Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CALORIMETRY”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,675 records · Page 93Linked to original sources
PubMed1998

Effect of metal ion substitutions in concanavalin A on the binding of carbohydrates and on thermal stability.

Isothermal titration calorimetry (ITC) measurements were performed on the binding of alpha methyl-D-mannopyranoside, D-mannopyranose, alpha methyl-D-glucopyranoside, and D-glucopyranose (Glu) to cobalt, nickel, and cadmium substituted concanavalin A (Con A) derivatives at pH = 6.9 and at 25 degrees C. The metal substituted Con A derivatives consisted of Co2+, Ni2+, and Cd2+ substituted for the Mn2+ ion in the S1 site of Con A which is about 12.8 A away from the center of the carbohydrate binding site of Con A. The thermodynamic quantities determined from the ITC measurements were the same for most of the binding reactions indicating that the structure of the binding site in solution is the same for all the Con A derivatives in solution and that the presence of different 2+ metal ions in the S1 site has little effect on the binding reactions. Differential scanning calorimetry scans of solutions of the metal ion derivatives of Con A show that the thermodynamics of the unfolding transition for the cobalt and nickel substituted derivatives are the same as for Con A: they dissociate from tetramers into monomers as they unfold around 85 degrees C. The cadmium substituted Con A derivative, however, exhibits an additional transition around 93 degrees C which also appears following the addition of Cd2+ to the Con A solutions. This transition results from the unfolding of a species of Con A with Cd2+ substituted into a third binding site at the monomeric interface of the Con A tetramer. The higher stability of this species is not only exemplified by the higher thermal transition temperature but also by the lack of dissociation as it unfolds. Cd2+ is released from the S3 site upon decreasing the pH from 6.9 to 6.4. ITC measurements on the binding reaction of Cd2+ to Con A show that the binding enthalpy is 40.2 +/- 0.4 kJ mol-1 at 23.4 +/- 0.2 degrees C and the binding reaction exhibits a large heat capacity change of 1.43 +/- 0.41 kJ mol-1 K-1.

Binding Sites↗
PubMed2001

Heterogeneous DNA binding modes of berenil.

Isothermal titration calorimetry (ITC) profiles of berenil bound to different DNAs show that, despite the strong preference of berenil for AT-rich regions in DNA, it can bind to other DNA sequences significantly. The ITC results were used to quantify the binding of berenil, and the thermodynamic profiles were obtained using natural DNAs as well as synthetic polynucleotides. ITC binding isotherms cannot be simply described when a single set of identical binding sites is considered, except for poly[d(A-T)2]. Ultraviolet melting of DNA and differential scanning calorimetry were also used to quantify several aspects of the binding of berenil to salmon testes DNA. We present evidence for secondary binding sites for berenil in DNA, corresponding to G+C rich sites. Berenil binding to poly[d(G-C)2] is also observed. Circular dichroism experiments showed that binding to GC-rich sites involves drug intercalation. Using a molecular modeling approach we demonstrate that intercalation of berenil into CpG steps is sterically feasible.

Animals↗
PubMed2000

Why has porcine VEG protein unusually high stability and suppressed binding ability?

Von Ebner gland protein (VEGP) and odorant-binding protein (OBP) were purified from porcine lingual epithelium and nasal mucosa, respectively. Both VEGP and OBP preparations were homogeneous as indicated by SDS-PAGE, isoelectric focusing, gel-filtration and electrospray mass spectrometry. However, high-sensitivity differential scanning calorimetry (HS-DSC) yielded multiphasic denaturation thermograms for both proteins indicating their conformational heterogeneity. The unfolding transition of VEGP is observed at extremely high temperatures (about 110 degrees C), which is unexpected for a protein with significant structural homology to OBP and other lipocalins. Isothermal titration calorimetry (ITC) did not detect the binding of either aspartame or denatonium saccharide to VEGP nor did it detect binding of 2-isobutyl-3-methoxypyrazine (IBMP) to OBP. Extraction of OBP with mixed organic solvents eliminated the conformational heterogeneity and the protein showed a reversible two-state transition in HS-DSC thereafter. ITC also showed that the extracted OBP was able to bind IBMP. These results imply that tightly bound endogenous ligands increase the thermal stability of OBP and block the binding of other ligands. In contrast to OBP, the extraction of VEGP with organic solvents failed to promote binding or to establish thermal homogeneity, most likely because of the irreversible denaturation of VEGP. Thus, the elucidation of the functional behaviour of VEGP is closely related to the exhaustive purging of its endogenous ligands which otherwise very efficiently mask ligand binding sites of this protein.

