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PubMed1997

Calorimetric validation of 13C bicarbonate and doubly labeled water method for determining the energy expenditure in goats.

The purpose of the present study was to validate the 13C bicarbonate method (13C-M) and the doubly labeled water method (DLWM) for the estimation of the CO2 production R(CO2) in goats as a ruminant model. Indirect calorimetry was chosen as the reference method. Studies were carried out in 2 male African dwarf goats at 3 different developing stages (age: 5, 10, and 14 months, body mass: 14.6, 20.3, and 21.7 kg). Animals were fed a balanced feed 14 days before and during the studies. The isotope tracers (4 mg/kg NaH13CO3, 120 mg/kg 2H2O, and 75 mg/kg H218O; 99 AT.-%) were simultaneously given as a single pulse injection into the jugular vein. Thereafter, the animals were kept for 8 days in two respiration chambers (volume of chamber: 2.85 m3, air flow rate: 25 1/min) for the estimation of CO2 production and O2 consumption. For the determination of R(CO2) using the 13C-M samples of exhaled breath were drawn from the respiration chambers. The 13C enrichment and CO2 concentration of breath samples were measured by means of an infrared isotope analyzer. In order to determine R(CO2) by means of the DLWM, blood serum was used. The 2H and 18O enrichments were measured by an isotope ratio mass spectrometer. Urine samples were collected over 24 h to quantify renal water losses. The R(CO2) was calculated by means of the 13C-M using the area under the 13C enrichment-time curve. The determination of R(CO2) by means of the DLWM was based on the slopes of the 2H and 18O disappearance curves and the body water pool obtained from the zero time intercept of the isotope curves. The values of R(CO2) resulting from the 13C-M were found to be comparable with those from the calorimetric measurement. Smaller (not statistically significant) values of R(CO2)--92% from 13C-M and 87% from DLWM--compared to the indirect calorimetry could indicate the incorporation of 13C and 2H into metabolites other than CO2 and H2O, respectively. The body water contents calculated from the zero time intercepts of the 2H and 18O disappearance curves amounted to 66% and 63%, respectively. The body water content was found to be not related to the age of animals. The renal water loss was calculated to be 35% of the total water loss (0.76 l/d.

Aging↗
PubMed1996

Relationship between pulmonary oxygen consumption, lung inflammation, and calculated venous admixture in patients with acute lung injury.

OBJECTIVE: To determine in patients with acute lung injury whether increased pulmonary oxygen consumption (VO2pulm), computed as the difference between oxygen consumption measured by indirect calorimetry (VO2meas) and calculated by the reverse Fick method (VO2Fick), would: (1) correlate with the degree of lung inflammation assessed by bronchoalveolar lavage (BAL); (2) lead to an overestimation of calculated venous admixture (Qva/Qt). DESIGN: Prospective study. SETTING: University hospital, medical intensive care unit. INTERVENTION: None. MEASUREMENTS AND RESULTS: In nine mechanically ventilated patients with acute lung injury (Apache II 12 +/- 5, lung injury score 2 +/- 0.6, mean +/- SD), whole-body VO2 (VO2wb) was determined simultaneously by indirect calorimetry and the reverse Fick technique, after which BAL was immediately performed. VO2meas was significantly higher than VO2Fick (128 +/- 24 and 102 +/- 18 ml/min per m2, respectively, p < 0.001). Median VO2pulm was 25.3 ml/min per m2 (range 1.98-51.5), thus representing 19 +/- 11% of VO2wb. Total BAL cellularity was increased in all patients (median 47, range 24-200 x 10(4)/ml), as was the total polymorphonuclear (PMN) count (median 78 range 5-93 x 10(4)/ml). Macrophage counts were in the normal range. There were raised BAL levels of interleukin-6 (IL-6) (median 945, range 23-1800 ng/ml) and elastase (median 391, range 5-949 ng/ml). Median protein levels were 270 micrograms/ml (range 50-505). There was no correlation between VO2pulm and BAL cellularity, PMNs, elastase, IL-6, or protein. Qva/Qt was 31.7 +/- 8%. Qva/Qt, corrected for the presence of VO2pulm, (Qva/Qtcorr), was 30.3 +/- 8% (P < 0.01 vs Qva/Qt), a 4.2% overestimation due to VO2plum. There was no correlation between Qva/Qt or Qva/Qtcorr and VO2pulm. CONCLUSIONS: In mechanically ventilated patients with acute lung injury, VO2pulm was increased and led to a 19% underestimation of VO2wb determined by the reverse Fick method, as well as to a 4.2% overestimation of calculated Qva/Qt. Lung inflammatory activity was increased, as assessed by BAL cellularity, IL-6 and elastase levels. However, there was no correlation between VO2pulm and the intensity of pulmonary inflammation.

