mol-1, whereas in the absence of corepressor, the free energy of interaction with a single site is -5.8 kcal.mol-1. Cooperative interactions between two repressor molecules bound to two adjacent sites contribute an additional free energy of -1.3 kcal.mol-1 to binding at the second site. Binding is enthalpically unfavorable in the absence of the corepressor with delta H = +2.6 kcal.mol-1 but becomes exothermic with delta H = -4.6 kcal.mol-1 when corepressor is present. The heat capacity for the system decreases significantly by delta Cp = -290 cal.mol-1.K-1 on a per site basis when the protein binds to DNA, and interactions between repressor molecules bound to adjacent sites contribute a delta Cp = -800 cal.mol-1.K-1, indicating that solvent exclusion plays a significant role in binding in this system. The corepressor binds to the unbound repressor protein with a free energy of delta G = -6.0 kcal.mol-1 and to the MetJ-operator complex with delta G = -6.95 kcal.mol-1. Repressor binding to random-sequence DNA was estimated to occur with a free energy of -5.7 kcal.mol-1 in the presence of corepressor. These data clearly indicate that MetJ repressor dimer binds specifically to the central region of its 8 bp cognate metbox operator but recognizes partial operator sequences as short as 6 bp. Cooperativity in binding of adjacent MetJ dimers to a double metbox sequence is demonstrated to be important in determining the energetics of the interaction. Finally, the corepressor S-adenosylmethionine enhances the affinity of MetJ for its recognition site DNA by a factor of 25 and contributes significantly to the net exothermicity of repressor binding.

Bacterial Proteins↗
PubMed1995

Thermodynamics of inhibitor binding to mutant forms of glucoamylase from Aspergillus niger determined by isothermal titration calorimetry.

We have investigated the binding of mutant forms of glucoamylase from Aspergillus niger to the inhibitors 1-deoxynojirimycin and acarbose. The mutants studied comprise a group of single amino acid replacements in conserved regions near the active site of the enzyme. For each mutant we have determined both the affinities for the two inhibitors and the thermodynamic state functions for binding using titration microcalorimetry. We find that acarbose binds to all the mutants with a wide range of binding constants (10(4) < Ka < 10(13) M-1). In contrast, 1-deoxynojirimycin shows either binding at near wild-type affinity (Ka approximately equal to 10(4) M-1) or no detectable binding. The changes in the affinities of the mutant enzymes are rationalized in terms of the known three-dimensional structure of the wild-type enzyme with subsites 1, 2, and 3 being important for acarbose binding while only subsite 1 is critical for 1-deoxynojirimycin binding. In most of the mutants studied the magnitudes of the enthalpies and the entropies of binding of the mutant enzymes differed from those of the wild-type enzyme with the mutant enzymes having a relatively large portion of their binding energy composed of enthalpy and a relatively small proportion composed of entropy. The pattern of changes in the enthalpy and entropy is hypothesized to be due to changes in the structural complementarity of the binding pocket and the inhibitor.

1-Deoxynojirimycin↗
PubMed1994

Thermodynamic effects of reduction of the active-site disulfide of Escherichia coli thioredoxin explored by differential scanning calorimetry.

Intramolecular disulfide bonds in protein molecules, whether present in the wild-type protein or engineered via site-directed mutagenesis, are capable of significantly increasing the stability. Establishing thermodynamic parameters associated with the redox formation of this linkage is often encumbered by other global structural changes within the protein molecule. The active site of Escherichia coli thioredoxin possesses a disulfide/dithiol in a short loop, oxidation/reduction of which is accompanied by little structural alteration of the protein. Data for the thermal denaturation of the reduced protein are presented, which on comparison to the data obtained for the oxidized form [Ladbury, J.E., Wynn, R., Hellinga, H.W., & Sturtevant, J.M. (1993) Biochemistry 32, 7526-7530] are used to establish thermodynamic parameters for the redox reaction in this molecule. Data for an isosteric double mutation in the active site of thioredoxin (Cys32Ser/Cys35Ser) are also presented. Although the wild-type and mutated proteins show a similar reduction in free energy compared to the oxidized form (-3.0 +/- 0.4 and -3.1 +/- 0.3 kcal mol-1, respectively), the enthalpic and entropic contributions to this destabilization are different for the two proteins.