Animals↗
PubMed1998

Energy expenditure during allogeneic and autologous bone marrow transplantation.

Resting energy expenditure (REE) was measured by indirect calorimetry during allogeneic and autologous bone marrow transplantation in order to evaluate the evolution in allogeneic and autologous recipient patients. REE values obtained with indirect calorimetry and compared with values using the Harris-Benedict formula were different. Evolution of REE during aplasia were significantly different in autologous and allogeneic recipients with an increase 11.5 ' 10.8 cent for autologous and a decrease of - 7.3 ' 8.9 cent in allogeneic bone marrow transplantation (BMT) patients. There were no differences in nutritional status and REE before BMT, at discharge and 1 month after discharge between the two groups but all patients had inflicted damage on their nutritional status at discharge from hospital after BMT. However, these patients differed because of the decrease in oral nutritional intake and an increase in the length of aplasia and hospital stay in allogeneic patients. Currently, there is no proof that recommendations for nutritional interventions or results of nutritional investigations in allogeneic BMT can be extrapolated in autologous patients. Harris-Benedict formula does not estimate the energy expenditure of patients submitted to massive chemotherapy and BMT with enough precision because of the great differences in individuals.

Adult↗
PubMed2001

Insights into the dynamics of DMSO in phosphatidylcholine bilayers.

The solvation effects of dimethyl sulfoxide (DMSO) on the phase stability of dimyristoylphosphatidylcholine (DMPC) have been fully characterized using differential scanning calorimetry (DSC) and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of the sub-, pre-, and main transitions of DMPC were found to increase linearly with increasing mole fraction of DMSO up to mole fraction X=0.13 DMSO/H(2)O. Beyond X=0.13, the pre-transition peak started to merge with the peak representing the main transition. Simultaneously, the subtransition peak began to disappear as its transition temperature also decreased. At X=0.18, with both the subtransition and pre-transition absent, the main transition between the planar gel and the liquid-crystalline phase was observed at 30.3 degrees C. Transition enthalpy values indicated that the subgel, planar gel and rippled gel phases are most stable at X=0.11, 0.16 and 0.20 DMSO/H(2)O, respectively. This demonstrates that DMSO exerts distinct effects on each respective phase and corresponding transition. Temperature-dependent fluorescence emission scans show an increase in hydration as the system proceeds from the subgel phase all the way to the liquid-crystalline phase and correlated well with the effects of DMSO on the transition temperatures of DMPC observed in our calorimetry data. Initial observations for the sub- and main transition are further confirmed by fluorescence anisotropy using DPH as a probe. The results illustrate the differences in the microviscosity of each phase and how DMSO affects the phase transitions. Ultimately, our results suggest the most likely mechanism governing the biological actions of DMSO may involve the regulation of the solvation effects of water on the phospholipid bilayer.

Calorimetry, Differential Scanning↗
PubMed2002

The alterations of lipid bilayer fluidity induced by newly synthesized phenothiazine derivative.

