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PubMed1995

Quantification of carbohydrate oxidation by respiratory gas exchange and isotopic tracers.

Estimates of glucose oxidation measured by indirect respiratory calorimetry and by [U-13C]glucose tracer were compared as a function of respiratory exchange ratio (RER) in 14 studies performed on 9 healthy adult subjects. RER was varied between 0.7 and 1.04, either by fasting or by infusing glucose. 13C enrichment of plasma glucose and expired CO2 were measured by mass spectrometry. The two methods gave similar results when the nonprotein respiratory quotient (NPRQ) was between 0.76 and 0.90. Glucose oxidation by the tracer method was quantified to be higher than that by respiratory calorimetry when NPRQ was < 0.76; it was lower than the respiratory calorimetry estimate when NPRQ was > 0.90. The discrepancy between the two methods at low RER may represent the contribution of gluconeogenesis, whereas, at high RER, the discrepancy may be the consequence of lipogenesis. We conclude that respiratory calorimetry and [13C]glucose tracer give comparable results only in a narrow range of RER. These data are important when the disposal of glucose is compared using these techniques in different metabolic states with varying respiratory quotients.

Adult↗

PubMed2006

Prediction equation of resting energy expenditure in an adult Spanish population of obese adult population.

OBJECTIVE: The aim of our study was to evaluate the accuracy of the equations to estimate REE in obese patents and develop a new equation in our obese population. SUBJECTS AND METHODS: A population of 200 obesity outpatients was analyzed in a prospective way. The following variables were specifically recorded: age, weight, body mass index (BMI), waist circumference, and waist-to-hip ratio. Basal glucose, insulin, and TSH (thyroid-stimulating hormone) were measured. An indirect calorimetry and a tetrapolar electrical bioimpedance were performed. REE measured by indirect calorimetry was compared with REE obtained by prediction equations to obese or nonobese patients. RESULTS: The mean age was 44.8 +/- 16.81 years and the mean BMI 34.4 +/- 5.3. Indirect calorimetry showed that, as compared to women, men had higher resting energy expenditure (REE) (1,998.1 +/- 432 vs. 1,663.9 +/- 349 kcal/day; p < 0.05) and oxygen consumption (284.6 +/- 67.7 vs. 238.6 +/- 54.3 ml/min; p < 0.05). Correlation analysis among REE obtained by indirect calorimetry and REE predicted by prediction equations showed the next data; Berstein's equation (r = 0.65; p < 0.05), Harris Benedict's equation (r = 0.58; p < 0.05), Owen's equation (r = 0.56; p < 0.05), Ireton's equation (r = 0.58; p < 0.05) and WHO's equation (r = 0.57; p < 0.05). Both the Berstein's and the Ireton's equations overpredicted REE and showed nonsignificant mean differences form measured REE. The Owen's, WHO's, and Harris Benedict's equations underpredicted REE. Our male prediction equation was REE = 58.6 + (6.1 x weight (kg)) + (1,023.7 x height (m)) - (9.5 x age). The female model was REE = 1,272.5 + (9.8 x weight (kg)) - (61.6 x height (m)) - (8.2 x age). Our prediction equations showed a nonsignificant difference with REE measured (-3.7 kcal/day) with a significant correlation coefficient (r = 0.67; p < 0.05). CONCLUSION: Previously developed prediction equations overestimated and underestimated REE measured. WHO equation developed in normal weight individuals provided the closest values. The two new equations (male and female equations) developed in our study had a good accuracy.

Adult↗

PubMedDate not supplied

Comparison of two systems of measuring energy expenditure.