Binding Sites↗
PubMed1994

Domain structure of Escherichia coli DNA gyrase as revealed by differential scanning calorimetry.

The domain structure of DNA gyrase from Escherichia coli has been examined using differential scanning microcalorimetry. The intact enzyme (an A2B2 tetramer) shows at least four transitions with apparent Tm's at 44.8, 53.3, 58.6, and 60.7 degrees C. Comparison with the thermal stabilities of the two separate subunits and genetically-engineered protein fragments has been used to assign these transitions to individual domains within the intact gyrase proteins. The thermal unfolding of DNA gyrase and all individual fragments are irreversible under the conditions of the calorimetric experiment. Further evidence for the assignment of transitions to particular domains has been obtained by studying the effects of tight-binding ligands such as novobiocin on the thermal stabilities of the various protein fragments.

Adenosine Triphosphate↗
PubMed1985

Clotting of fibrinogen. 2. Calorimetry of the reversal of the effect of calcium on clotting with thrombin and with ancrod.

When clotting is effected by thrombin in the presence of calcium, the endotherm for the D nodules of fibrinogen broadens significantly and then becomes narrow again, while increasing in size. Clotting effected by the snake venom enzyme Ancrod, which releases only the A fibrinopeptides from the E nodule, shows only the broadening of the D endotherm. Accordingly, significant interactions of the D nodules of fibrinogen become possible only when the B fibrinopeptides of the E nodule are released on clotting. When calcium present during clotting is removed from the fibrin clot with ethylenediaminetetraacetic acid, the endotherm for the D nodules of fibrin shows nearly complete reversal if clotting was effected with Ancrod but appears to be divided into two endotherms if clotting was effected with thrombin. At neutral pH, new endotherms were observed for fibrinogen in the temperature range 105-140 degrees C.

Ancrod↗
PubMed1990

Differential scanning calorimetry study of mixed-chain phosphatidylcholines with a common molecular weight identical with diheptadecanoylphosphatidylcholine.

To examine the thermotropic phase behavior of various mixed-chain phosphatidylcholines in excess water and to compare it with the known behavior of identical-chain phosphatidylcholines, we have carried out high-resolution differential scanning calorimetric (DSC) studies on aqueous dispersions of 10 different mixed-chain phosphatidylcholines. These lipids, C(16):C(18)PC, C(18):C(16)PC, C(15):C(19)PC, C(19):C(15)PC, C(14):C(20)PC, C(20):C(14)PC, C(13):C(21)PC, C(21):C(13)PC, C(12):C(22)PC, and C(22):C(12)PC, have a common molecular weight which is the same as that of C(17):C(17)PC, an identical-chain phosphatidylcholine with a molecular weight of 762.2. When the values of any of the thermodynamic parameters (Tm, delta H, and delta S) of the mixed-chain phosphatidylcholines and C(17):C(17)PC are plotted against the normalized chain-length difference (delta C/CL), a linear function with negative slope is obtained provided that the value of delta C/CL is within the range of 0.09-0.4. The linear relationship suggests that these mixed-chain phospholipids are packed in the gel-state bilayer similar to the bilayer structure of C(17):C(17)PC at T less than Tm; however, the negative slope suggests that the conformational statistics of the hydrocarbon chain and the lateral lipid-lipid interactions of these phosphatidylcholines in the gel-state bilayer are perturbed proportionally by a progressive increase in the chain-length inequivalence between the two acyl chains within each lipid molecule. When the value of delta C/CL for mixed-chain phosphatidylcholines reaches the range of 0.44-0.55, the thermotropic phase behavior deviates markedly from that of less asymmetric phosphatidylcholines, suggesting that these highly asymmetric lipids are packed into mixed interdigitated bilayers at T less than Tm. The heating and cooling pathways of aqueous dispersions prepared from the 10 mixed-chain phospholipids are also discussed.