Using fluorescence spectroscopy, calorimetry and ESR the interactions of the phenothiazine derivative 2-trifluoromethyl-10-(4-[methylsulfonylamid]buthyl)-phenothiazine (FPhMS) with lipids were studied. Calorimetry showed biphasic effect of FPhMS on main phase transition of DPPC. At molar ratios up to 0.06 drug induced decrease of transition temperature and enthalpy, while at higher concentrations it caused subsequent increase of these parameters. For all concentrations studied we observed gradual broadening of transition peaks. Fluorescence polarization revealed that in FPhMS/lipid mixtures, order in bilayers is decreased in the gel state and increased in the liquid crystalline state. ESR experiment showed that at molar ratio of 0.06, FPhMS reduces the mobility of spin probes located in both polar and hydrophobic regions. Comparing observed effects with those reported for cholesterol/lipid mixtures, we conclude that at higher concentrations FPhMS presumably induces a new mode of bilayer packing. This structure is less co-operative than an unperturbed bilayer, but locally the mobility of lipid molecules is decreased.

Calorimetry, Differential Scanning↗
PubMed2003

Physico-chemical studies of a DNA triplex containing a new ferrocenemethyl-thymidine residue in the third strand.

The stability of a 16-mer DNA triple helix containing a 3-N(ferrocenemethyl)-thymidine residue in the third strand has been investigated in comparison with the unmodified triplex of the same sequence. A complete physico-chemical characterization of the two triple helices on changing the pH by means of calorimetry, circular dichroism and molecular modeling is therefore reported. The thermodynamic parameters were obtained in the pH range 5.5-7.2 by differential scanning calorimetry (DSC). For both triplexes the T(m) and Delta H degrees (T(m)) values increase on decreasing the pH. In the pH range 7.2-6.0 the triplex containing the ferrocenemethyl nucleoside is less stable than the unmodified one, whereas the modified triplex becomes more stable at pH 5.5. Such difference in stability at each pH value is overwhelmingly enthalpic in origin. CD spectra show conformational changes on decreasing the pH for both the triplexes. By spectroscopic pH titration the apparent pK(a) values of the cytosines in the two triplexes could be estimated, with the cytosines in the TFO containing the ferrocenemethyl residue having lower apparent pK(a) values. These results are consistent with the calorimetric data, showing a decrease of the thermodynamic parameters in the pH range 7.2-6.0 and an increase at pH 5.5 for the ferrocenylated triplex with respect to the unmodified one. The thermodynamic and spectroscopic data are also discussed in relation to molecular models.

Calorimetry, Differential Scanning↗
PubMed1999

A thermodynamic study on the formation and stability of DNA duplex at transcription site for DNA binding proteins GCN4.

Using isothermal titration calorimetry (ITC), we studied the thermodynamic parameters of the 15-mer duplex dsDNA [d(GAGATGACTCATCTC)].[d(GAGATGAGTCATCTC)] formation from its two complementary single strands (S1 and S2) over a range of temperatures. The two complementary single strands d(GAGATGACTCATCTC) (herein called S1) and d(GAGATGAGTCATCTC) (herein called S2) containing palindromic sequences may assume ordered structures at low temperatures, which made the duplex dsDNA formation rather complicated. The thermodynamic parameters for the duplex formation, such as the binding constants (Kb), the enthalpies (delta H0), the free energies (delta G0), the entropies (delta S0) are strongly temperature-dependent. The thermally-induced disruptions of the duplex and its two complementary single strands, S1 and S2, were measured using differential scanning calorimetry (DSC) and CD spectroscopy, the results demonstrate that the DNA duplex is very stable, and its component single strands have an ordered structure at low temperature. This 15-mer specific sequence DNA may act as recognition site for DNA binding proteins GCN4 and plays a key role in transcription regulation of gene expression. Our analyses of the thermodynamic data suggest that the duplex formation is a coupled process between conformational transitions in the two single strands and their binding to form duplex dsDNA.

Binding Sites↗
PubMed2002

Investigation of the stability of a mixture of single-based propellants and resins or polymers.