BACKGROUND: Health care professionals typically use resting metabolic rate (RMR) via indirect calorimetry to determine a person's energy expenditure. Traditional indirect calorimetry measurements involve an expensive, cumbersome piece of equipment that requires careful calibration. The recent development of a handheld indirect calorimeter makes it easier to measure RMR. The purpose of this study was to compare simultaneous measurements of RMR with handheld and traditional indirect calorimeters. METHODS: Healthy, free-living subjects (n = 50) age 18 years and older were tested simultaneously with both indirect calorimeters. All subjects breathed through the handheld device using a mouthpiece while wearing noseclips to prevent leaks. The handheld indirect calorimetry device was placed inside a canopy. The exhaled gas from the handheld was positioned directly over the inlet to the port delivering gases to the traditional device's mixing chamber. The canopy facilitated the simultaneous collection of all expired gases into the traditional device. During the measurement, oxygen consumption and RMR were continuously recorded on a personal computer. RESULTS: Mean oxygen consumption and RMR did not significantly differ between the two devices, with a mean difference of 0.58 +/- 15.33 mL/min (p = .790) and 4.66 +/- 113.39 kcal/day (p = .773) and an absolute difference of 12.3 +/- 8.99 mL/min and 86.58 +/- 72.32 kcal/day, respectively. Correlation coefficients for oxygen consumption and RMR were 0.945 and 0.941, respectively. CONCLUSIONS: No significant difference was found between the measurements of indirect calorimetry with the MedGem (HealtheTech, Golden, CO) device compared with the DeltaTrac device (Datex-Ohmeda, Madison, WI). These findings suggest that the handheld indirect calorimeter may provide an accurate measure of oxygen consumption and RMR measurements for spontaneously breathing subjects.

Adult↗

PubMed2000

Validation of a [13C]bicarbonate tracer technique to measure neonatal energy expenditure.

The use of a stable isotope-labeled [13C]bicarbonate infusion to measure energy expenditure is advantageous, as a complete collection of expired air is not required. This technique allows for facile measurements of energy expenditure in intubated neonates. The aim of the present study was to determine the accuracy of energy expenditure estimates in postsurgical neonates by using the [13C]bicarbonate method compared with the current standard, indirect calorimetry. Eight neonates who were receiving total parenteral nutrition [98 +/- 21 (SD) kcal x kg(-1) x d(-1); 3.1 +/- 0.7 (SD) protein g x kg(-1) x d(-1)] were studied on postoperative d 15.5 +/- 11.9. A primed continuous 3-h intravenous infusion of NaH13CO3 and indirect calorimetry were performed simultaneously. Energy expenditure was calculated separately from the Weir equation and from the dilution of 13CO2 in the breath in combination with the individual energy equivalents of CO2 from the diet. The rate of CO2 appearance and energy expenditure calculated from the bicarbonate method (0.725 +/- 0.021 mol x kg(-1) x d(-1); 89.5 +/- 2.5 kcal x kg(-1) x d(-1)) highly correlated (r = 0.94 and 0.98, respectively) with the CO2 excretion and energy expenditure determined by indirect calorimetry (0.489 +/- 0.016 mol x kg(-1) x d(-1); 60.2 +/- 2.0 kcal x kg(-1) x d(-1)) when analyzed nonproportionately to weight. Bland-Altman analysis demonstrated the 95% confidence interval to be +/- 8.2 kcal x kg(-1) x d(-1). Linear regression analysis revealed a highly statistically significant equation relating the two energy expenditures: Indircal (kcal/d) = -9.341 + [0.705 x Bicarb (dcal/d)]; p < 0.001, r2 = 96.4%. We conclude that energy expenditure in neonates can be accurately determined using the [13C]bicarbonate method and a regression equation. Therefore, the bicarbonate method may be useful for determining energy expenditure in neonates not readily accessible to indirect calorimetry, such as those being mechanically ventilated or on extracorporeal life support.

Bicarbonates↗

PubMed2000

Factors affecting the measurement of energy expenditure during energy balance studies in preterm infants.

Our objective was to examine factors that affect the accuracy of energy expenditure measurements (EE), when using flow-through indirect calorimetry (IC), to determine the minimum length of time needed to measure 24- and 48-h EE and to compare cross-over and parallel designs as methods of investigation during energy balance collections (EB) in preterm infants. A baby doll manikin was used to determine equilibration times and to compare VCO(2) and VO(2) as measured by flow meter and indirect calorimetry under different study conditions, one of which simulated an EB. "Continuous" EE was measured to determine the minimum length of time needed to accurately reflect 24- and 48-h EE and to compare parallel and cross-over studies as methods of study design in a group of "normal" enterally fed preterm infants. The mean (+/-SD) errors between flow meter and indirect calorimetry determinations for VCO(2) and VO(2) were -1.9 +/- 2.5 and -1.8 +/- 4.3% under conditions that simulated an EB. Cumulative 6-h EE accurately predicted 24- and 48-h EE. Expressed in absolute terms (kcal/d), EE did not change on a day-to-day basis but did increase over the 2-wk study period. Expressed on body weight basis (kcal/kg/d), EE did not change on a day-to-day or week-to-week basis. The variance in EE due to biologic variability; i.e. the parallel design, was approximately 6 times greater than that due to age, weight, and weight gain; i.e. the cross-over design. Indirect calorimetry, therefore, accurately measures EE in conditions simulating an energy balance collection. Six-hour EE determinations are valid estimate of EE during a 48-h balance collection, while cross-over studies may be the preferred method of study design during short-term studies of EE in preterm infants.