Adult↗
PubMed1994

Evaluation of oxygen uptake and delivery in critically ill patients: a statistical reappraisal.

OBJECTIVE: The evaluation of oxygen consumption (VO2) and oxygen delivery (DO2) has gained increasing importance in the monitoring of critically ill patients. They can be obtained from either direct measurements or by indirect calculations based on the Fick principle. However the choice between these two approaches remains controversial. The aim of the study was to investigate whether these 2 methods provide similar results, and if not, to define the best one in terms of reproducibility. DESIGN: Oxygen delivery and oxygen consumption were prospectively analyzed in 171 consecutive critically ill patients. Metabolic data were obtained simultaneously. SETTING: The study was completed in the intensive care unit as part of the management of the patients studied. PATIENTS: A first "group" of 279 evaluations was carried out in 73 consecutive critically ill patients. The results were subsequently validated by 423 observations performed in the 98 following patients. INTERVENTIONS: Before and during each evaluation, the patients were kept in stable hemodynamic and metabolic conditions. All were mechanically ventilated. MEASUREMENTS AND RESULTS: VO2 was evaluated by calculation (Fick principle) and direct measurement using indirect calorimetry. Cardiac output was both measured by the thermodilution technique and calculated (Fick principle) and the data were used for the evaluation of the directly measured and indirectly calculated DO2. For both VO2 and DO2 the agreement between direct and indirect evaluations was not satisfactory. Differences as great as 55 ml/min.m2 and 267 ml/min.m2 between simultaneously measured and calculated VO2 and DO2 respectively may be expected. Finally, the indirect calculated methods were less reproducible than the measured ones. These observations resulted mainly from the cumulative effects of the random errors in the metabolic data entering into the calculation of VO2 and DO2. CONCLUSIONS: Our data suggested that the indirect calculation (Fick equation) and the direct measurement (indirect calorimetry, thermodilution) of both VO2 and DO2 did not provide similar results. Direct measurements are more reproducible methods and must be preferred.

Adolescent↗
PubMed2000

Polymorphism of clarithromycin.

It is well recognized that physicochemical properties of drugs are affected by the type of polymorphic crystalline form of drugs. Clarithromycin is known to exist in at least three polymorphic crystalline forms. Since conventional means to obtain the most thermodynamically stable form (Form II) for the antibiotics is known to be associated with a low purity of the stable form, we developed a novel method to improve the purity of the crystalline form by a modification of the preparation process. The new method involved a simple recrystallization of clarithromycin in solvents having 5-12 carbon atoms (e.g., hexane and heptane) or ethers with 4-10 carbon atoms (e.g., isopropyl ether) and, thus, less likely to be associated with the problem in purity of resulting crystal. Differential scanning calorimetry and powder X-ray diffraction were used to compare the crystalline form of the resultant powder with Form II crystal prepared by the conventional method. The crystal prepared by the new method was identical to Form II crystal of the conventional method as evidenced by the lack of the exothermic peak near 110 degrees C in differential calorimetry scan, indicating that Form II crystal could be readily prepared by the new process. Therefore, these data indicated that the improvement in the purity of the Form II crystal for clarithromycin as well as a significant cost reduction is likely by the novel method.

Anti-Bacterial Agents↗
PubMed1994

Malignant hyperthermia and calcium-induced heat production.