The stabilisation of single-based propellants (SBP) with equal quantities of bisphenol A-based epoxy resin (ER), bromine-containing epoxy resin (BER), chlorinated polyvinylchloride (PCV) and chlorinated rubber (CR) were investigated by viscosimetry and calorimetry. All compounds were thermodynamically compatible with SBP. The degree of stabilisation of SBP was measured after accelerated ageing and was evaluated by the loss of weight and by means of differential scanning calorimetry (DSC). All compounds produced a significant decrease in the loss of weight, typically by more than a factor of 15 compared with unstabilised SBP. The stability of the compounds decreased in the order: CR>BER>PCV>ER.

Aerosol Propellants↗
PubMed2002

Solid state characterization of an neuromuscular blocking agent--GW280430A.

GW280430A is an ultrashort-acting neuromuscular blocking agent and is targeted for muscle relaxation as part of the intubation surgical procedure. The objective of this work was to perform solid state characterization on GW280430A and to evaluate the relationship between water content and glass transition temperature (Tg). GW280430A was characterized by differential scanning calorimetry, thermogravimetric analysis, powder X-ray diffraction (PXRD), microscopy and moisture sorption. The effect of water content on the Tg of GW280430A was evaluated by equilibrating the material over saturated salt solutions at a range of relative humidities (6.4-72.6%) and determining the Tg by DSC using hermetically sealed aluminum pans. GW280430A undergoes dehydration at 40 degrees C, glass transition at 130 degrees C and decomposition at 190 degrees C by differential scanning calorimetry. By PXRD and moisture sorption, GW280430A is an amorphous material and deliquesces at about 70% RH at room temperature. Water acts as a potent plasticizer for GW280430A and the Tg decreases significantly as the water content increases. No measurable decomposition of GW280430A was observed after 4 weeks at 40 degrees C/75% RH.

Algorithms↗
PubMed2003

Crystal modification of phenytoin using different solvents and crystallization conditions.

Phenytoin crystals having different types of habits, were prepared by recrystallization from ethanol and acetone solutions under different conditions (cooling rate or crystallization temperature, solvent evaporation and watering-out techniques). Scanning electron microscopy, X-ray powder diffractometry, FT-IR spectrometry and differential scanning calorimetry were used to investigate the physical characteristics of the crystals. The dissolution behavior and compaction properties of various batches of crystals were also studied. It was found that using watering-out technique as a crystallization method, produced thin plate crystals, while the crystals obtained by other methods were needle shape for alcoholic solutions and rhombic for acetone solutions. X-ray diffraction spectra and differential scanning calorimetry studies, did not show any polymorphic change. The dissolution rate of different crystals was lower than that of untreated samples. The compacts of phenytoin crystals produced from alcohol or acetone (especially those made by watering-out method) had higher crushing strengths than untreated phenytoin compacts due to the lower porosity and the lower elastic recovery.

Calorimetry, Differential Scanning↗
PubMed2003

A kinetic and thermodynamic study of seratrodast polymorphic transition by isothermal microcalorimetry.

The development of isothermal microcalorimetry to a study of the kinetic and thermodynamics of polymorphic transitions in seratrodast ((+/-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoic acid) Form II is reported. Sieved samples of Form II were allowed to convert to Form I, in a reaction vessel of an isothermal microcalorimeter, under 13, 31, 63 and 93% relative humidity (RH) between 48 and 65 degrees C. The power (Phi, in Watts) versus time curves from the microcalorimeter were integrated into the heat output (q, in Joules) versus time curves to yield fractional extent of Form I converted versus time curves. The change in enthalpy (-5.70 kJmol(-1)) agreed very closely with that obtained by differential scanning calorimetry and solution calorimetry, which indicated that the power measured by the microcalorimeter was due only to the Form II-to-Form I transition. Application of the theoretical kinetic method [J. Am. Ceram. Soc. 55 (1972) 74] revealed that the transition took place via a two-dimensional growth of nuclei mechanism at all the studied relative humidities and temperatures. The rate constant increased with increasing RH and temperature, and with decreasing the particle size of sample. The activation energies obtained from Arrhenius plots were 292, 290, 280 and 284 kJmol(-1), and the extrapolated rate constants at 25 degrees C were also 3.01 x 10(-10), 3.11 x 10(-10), 9.65 x 10(-10) and 3.84 x 10(-9)s(-1) for 13, 31, 63 and 93% RH, respectively.