Aging↗

PubMed1999

A method for the measurement of glucose oxidation using the constant infusion of stable isotope.

We developed a method to measure the oxidation of glucose using the primed constant infusion of [U-13C] glucose in critically ill patients fed by total parenteral nutrition. The results obtained from the isotopic method were compared to those from indirect calorimetry in the critically ill patients. A patient with esophageal carcinoma was used for the preliminary study. The study was performed on the third postoperative day, assuming severely stressed state. Priming doses of NaH13CO3 at a dosage of 0.32 mg/kg and D-[U-13C] glucose at a dosage of 0.32 mg/kg were injected. D-[U-13C] glucose was then infused at an infusion rate of 0.004 mg/kg/min. It was revealed that the time required for an isotopic plateau was approximately 45 min in plasma glucose and 120 min in an expired air in highly stressed state. Isotopic measurement and indirect calorimetry were performed simultaneously pre- and postoperatively on three patients who underwent surgery for esophageal carcinoma. Increased fat oxidation was obtained by the isotopic method, whereas indirect calorimetry indicated nonprotein RQ above 1.0. Isotopic measurement offered a useful information that cannot be obtained from indirect calorimetry concerning the energy metabolism in the critical illness. Thus our method for the measurement of glucose oxidation is both simple and useful in investigating the energy metabolism in critically ill patients.

Blood Glucose↗

PubMedDate not supplied

[Study of energy expenditure in anorexia nervosa: agreement between indirect calorimatry and several equations].

UNLABELLED: Nutritional management is essential in anorexia nervosa (AN), although nutrient replenishment must be done progressively to prevent the occurrence of re-alimentation syndrome. OBJECTIVE: to compare resting enengy expenditure (REE) by means of indirect calorimetry and by different equations in AN female patients. MATERIAL AND METHODS: we studied 21 women admitted for AN (DSM-IV), mean age 17 years (SD 5.9), range 12-34 years. Admission stay was 55.1 +/- 20.7 days (21-91). Initial nutritional assessment included anthropometrics (BMJ, TSF, SSE, MAC, MAMC) and tetrapolar bioimpedance (HoltainBC). Indirect calorimetry (IC) was done after overnight fasting (Deltatrac II MBM-200). In 9 patients, the same study was repeated before hospital discharge. We compared REE (Kcal/24 h) measured by IC with that obtained by several equations [Fleish, Harris- Benedict, FAO, Schofield-HW (SHW), Schebendach] through the intraclass correlation coefficient (ICC) and the Bland-Altman method. RESULTS: Nutritional status significantly improved during hospital admission. Fifty percent of the recovered weight was fat mass. REE significantly increased during admission. The equations overestimated REE as compared to IC (p < 0.05), except for the Schebendach equation that underestimated REE. The agreement between direct measurement of REE by IC and its estimation through equations was poor [Fleish (ICC = 0.21); HB (ICC = 0.21), SHW (ICC = 0.19), Schebendach (ICC = 0.15)]. Through the Bland-Altman method, we observed that there was a variable bias between IC and equations, with a clinically acceptable agreement for REW values of around 1200 Kcal/day. CONCLUSIONS: 1) In our study we obtained a poor agreement between REW values measured by indirect calorimetry and those estimated by equations. 2) Through the Bland-Altman method, we observed that all equations present a variable bias as for IC, the agreement being clinically acceptable for REE values of around 1200 Kcal/day. 3) Thus, indirect calorimetry seems to be a very useful tool to calculate the energy requirements of anorexia nervosa patients.

Adolescent↗

PubMed1991

Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus).

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.

Calorimetry, Differential Scanning↗

PubMed1998

[The heart of anorexic adolescents].