The abnormal increase in intracellular Ca++ in malignant hyperthermia (MH) is well documented, but the link between the increased Ca++ concentration and high temperature remains speculative. We investigated the possibility that the Ca(++)-induced change in the state of cell membranes may contribute to the temperature elevation. Calcium ion transforms phospholipid membranes from the fluid to solid state. This is analogous to the freezing of water, and liberates latent heat. Differential titration calorimetry (DTC) measures heat production or absorption during ligand binding to macromolecules. When CaCl2 solution was added to anionic dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidylglycerol (DMPG) vesicle membranes in incremental doses, DTC showed that the heat production suddenly increased when the Ca++ concentration exceeded about 120 microM. At this Ca++ concentration range, these lipid membranes underwent phase transition. The latent heat of transition was measured by differential scanning calorimetry (DSC). The values were 7.1 +/- 0.7 (SD, n = 4) kcal.mol-1 of DMPA and 6.8 +/- 0.7 (SD, n = 4) kcal.mol-1 of DMPG. The study shows that Ca++ produces heat when bound to lipid membranes. We are not proposing, however, that this is the sole source of heat. We contend that the lipid phase transition is one of the heat sources and it may trigger a hypermetabolic state by elevating the temperature of cell membranes. Because Ca++ is implicated as the second messenger in signal transduction, multiple systems may be involved. More studies are needed to clarify how Ca++ increases body temperature.

Body Temperature↗
PubMed1999

Long-term measurements of energy expenditure in severe burn injury.

The objective of this study was to evaluate resting energy expenditure (REE) in spontaneously breathing and artificially ventilated burn patients during the entire intensive care period. In 27 patients with 51 +/- 20% body surface area burned (BSAB) the REE was determined via indirect calorimetry. Three groups were formed according to the mortality prognosis index of Zawacki et al. In groups A, B, and C the predicted mortality rates were <20%, 20% to 80%, and >80%, respectively. The frequency of acute respiratory distress syndrome (ARDS), sepsis, renal failure, and mortality increased from group A toward group C. The REE test revealed wide individual variation and was usually overestimated by all tested formulas. The mean REE was comparable in groups A, B, and C during the first 20 days (49 +/- 16% vs. 59 +/- 21% vs. 57 +/- 18% above the REE calculated by the Harris-Benedict equation, or HBEE). The REE of patients in groups A and B declined after this period, whereas the long-term ventilated patients in the prognostically unfavorable group C showed a high REE up to the 45th day, usually accompanied by severe organ dysfunction and major metabolic disorders. During this time a nutritional regimen meeting the actual REE could not be achieved. In the clinical situation when indirect calorimetry is not available, REE can be stated to be 50% to 60% above HBEE in patients with >20% BSAB for at least 20 days. Expecting a stable clinical course in patients with a predicted mortality of <20% (group A), oral nutrition usually seems sufficient after a short period of artificial nutritional support (1 week). Patients with a predicted mortality of more than 20% have a complication-burdened clinical course and a prolonged period of ventilation (groups B and C). These patients need parenteral and enteral nutrition for at least 20 days after trauma to prevent severe malnutrition.

Adolescent↗
PubMed2003

Energy expenditure in ischemic stroke patients treated with moderate hypothermia.

OBJECTIVE: To determine total energy expenditure (TEE) in patients with acute ischemic stroke in the territory of the middle cerebral artery (MCA), treated with moderate hypothermia (33 degrees C). DESIGN AND SETTING: Prospective study in a neurological ICU. PATIENTS: Ten consecutive patients with severe MCA infarction undergoing moderate hypothermia. MEASUREMENTS AND RESULTS: Indirect calorimetry was performed continuously over the first 6 days after admission. Mean daily TEE decreased significantly from 1549 before initiation of hypothermia to 1099, 1129, and 1157 on the first, second, and third days of hypothermia, respectively and returned to baseline values after hypothermia was terminated. The ratio of TEE to predicted basal energy expenditure declined from 1.01 before induction of hypothermia to an average of 0.74 during steady state of hypothermia and increased to 1.16 after rewarming. CONCLUSIONS: We found a significant fall in TEE in sedated, curarized, and ventilated ischemic stroke patients during moderate hypothermia. Indirect calorimetry appears to be a useful tool for measuring energy expenditure in these patients, as predicted basal energy expenditure overestimates the caloric requirements during hypothermia.

Adult↗
PubMed2000

Different dosages of dobutamine in septic shock patients: determining oxygen consumption with a metabolic monitor integrated in a ventilator.