Benzoquinones↗
PubMedDate not supplied

Energy expenditure measured by doubly labeled water, activity recall, and diet records in the rural elderly.

OBJECTIVE: The purpose of this study was to determine whether energy expenditure estimated from physical activity and energy intake were equivalent to total daily energy expenditure in an elderly rural population. METHODS: Twenty-seven elderly male (n = 14) and female (n = 13) subjects (mean age, 74 y) were recruited from a rural Pennsylvania population. Over a 2-wk period, total daily energy expenditure was measured by doubly labeled water (TEE) and estimated from 7-d physical activity recall factors multiplied by weight (PA(WT)), estimated basal metabolic rate (PA(BMR)) and resting energy expenditure from indirect calorimetry (PA(REE)), and energy intake from 3-d self-reported diet records (EI). Analysis of variance was used to determine significant within-subject differences in physical activity, energy intake, and energy expenditure. RESULTS: PA(REE) (men: 13.69 +/- 3.23 MJ, women: 9.51 +/- 2.40 MJ) and PA(BMR) (men: 13.69 +/- 2.99 MJ, women: 10.15 +/- 2.21 MJ) were not significantly different from TEE (men: 12.43 +/- 1.63 MJ, women: 9.44 +/- 0.90 MJ). EI (men: 8.66 +/- 2.34 MJ, women: 7.12 +/- 0.93 MJ) was significantly less than TEE, and PA(WT) (men: 17.03 +/- 4.07 MJ, women: 12.86 +/- 3.41 MJ) was significantly greater than TEE. CONCLUSIONS: Whereas 7-d physical activity recall determined with an age- and gender-specific estimate of resting metabolic rate or measured using indirect calorimetry accurately estimated TEE for this group of rural elderly, self-reported diet records consistently underestimated and physical activity recall determined with weight alone consistently overestimated energy expenditure measured by doubly labeled water.

Aged↗
PubMed2003

Effect of hypocaloric meals with different macronutrient compositions on energy metabolism and lung function in obese women.

OBJECTIVE: We investigated the effect of hypocaloric mixed diets with different proportions of carbohydrate, protein, and fat on resting metabolic rate and the thermic effect of food in obese women. METHODS: Three mixed hypocaloric diets were consumed in random order during separate periods lasting 7 d each. Between each dietary period there was a washout period of 10 d. Diet 1 had a higher proportion of energy from carbohydrate (72%), diet 2 had a higher proportion of energy from protein (43%), and diet 3 had a higher proportion of energy from fat (68%). Indirect calorimetry and lung function tests were done after the completion of each 7-d diet. Seven obese women, ages 22 to 45 y and with body mass indexes of 32 to 59 kg/m(2), participated in the study. Oxygen consumption, carbon dioxide production, resting metabolic rate, and the thermic effect of food by indirect calorimetry were measured. Lung function tests included spirometry in the seated and upright positions, arterial blood gas analysis, and maximal inspiratory and expiratory pressures. RESULTS: There were no statistically significant differences in the resting metabolic rate and the thermic effect of food resulting from the three diets. The mean resting metabolic rates (kJ/d) were 7453 +/- 1446 for diet 1, 7461 +/- 1965 for diet 2, and 7076 +/- 2048 for diet 3. The mean thermic effects of food (kcal/min) were -0.02 +/- 0.07 for diet 1, -0.01 +/- 0.25 for diet 2, and 0.05 +/- 0.13 for diet 3. Lung function tests were normal before and after the hypocaloic diets: partial pressure of oxygen (mmHg) values were 81 +/- 13, 77 +/- 8, and 78 +/- 11 for diets 1 to 3, respectively; and partial pressure of carbon dioxide (mmHg) were 37 +/- 4, 37 +/- 3, and 37 +/- 4 for diets 1 to 3, respectively. CONCLUSIONS: Obese women with normal lung function tests and consuming mixed hypocaloric diets showed no alteration in resting metabolic rate and a reduced or absent thermic effect of food independently of the macronutrient composition.