BACKGROUND: Anorexia nervosa (AN) is often associated with cardiac changes, such as thinning of the left ventricle (LV), reduction of LV mass, abnormalities of mitral valve function and systolic dysfunction. Some authors have reported QT interval prolongation and sudden death in these patients. METHODS: We studied 23 adolescent females, aged 14.7 +/- 2 years (mean +/- SD), with AN. Serum electrolytes, proteins and albumin were measured in all patients. Electrocardiogram, Doppler-echocardiogram and chest X-rays were also performed on the same day. Eighteen patients were also examined via indirect calorimetry (difference from basal metabolic rate) and 21 underwent dosage of thyroid hormones. RESULTS: The patients, who were of normal height (159 +/- 7.4 cm), were underweight (36 +/- 4.8 kg) and had a body mass index (BMI) of less than 19 (14.2 +/- 1.3). Serum electrolytes, proteins, albumin and chest X-rays were substantially normal in all patients; 74% of them showed reduction of FT3. The calorimetry was reduced (-27.1 +/- 10.6%) with the exception of one patient. Resting heart rate was 58 +/- 12 bpm. We found normal values for PR, QRS, QT (0.41 +/- 0.03 s1/2) and QTc intervals (0.40 +/- 0.03 s1/2) and QT dispersion (40.9 +/- 14.1 ms). Echocardiography showed a reduction in the dimensions of the interventricular septum (52% of patients), LV free wall (61%), left atrium (31%) and LV mass (61%). Fractional shortening was normal in all but one patient. In 61% of cases, there was mild or moderate pericardial effusion that was clinically silent and inversely related to BMI (r = -0.38, p 0.08, ns), to calorimetry (r = -0.56, p < 0.0055), to FT3 (r = -0.53, p < 0.05) and to sodium concentration (r = -0.43, p 0.04). CONCLUSIONS: Teen-agers with AN often show a reduction in LV thickness and mass, as well as clinically silent pericardial effusion that is inversely related to BMI, calorimetry, FT3 and sodium serum concentrations. We did not find any prolongation of QTc interval or of QT dispersion.

Adolescent↗

PubMed2003

Validity of uniaxial accelerometry during activities of daily living in children.

The purpose of this study was to examine the validity of treadmill-based equations of two commonly used uniaxial accelerometers to estimate energy expenditure (EE) during activities of daily living in children. Twelve subjects with mean (SD) age11.4 (0.4) years engaged in a choreographed routine consisting of three activities (sweeping, bowling, and basketball) of 4min duration while wearing a Manufacturing Technology, Inc. (MTI) accelerometer, Caltrac accelerometer, and a portable gas analyzer (Cosmed K4b(2)). The equations of Trost et al. and Sallis et al. were used to convert activity counts to estimations of EE for the MTI and Caltrac, respectively. Correlation coefficients between Caltrac predictions of EE and measured EE from indirect calorimetry ranged from r=0.22 to 0.72 for individual activities. Correlations between MTI EE predictions and indirect calorimetry ranged from r=0.50 to 0.68 for individual activities. When the activities were pooled the correlations between EE from uniaxial accelerometers and EE from indirect calorimetry were moderately strong (MTI, r=0.78 and Caltrac, r=0.82). Inter-accelerometer (counts min(-1)) correlations were 0.08, -0.54, 0.63, and 0.79 for sweeping, bowling, basketball, and pooled data, respectively. The overall mean difference, or bias, and 95% confidence intervals (CI) for each uniaxial accelerometer compared to indirect calorimetry were as follows: Caltrac, bias = 2.80 (2.36, 3.24) kcal min(-1); MTI, bias = 0.88 (0.23, 1.53) kcal min(-1). Both accelerometers significantly underestimated measured EE ( P<0.05). Uniaxial accelerometers provide potential for the measurement of physical activity (PA) and EE in children. Future studies refining accelerometry predictions of PA and EE are warranted.

Acceleration↗

PubMed2004

Effect of alcohol addition to the aqueous phase on the thermal effects of micellization of cationic benzyldimethyldodecylammonium bromide and its adsorption onto porous and nonporous silicas.