OBJECTIVE: Oxygen consumption (VO2) obtained from respiratory gases by indirect calorimetry (VO2,IC) with a metabolic monitor integrated in a ventilator were to be compared to VO2 obtained by the Fick principle (VO2,Fick) in septic patients following an increase in oxygen delivery (DO2) induced by positive inotropic support. DESIGN: Prospective clinical study. SETTING: University Hospital, Surgical Intensive Care Unit (ICU). PATIENTS: Thirty patients suffering from sepsis. INTERVENTIONS: DO2 was increased by dobutamine infusion, starting with an initial dosage of 5 microg x kg x min, increased to a maximum of 10 microg x kg x min. MEASUREMENTS AND MAIN RESULTS: Dobutamine infusion induced a dosage-related increase in DO2 (from 577 +/- 192 to 752 +/- 202 ml x min x m2, p < 0.01), which was associated with a statistically significant increase in VO2,IC (from 173 +/- 30 to 188 +/- 28 ml x min x m2, p < 0.01) and in VO2,Fick (from 140 +/- 25 to 156 +/- 24 ml x min x m2, p < 0.01). The comparison between VO2,IC and VO2,Fick revealed differences (bias and precision--33 +/- 32 ml x min x m2). CONCLUSIONS: With a metabolic monitor integrated in a ventilator it was possible to carry out continuous monitoring of calorimetric data under clinical conditions. In contrast to previous studies using indirect calorimetry, this study showed a moderate correlation between VO2 and DO2 in septic patients using either method. The clinical relevance of this finding requires further investigation. Different factors (e. g. injectant temperature, pulmonary VO2) produced substantial differences between VO2,IC and VO2,Fick as previously shown.

Adult↗
PubMed1996

Effect of intraperitoneal injection of glucose on glucose oxidation and energy expenditure in the mdx mouse model of duchenne muscular dystrophy.

Previous studies suggesting that glucose metabolism could be impaired in the skeletal muscles of the mdx mouse led us to study the metabolic response to i.p. injection of either glucose or glucose and insulin in the free-moving mdx mouse. In the first study, changes in blood glucose and plasma insulin levels were measured in mice with chronic venous cannulae. In the second study, the thermogenic response to glucose and the changes induced on glucose oxidation (Gox) and lipid oxidation (Lox) were assessed by indirect calorimetry. The experiments showed that insulin response, as well as whole body glucose uptake, were normal in mdx mice. Addition of exogenous insulin abolished the increase in blood glucose level similarly in mdx and control mice. The thermogenic response to glucose was identical in mdx and control mice but, when insulin was injected with glucose, it increased significantly in mdx mice. Exogenous glucose increased Gox less and decreased Lox less in mdx than in control mice. Addition of exogenous insulin reduced the difference between mdx and control mice but affected Gox and Lox less in mdx than in control mice. Key words Duchenne muscular dystrophy middle dot mdx Mouse middle dot Glucose tolerance test middle dot Respiratory quotient middle dot Energy metabolism middle dot Indirect calorimetry

Animals↗
PubMed2005

Combined spectroscopic and calorimetric characterisation of rubredoxin reversible thermal transition.

Rubredoxins are small iron proteins containing the simplest type of iron-sulphur centre, consisting of an iron atom coordinated by the thiol groups of four cysteines. Here we report studies on the conformational stability of a new type of rubredoxin from the hyperthermophile Methanocaldococcus jannaschii, having an atypical metal site geometry resulting from a modified iron-binding motif. Absorption and fluorescence spectroscopies were used in combination with differential scanning calorimetry to probe different aspects of the thermal unfolding transition: iron site degradation (absorption at 380 nm), tertiary structure unfolding (Trp emission), exposure of hydrophobic regions (1-anilinonaphalene-8-sulphonate fluorescence enhancement) and iron release. Thermal denaturation was found to be irreversible and caused by decomposition of the metal centre. The protein is hyperstable and between pH 4 and 10 it is only thermally denatured in the presence of a strong chemical denaturant. The study of the heating rate dependence of the melting temperature allowed us to determine the reaction equilibrium thermodynamic parameters. At pH 2 the protein is destabilised owing to the absence of salt bridges and it has a T(m) of 65 degrees C. In these conditions, there is excellent agreement between the parameters determined by the different spectroscopic methods and calorimetry. The highest stability was found to be at pH 8, and a detailed study of the heating rate dependence in the presence of guanidine thiocyanate in this condition allowed the determination of a reversible T(m) of 118 degrees C.

Binding Sites↗
PubMed1999

Quantitative evaluation of metal ion binding to PvuII restriction endonuclease.