Adult↗
PubMed2000

Energy metabolism and substrate oxidation in patients with Crohn's disease.

Weight loss and malnutrition are common features in patients with Crohn's disease. This study was designed to evaluate diet-induced thermogenesis and substrate oxidation in patients with Crohn's disease. Twenty-three patients (17 women, 6 men; age 34 +/- 2 y) and 17 healthy control subjects (13 women, 4 men; age 36 +/- 3 y) were studied. Resting energy expenditure and fasting substrate oxidation were measured by indirect calorimetry in the morning after an overnight fast. After a standard homogenized test meal (10 kcal/kg), indirect calorimetry was performed every 30 min for 3 h to measure the diet-induced thermogenesis and the postprandial substrate oxidation. In the fasting state, resting energy expenditure was significantly higher in patients than in control subjects (1433 +/- 43 versus 1279 +/- 53 kcal/24 h). Lipid oxidation was higher in patients with Crohn's disease than in control subjects (1.17 +/- 0. 07 versus 0.61 +/- 0.11 mg. kg(-1). min(-1), P < 0.01). Postprandially, diet-induced thermogenesis was significantly lower in patients with Crohn's disease than in control subjects (4.6% +/- 0.5 versus 6.3% +/- 0.5 of energy intake, P < 0.01). Lipid oxidation was significantly higher in patients with Crohn's disease than in control subjects (0.78 +/- 0.05 versus 0.56 +/- 0.08 mg. kg(-1). min(-1), P < 0.05), and glucose oxidation was lower in patients with Crohn's disease than in control subjects. In patients with Crohn's disease, lipid oxidation positively correlates with the disease activity evaluated by the Crohn's Disease Activity Index (r = 0.48, P150), fasting and postprandial lipid oxidation was significantly higher than in patients with inactive Crohn's disease (P < 0.05). In conclusion, patients with Crohn's disease have increased fat oxidation, which correlates with disease activity and this may explain the reduced fat stores in patients with Crohn's disease.

Adult↗
PubMed1998

Preparation and characterization of nanoparticles containing an antihypertensive agent.

Isradipine, an antihypertensive agent, was encapsulated by the nanoprecipitation method using polymers including poly(epsilon-caprolactone), poly(D,L-lactide) and poly(d, L-lactide-co-glycolide). In vitro scanning electron microscopy and differential scanning calorimetry were used to characterize the nanoparticles. The average diameters of the nanoparticles ranged from 110 nm to 208 nm. PCL nanoparticles were larger than nanoparticles prepared with the other polymers. The zeta potential of the nanoparticles was negative, with values of about -25 mV which promoted good stabilization of the particles. The amorphous state of PLA and PLAGA non-loaded nanoparticles and the semi-crystalline state of PCL were demonstrated with X-ray diffraction and differential scanning calorimetry. For all nanoparticles, isradipine was found to be totally amorphous in the polymer which suggested that the drug was molecularly dispersed in the matrix. The colloidal suspensions displayed a sustained release profile in comparison with the drug release profile of isradipine in a PEG solution. Results from this investigation suggest that these nanospheres will be a good candidate delivery system for oral administration, to reduce the initial hypotensive peak and to prolong the antihypertensive effect of the drug.

Antihypertensive Agents↗
PubMed1996

Effects of physicochemical factors on the secondary structure of beta-lactoglobulin.