Calorimetry, Differential Scanning↗
PubMed1981

Dilatometry and calorimetry of saturated phosphatidylethanolamine dispersions.

The specific volumes of a series of saturated phosphatidylethanolamine dispersions with 12, 14, and 16 carbon atoms per chain have been measured in the region of the chain melting transition, Tm. The change in specific volume at Tm for the 12 and 14 carbon compounds are 0.0160 and 0.0204 mL/g, respectively. Comparisons are drawn between this class of lipids and phosphatidylcholines. In both cases, Tm extrapolates with increasing chain length to the melting point of polyethylene. Both types of lipids appear to be packed in a similar way below Tm. One major difference is that dilaurylphosphatidylethanolamine undergoes a second transition above Tm.

Calorimetry↗
PubMed2006

An isothermal titration calorimetry study of the binding of substrates and ligands to the tartrate dehydrogenase from Pseudomonas putida reveals half-of-the-sites reactivity.

An isothermal titration calorimetric study of the binding of substrates and inhibitors to different complexes of tartrate dehydrogenase (TDH) from Pseudomonas putida was carried out. TDH catalyzes the nicotinamide adenine dinucleotide (NAD)-dependent oxidative decarboxylation of d-malate and has an absolute requirement for both a divalent and monovalent metal ion for activity. The ligands Mn(2+), meso-tartrate, oxalate, and reduced nicotinamide adenine dinucleotide (NADH) bound to all TDH complexes with a stoichiometry of 1 per enzyme dimer. The exception is NAD, which binds to E/K(+), E/K(+)/Mn(2+), and E/K(+)/Mg(2+) complexes with a stoichiometry of two per enzyme dimer. The binding studies suggest a half-of-the-sites mechanism for TDH. No significant heat changes were observed for d-malate in the presence of the E/K(+)/Mn(2+) complex, suggesting that it did not bind. In contrast, meso-tartrate does bind to E/K(+)/Mn(2+) but gives no significant heat change in the presence of E/Mn(2+), suggesting that K(+) is required for meso-tartrate binding. meso-Tartrate also binds with a large DeltaC(p) value and likely binds via a different binding mode than d-malate, which binds only in the presence of NAD. In contrast to all of the other ligands tested, the binding of Mn(2+) is entropically driven, likely the result of the entropically favored disruption of ordered water molecules coordinated to Mn(2+) in solution that are lost upon binding to the enzyme. Oxalate, a competitive inhibitor of malate, binds with the greatest affinity to E/K(+)/Mn(2+)/NADH, and its binding is associated with the uptake of a proton. Overall, with d-malate as the substrate, data are consistent with a random addition of K(+), Mn(2+), and NAD followed by the ordered addition of d-malate; there is significant synergism in the binding of NAD and K(+). Although the binding of meso-tartrate also requires enzyme-bound K(+) and Mn(2+), the binding of meso-tartrate and NAD is random.

Alcohol Oxidoreductases↗
PubMed2007

Thermodynamic characterization of the interaction between CAR-RXR and SRC-1 peptide by isothermal titration calorimetry.

The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer (CAR-RXR) with the retinoid X receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR-RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, binding of coactivator peptide to the CAR-RXR heterodimer is characterized by a favorable enthalpy change and an unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases the affinity for coactivator by decreasing the unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis-RA, generates a second SRC-1 site and increases the affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (deltaCp) is also investigated. A 2-fold difference in deltaCp is observed between liganded and unliganded CAR-RXR. The observed thermodynamic parameters for binding of SRC-1 peptide to liganded and apo CAR-RXR as well as the difference in the deltaCp data provide evidence that the apo CAR-RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR-RXR indicates that preformation of the binding site improves the complementarity of the coactivator-receptor interaction.

Alitretinoin↗