Restriction enzymes are important examples of phosphodiester hydrolysis activity and as such have been of increasing interest to structural biologists. Much of the architecture of endonuclease active sites has been derived from X-ray crystallographic studies. These structures implicate conserved active site acidic residues and the scissile bond of the substrate as coordination ligands of required metal ions. Central to the development of restriction enzyme mechanism is our understanding of the role of metal ion binding in the reaction, an important feature of which is identifying the energetic contributions of the enzyme and the substrate to metal ion affinity. To begin to address this issue, isothermal titration calorimetry (ITC) and 19F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with Kd values of approximately 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with Kd approximately 2 mM. Mn(II) paramagnetic broadening in 19F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease.

Calcium↗
PubMed2006

Blue cooperative luminescence in Yb3+-doped barium gallogermanate glass excited at 976 nm.

The Yb3+-doped barium gallogermanate glass has been prepared via the conventional melt method. The absorption spectra, the near infrared emission spectra, the upconversion emission spectra and the differential scanning calorimetry have been measured. Bright blue upconversion emission centered at 476 nm has been observed under the 976 nm laser diode excitation at room temperature. The blue upconversion emission mechanism has been discussed on the blue emission intensity and the measured lifetime. The slope of the log-log plot of the blue emission intensity versus the pump power is equal to 1.98, and the blue luminescence decay time is half of the near infrared fluorescence decay time, confirming that the blue emission comes from Yb3+-Yb3+ pairs cooperative upconversion mechanism. The result of differential scanning calorimetry suggests that this type of glass is suitable as a potential candidate for fiber drawing.

Calorimetry, Differential Scanning↗
PubMed2005

A quantitative model to evaluate solubility relationship of polymorphs from their thermal properties.

PURPOSE: The objective of the study is to develop a model to estimate the solubility ratio of two polymorphic forms based on the calculation of the free energy difference of two forms at any temperature. This model can be used for compounds with low solubility (a few mole percent) in which infinite dilution can be approximated. METHODS: The model is derived using the melting temperature and heat of fusion for apparent monotropic systems, and the solid-solid transition temperature and heat of transition for apparent enantiotropic systems. A rigorous derivation also requires heat capacity (Cp) measurement of liquid and two solid forms. This model is validated by collecting thermal properties of polymorphs for several drugs using conventional or modulated differential scanning calorimetry. From these properties the solubility ratio of two polymorphs is evaluated using the model and compared with the experimental value at different temperatures. RESULTS: The predicted values using the full model agree well with the experimental ones. For the purpose of easy measurement, working equations without Cp terms are also applied. Ignoring Cp may result in an error of 10% or less, suggesting that the working equation is applicable in practice. Additional error may be generated for the apparent enantiotropic systems due to the inconsistency between the observed solid-solid transition temperature and the true thermodynamic transition temperature. This inconsistency allows the predicted solubility ratios (low melt/high melt) to be smaller. Therefore, a correction factor of 1.1 is recommended to reduce the error when the working equation is used to estimate the solubility ratio of an enantiotropic system. CONCLUSIONS: The study of the free energy changes of two crystalline forms of a drug allows for the development of a model that successfully predicts the solubility ratio at any temperature from their thermal properties. This model provides a thermodynamic foundation as to how the free energy difference of two polymorphs is reflected by their equilibrium solubilities. It also provides a quick and practical way of evaluating the relative solubility of two polymorphs from single differential scanning calorimetry runs.

Calorimetry, Differential Scanning↗
PubMed2006

Screening of lipid carriers and characterization of drug-polymer-lipid interactions for the rational design of polymer-lipid hybrid nanoparticles (PLN).

PURPOSE: The thermodynamics and solid state properties of components and their interactions in a formulation for polymer-lipid hybrid nanoparticles (PLN) were characterized for screening lead lipid carriers and rational design of PLN. METHODS: Verapamil HCI (VRP) was chosen as a model drug and dextran sulfate sodium (DS) as a counter-ionic polymer. Solubility parameters of VRP, VRP-DS complex, and various lipids were calculated and partition of VRP and VRP-DS in lipids was determined. Thermodynamics of VRP binding to DS was determined by isothermal titration calorimetry (ITC). The solid state properties of individual components and their interactions were characterized using differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). RESULTS: Dodecanoic acid (DA) was identified as the best lipid carrier among all lipids tested based on the solubility parameters and partition coefficients. VRP-DS complexation was a thermodynamically favorable process. Maximum binding capacity of DS and the highest drug loading capacity of DA were obtained at an equal ionic molar ratio of DS to VRP. In the PLN formulation, DA remained its crystal structure but had a slightly lower melting point, while VRP-DS complex was in an amorphous form. CONCLUSIONS: Drug loading efficiency and capacity of a lipid matrix depend on the VRP-DS binding and the interactions of the complex with the lipid. A combined analysis of solubility parameters and partition coefficients is useful for screening lipid candidates for PLN preparation.