Fourier transform infrared spectroscopy and differential scanning calorimetry were used as complementary techniques to study changes in the secondary structure of beta-lactoglobulin under various physicochemical conditions. The effects of pH (3-9), NaCl (0-2 M), and lactose, glucose and sucrose (100-500 g/l) in the temperature range 25-100 degrees C on the conformation sensitive amide I band in the i.r. spectrum of beta-lactoglobulin in D2O solution were examined. The 1692 cm-1 band in the amide I band profile had not been definitively assigned in previous studies of the i.r. spectrum of beta-lactoglobulin. The decrease in this band at ambient temperature with time or upon mild heating was attributed to slow H-D exchange, indicating that it was due to a structure buried deep within the protein. The disappearance of the 1692 cm-1 band on heating was accompanied by the appearance of two bands at 1684 and 1629 cm-1, assigned to beta-sheets. The 1692 cm-1 band was therefore attributed to a beta-type structure. beta-Lactoglobulin showed maximum thermal stability at pH 3 and was easily denatured at pH 9. On denaturation, the protein unfolded into more extensive random coil structures at pH 9 than at pH 3. After 10 h at pH 9 (25 degrees C), beta-lactoglobulin was partly denatured. Heating to 60-80 degrees C generally resulted in the loss of secondary structure. At all pH values studied, two new bands at 1618 and 1684 cm-1, characteristic of intermolecular beta-sheet structure and associated with aggregation, were observed after the initial denaturation. Differential scanning calorimetry studies indicated that the thermal stability of beta-lactoglobulin was enhanced in the presence of sugars. The Fourier transform i.r. results obtained provide evidence that sugars promoted the unfolding of beta-lactoglobulin via multiple transition pathways leading to a transition state resisting aggregation.

Calorimetry, Differential Scanning↗
PubMed1995

Calorimetric analysis of lambda cI repressor binding to DNA operator sites.

Enthalpies and heat capacities were determined by isothermal titration calorimetry for bacteriophage lambda cI repressor binding to DNA containing various combinations of the three operator sites OR1, OR2, and OR3 (each comprising a consensus half-site and a specific nonconsensus half-site). Differential scanning calorimetry was employed to evaluate the effects of specific DNA binding on thermal melting of the N-terminal and C-terminal repressor domains. Principal findings of this study are as follows: (1) Binding of repressor to each of the DNA operators is dominated by a large negative enthalpy, in agreement with earlier van't Hoff analyses of quantitative footprint titration data [Koblan & Ackers (1992) Biochemistry 31, 57-65]. The calorimetric data also revealed negative heat capacities for cI binding that are of comparable magnitude with many other systems [Spolar & Record (1994) Science 263, 777-784]. However, this feature in combination with the large negative values of binding enthalpies leads to an enthalpic dominance throughout the physiological temperature range. The resulting thermodynamic profile is opposite to the entropically dominated binding observed for many systems, including lambda cro repressor which binds to the same sites as cI and also employs a helix-turn-helix binding domain [Takeda et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8180-8184]. It is suggested that these thermodynamic differences may arise from interactions of the cI repressor's N-terminal "arm" with the DNA. (2) Repressor monomers do not bind significantly to DNA containing either a consensus half-site or a nonconsensus half-site. Binding affinity to the double-consensus operator is much weaker than to any of the natural full-site operators. The same was found with other combinations of half-sites. A mutant repressor (PT158) which is severely defective in dimerization [Burz et al. (1994) Biochemistry 33, 8399-8405] was also found to bind only full-site operators and showed dimeric stoichiometry. (3) The thermal melting unit for N-terminal domains in the absence of DNA was found to reach values of 6-8 (monomer units) at concentrations where high-order oligomers of wild-type protein are formed [Senear et al. (1993) Biochemistry 32, 6179-6189]. However, in the presence of DNA operator sites, the cooperative unit for thermal unfolding was reduced to precisely two monomers, indicating that the N-terminal domain binds strictly as a dimer. (4) Significant nonadditivity was observed for the repressor binding enthalpies and heat capacities determined with multiple combinations of full-site operators.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