Titration calorimetry has been used to study the effect of the addition of two primary alcohols, 1-butanol and 1-heptanol, to the aqueous phase on the thermal effects of micellization of benzyldimethyldodecylammonium bromide (BDDAB) as well as its adsorption onto nonporous Spherosil XO15M and onto porous aluminosilicate SiAl32d22 possessing uniformly sized mesopores. A linear decrease of the critical micelle concentration (CMC) of the cationic surfactant with the additive content was inferred from the specific conductivity measurements. All adsorption and calorimetry experiments were carried out at 298 K and at a fixed alcohol content (0.01 mol kg(-1)) either in deionized water or in a 0.01 mol kg(-1) NaBr solution. Dilution calorimetry measurements allowed determination of the cumulative molar enthalpy changes and a new analysis of these data was proposed to calculate easily the enthalpy of micellization per mole of BDDAB, Delta(mic)h, and the CMC value. The alcohol addition was shown to render the micellization phenomenon more exothermic, the effect being larger as the chain length of alcohol increased. These effects were attributed to the location of alcohol molecules between the surfactant units, their hydroxyl groups close to the surfactant head-groups, in competition with the surfactant counterions. The individual isotherms of alcohol and surfactant adsorption onto XO15M and SiAl32d22 were determined. The plots of the pseudo-differential molar enthalpy of displacement, Delta(dpl)h, against the surface coverage by the surfactant cation, Theta(BDDA+), were derived from the titration calorimetry data. The formation of surface-bound aggregates was thought to be a prerequisite for alcohol coadsorption at the solid-solution interface. At least two different types of adsolubilization sites were postulated, one of the sites being the same as in micelles and the other related to the contact area between the hydrophobic surfactant tails and the equilibrium bulk solution. Coadsorption (adsolubilization) of alcohol molecules at such sites was found to increase the exothermic contribution to the enthalpy of displacement per mole of the BDDA+ adsorbed.

Adsorption↗

PubMed2001

On relationships between molecular structure, interaction and surface behavior in mixture: small-molecule surfactant+protein.

We report on the effect of distinct in nature small-molecule surfactants (model, a sodium salt of capric acid, Na-caprate; and commercially important, a citric acid ester of monoglyceride, CITREM; a sodium salt of stearol-lactoyl lactic acid, SSL (Na(+)); polyglycerol ester, PGE (080)) on molecular properties in a bulk and at the air-water interface of globular legumin and random-coiled micellar sodium caseinate. The role of the structure of both proteins and small-molecule surfactants in the effect studied has been elucidated by measurements in a bulk aqueous medium of the enthalpy of their interaction from mixing calorimetry, the change in value of weight average molecular weight of the proteins and the thermodynamics of the pair protein-protein interactions from laser static light scattering as well as, in addition, by measurements of the change in hydrodynamic radius for micellar sodium caseinate from laser dynamic light scattering. The effect of the small-molecule surfactants on the thermodynamics of the protein heat denaturation and thereby on the protein conformational stability has been studied by differential scanning calorimetry in the case of globular legumin. The interrelation between the effects of the small-molecule surfactants on the properties of the proteins in a bulk and at the planar air-water interface has been elucidated by tensiometry. The combined data of mixing calorimetry, differential scanning calorimetry and laser light scattering suggest some complex formation between the small-molecule surfactants and the proteins in a bulk aqueous medium. Predominantly hydrophobic interaction along with electrostatic and hydrogen bonding form the basis of the complex formation. The found effect of the small-molecule surfactants on the surface activity of their mixtures with proteins is governed primarily by both the extent of the protein association, resulting in specific hydrophobicity/hydrophilicity of the surface of the protein associates, and the specific protein conformational stability, for the globular protein, produced by the interaction between the proteins and the small-molecule surfactants.

Journal Article↗

PubMed1987

Calorimetric versus Growth Microbial Analysis of Cellulase Enzymes Acting on Cellulose.

ASSAY OF CELLULASE ENZYMOLOGY ON CELLULOSE WAS INVESTIGATED BY TWO METHODS: (i) plate colony counting to determine microbial growth and (ii) microbial calorimetry. These methods were chosen because they accept raw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays. Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 muM concentrations of cellulolytic lower sugars (glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5 mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requires completion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetric method to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated with Escherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiella sp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heat proportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- and endocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetric means for demonstrating enzyme synergism.

Journal Article↗

PubMed2006

Biophysical studies of the c-MYC NHE III1 promoter: model quadruplex interactions with a cationic porphyrin.