Calcium Channel Blockers↗
PubMed1997

Thermotropic phase behavior of in vivo extracted human stratum corneum lipids.

The thermotropic phase behavior of lipids extracted either in vivo from inner forearm (SCLE) or plantar callus (PC) was investigated by differential scanning calorimetry and small angle X-ray diffraction. PC composition was chromatographically modified (MPC) by eliminating the more polar lipids in order to evaluate their role. Analysis of composition confirms the potential use of PC as a source of stratum corneum lipids. MPC and SCLE exhibit similar differential scanning calorimetry (DSC) profiles with a main transition around 50 degrees C attributed to the solid-to-liquid phase transition of the ceramides. The absence of a transition around 50 degrees C for PC suggests the possible perturbation of ceramide packing by the significantly high proportion of phospholipids. X-ray data suggest a high miscibility of sebum components in stratum corneum lipids with possible modification of chain packing. The MPC patterns show a lipid phase separation which underscores the role of polar lipids in cholesterol/free fatty acids/sterol esters/ceramides structural cohesion.

Adult↗
PubMed1991

Calorimetric determination of cooperative interactions in high affinity binding processes.

It is demonstrated that isothermal titration calorimetry can be used to determine cooperative interaction energetics even for extremely tight binding processes in which the binding affinity constants are beyond the limits of experimental determination. The approach is based on the capability of calorimetry to measure the apparent binding enthalpy at any degree of ligand saturation. When calorimetric measurements are performed under conditions of total association at partial saturation, the dependence of the apparent binding enthalpy on the degree of saturation is a function only of the cooperative binding interactions. The method developed in this paper allows an independent estimation of cooperative energetic parameters without the need to simultaneously estimate or precisely know the value of the association constants. Since total ligand association at partial saturation is achieved only at macromolecular concentrations much larger than the dissociation constants, the method is especially suited for high and very high affinity processes. Biological associations in this category include fundamental cellular processes like cell surface receptor binding or protein-DNA interactions.

Binding Sites↗
PubMed1983

Calorimetric studies of flavin binding protein: flavin analog binding.

The effect of flavin structure variation upon the overall binding reaction of flavin to hen egg white riboflavin binding protein (WRBP) was correlated to thermodynamic parameters obtained via titration calorimetry. This effect was measured by determining a reference binding enthalpy (delta Href) for 3-carboxymethylriboflavin and subsequently comparing delta Href to the binding enthalpies determined for all other flavin analogs in this study. The reference enthalpy change was measured in detail under a variety of temperatures (14, 25, and 38 degrees C), pH's (5.5, 6.5, 7.4, 8.5), and buffer types. The reference enthalpy for the binding of N(3)-carboxymethylriboflavin to WRBP was measured as delta H = -20.9 kcal/mol at 25.0 degrees C and pH 7.4. Evaluation of the binding thermodynamic values for the reference flavin indicates that there is no H+ flux concomitant with binding. A small negative change in heat capacity (delta Cp) occurs during the binding process suggesting a contraction of the protein. Finally, the reaction appears to be independent of pH and buffer type indicating little, if any, involvement of charged residues in the binding of flavin to apoWRBP. Thermodynamic values for the binding of an additional eight flavin analogs were then measured at pH 7.4 and 25.0 degrees C. The thermodynamic binding parameters for these analogs were evaluated by comparison to those determined for the reference flavin. The results indicate that the ribityl C2' position is a major influence in the interaction between the ribityl side chain and the protein; the C9 position of the isoalloxazine ring is sterically restricted in a manner similar to positions C7 and C8; and the positions C7 and C8 appear to be unequal with regard to enthalpy release, suggesting that the C8 position is the most restricted region in the aromatic ring. These various findings indicate the unique ability of titration calorimetry to evaluate structural variation of ligands to their corresponding binding site interactions.

Animals↗
PubMed1987

Thermal inactivation of electron-transport functions and F0F1-ATPase activities.

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