Regulation of the structural equilibrium of G-quadruplex-forming sequences located in the promoter regions of oncogenes by the binding of small molecules has shown potential as a new avenue for cancer chemotherapy. In this study, microcalorimetry (isothermal titration calorimetry and differential scanning calorimetry), electronic spectroscopy (ultraviolet-visible and circular dichroism), and molecular modeling were used to probe the complex interactions between a cationic porphryin mesotetra (N-methyl-4-pyridyl) porphine (TMPyP4) and the c-MYC PU 27-mer quadruplex. The stoichiometry at saturation is 4:1 mol of TMPyP4/c-MYC PU 27-mer G-quadruplex as determined by isothermal titration calorimetry, circular dichroism, and ultraviolet-visible spectroscopy. The four independent TMPyP4 binding sites fall into one of two modes. The two binding modes are different with respect to affinity, enthalpy change, and entropy change for formation of the 1:1 and 2:1, or 3:1 and 4:1 complexes. Binding of TMPyP4, at or near physiologic ionic strength ([K(+)] = 0.13 M), is described by a "two-independent-sites model." The two highest-affinity sites exhibit a K(1) of 1.6 x 10(7) M(-1) and the two lowest-affinity sites exhibit a K(2) of 4.2 x 10(5) M(-1). Dissection of the free-energy change into the enthalpy- and entropy-change contributions for the two modes is consistent with both "intercalative" and "exterior" binding mechanisms. An additional complexity is that there may be as many as six possible conformational quadruplex isomers based on the sequence. Differential scanning calorimetry experiments demonstrated two distinct melting events (T(m)1 = 74.7 degrees C and T(m)2 = 91.2 degrees C) resulting from a mixture of at least two conformers for the c-MYC PU 27-mer in solution.

Binding Sites↗

PubMed2005

G148-GA3: a streptococcal virulence module with atypical thermodynamics of folding optimally binds human serum albumin at physiological temperatures.

The third albumin binding domain of streptococcal protein G strain 148 (G148-GA3) belongs to a novel class of prokaryotic albumin binding modules that is thought to support virulence in several bacterial species. Here, we characterize G148-GA3 folding and albumin binding by using differential scanning calorimetry and isothermal titration calorimetry to obtain the most complete set of thermodynamic state functions for any member of this medically significant module. When buffered at pH 7.0 the 46-amino acid alpha-helical domain melts at 72 degrees C and exhibits marginal stability (15 kJ/mol) at 37 degrees C. G148-GA3 unfolding is characterized by small contributions to entropy from non-hydrophobic forces and a low DeltaCp (1.1 kJ/(deg mol)). Isothermal titration calorimetry reveals that the domain has evolved to optimally bind human serum albumin near 37 degrees C with a binding constant of 1.4 x 10 7 M(-1). Analysis of G148-GA3 thermodynamics suggests that the domain experiences atypically small per residue changes in structural dynamics and heat capacity while transiting between folded and unfolded states.

Bacterial Proteins↗

PubMed2002

Triton promotes domain formation in lipid raft mixtures.

Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds.

Calorimetry↗

PubMed2003

Applications of calorimetric methods to drug discovery and the study of protein interactions.

Recent studies report the application of isothermal titration calorimetry and differential scanning calorimetry to the study of protein-ligand interactions, allosteric cooperativity and aspects of protein folding. New methods of data analysis compare alternative methods for determining ligand binding enthalpy and analyze potential sources of error in the experimental measurement of other thermodynamic parameters. Several reports examine issues concerning drug design and the correlation of thermodynamic and X-ray structural data. New instruments allow volumetric effects in biochemical systems to be evaluated calorimetrically and to substantially expand the throughput of differential scanning calorimetry measurements in drug discovery and other high-throughput applications.

Calorimetry↗

PubMed1992

Titration calorimetric and differential scanning calorimetric studies of the interactions of n-butanol with several phases of dipalmitoylphosphatidylcholine.

The interactions of n-butanol with dipalmitoylphosphatidylcholine (DPPC) were studied using titration calorimetry and differential scanning calorimetry (DSC). DSC results indicated that n-butanol induces the interdigitated phase in DPPC above 10 mg/mL butanol. A new application of titration calorimetry for measuring partition coefficients of nonsaturating solutes into lipids was developed. The partition coefficients and the heat of binding of n-butanol into DPPC were measured for the L beta', P beta', L alpha, and L beta I phases of DPPC. The partition coefficients were temperature dependent and ranged from 70 to 110 for the L beta I phase, from 170 to 183 for the L alpha phase, and similar to that for the L beta I phase in the P beta' phase. The binding to the L beta' phase could not be detected, giving an upper limit for this partition coefficient of 23. The enthalpies for binding to the L beta I and L alpha phases were 1.0 and 1.5 kcal/mol, respectively. The van't Hoff enthalpy was in good agreement with the calorimetric enthalpy for the partitioning into the L alpha phase; however, it was greater than the calorimetric enthalpy for the L beta I phase, suggesting that the interaction of n-butanol with this phase is cooperative in some way.

1,2-Dipalmitoylphosphatidylcholine↗