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PubMed2004

A mechanistic investigation of an amorphous pharmaceutical and its solid dispersions, part I: a comparative analysis by thermally stimulated depolarization current and differential scanning calorimetry.

PURPOSE: To explore using thermally stimulated depolarization current (TSDC), in comparison to differential scanning calorimetry (DSC), for the characterization of molecular mobility of an amorphous pharmaceutical new chemical entity (LAB687), an amorphous polymer (PVPK-30), and their combination as solid dispersions at different % drug loadings. METHODS: Amorphous drug was prepared by quenching from the melt. Solid dispersions containing 10-60% of drug in polymer were prepared by solvent evaporation method. Glass transition temperatures (Tg) were determined by DSC and TSDC. RESULTS: In comparison to a single T. obtained from DSC for the drug substance, TSDC shows two overlapping relaxations. Both peaks correspond to a-relaxations that are associated with the glass transition, with the second peak corresponding to the rigid fraction that is difficult to be detected by DSC because it is associated with only small changes in heat capacity. Two overlapping relaxations were also observed for the polymer vs. one Tg by DSC. The lower temperature relaxation is believed to be a beta-relaxation, whereas the higher temperature transition corresponds to an alpha-relaxation. For the solid dispersions, one single peak was obtained for each of the 20% and 30% dispersions in excellent agreement with the DSC results. However, at the 40% drug load, a small shoulder was observed by TSDC at the low temperature of the main peak. This shoulder becomes more pronounced and overlaps with the main peak as the drug load increases to 50% and 60%. Agreement between the Tg values calculated by the Gordon-Taylor equation and the DSC and TSDC experimental data, especially for the 20% and 30% drug loading, indicate ideal miscibility. At higher drug loads, only by TSDC was it possible to detect the saturation level of the drug in the polymer. CONCLUSIONS: TSDC proved to be very sensitive in detecting small reorientational motions in solids and in separating overlapping events with only slight differences in molecular motion exhibited as broad events in DSC. This allowed for detection of the rigid fraction of the amorphous drug, the sub-glass transition beta- relaxation in the polymer, and the limit of miscibility between the drug and the polymer in the solid dispersions.

Calorimetry, Differential Scanning↗
PubMed2004

Estimation of daily energy needs with the FAO/WHO/UNU 1985 procedures in adults: comparison to whole-body indirect calorimetry measurements.

OBJECTIVES: To measure daily energy expenditure (DEE) with indirect calorimetric facilities in sedentary and active subjects. To estimate daily energy needs with the FAO/WHO/UNU (1985) procedures (EDEE) and estimated energy requirement (EER) with the dietary reference intakes 2002 (DRI) in healthy adults with sedentary or high-activity conditions. To compare estimated daily energy needs with their measured values. DESIGN: Two groups of healthy subjects were tested under sedentary or high-activity conditions. In both groups, resting energy expenditure was measured after a 12-h overnight fast. DEE and basal metabolic rate (BMR) values were also measured with indirect calorimetry and compared to the relevant predicted values. Physical activity level and BMR were also estimated. SUBJECTS: A total of 45 sedentary (26 men and 19 women) and 69 active subjects (43 men and 26 women) aged 18-30 and 30-60 y. RESULTS: Measured daily energy expenditure (MDEE) was significantly lower than EDEE in sedentary men and women and in active men for the two age groups considered (P<0.05). EER was significantly lower than EDEE in both sedentary and active subjects of each subgroup (P<0.05). CONCLUSIONS: The FAO/WHO/UNU (1985) procedures may overestimate daily energy needs, particularly in sedentary individuals. However, DRI (2002) are probably more adapted to estimate real daily energy needs in sedentary and active subjects in comparison to the FAO/WHO/UNU (1985) procedures.

Adolescent↗
PubMed2003

Measurement of total energy expenditure in grossly obese women: comparison of the bicarbonate-urea method with whole-body calorimetry and free-living doubly labelled water.

OBJECTIVE: To establish validity of the bicarbonate-urea (BU) method against direct measurements of gaseous exchange (GE) in a whole-body indirect calorimeter and to compare BU and doubly labelled water (DLW) measurements in free-living conditions in the same group of grossly obese women. DESIGN: Energy expenditure (EE) was estimated by the BU method over 24 h concurrently with whole-body indirect calorimetry and subsequently over 5 consecutive days at home concurrently with 14 day DLW. Six women, body mass index (BMI) 52.4+/-10.4 kg/m(2) (s.d.), were studied. RESULTS: Total energy expenditure (TEE) measurements by BU and GE within the metabolic chamber were not significantly different (BU=11.79+/-1.89 MJ/day and GE=11.64+/-1.86 MJ/day; mean difference, 0.25+/-0.49 MJ/day, P>0.05). Free-living TEE derived from BU and DLW was also similar (13.28+/-1.86 and 13.86+/-2.25 MJ/day, respectively; mean difference 0.17+/-1.33 MJ/day, P<0.05). The measured physical activity level (PAL) in these very obese subjects was within the range reported in other free-living studies in less obese individuals (1.62+/-0.14 using DLW and 1.56+/-0.20 using BU). The BU method was well tolerated by the subjects. CONCLUSIONS: This study in grossly obese subjects, heavier than those participating in previous studies involving tracer methods, demonstrates validity of the BU against GE under controlled metabolic conditions, and the equivalence between BU and DLW under free-living conditions. The results suggest that both tracer methods are valid in this population group. This study also demonstrates the practicalities of using the BU method over 5 days, the longest application of the method so far.

Adult↗
PubMed2006

Relationship between severity of nocturnal desaturation and adaptive thermogenesis: preliminary data of apneic patients tested in a whole-body indirect calorimetry chamber.

The purpose of this study was to investigate the possibility of a relationship between the severity of obstructive sleep apnea syndrome (OSAS) and adaptive thermogenesis. Daily energy expenditure (DEE) and sleeping metabolic rate (SMR) were measured in apneic and a priori nonapneic subjects who were tested in a whole-body indirect calorimetry chamber for 24 h. The apneic patients were diagnosed by nocturnal home oximetry to determine the percentage of total recording time spent below 90% arterial oxygen saturation (% TRT <90% SaO(2)). Reference equations established from body weight and age in nonapneic subjects were used to predict DEE and SMR in apneic patients. The predicted values of the apneic patients were then compared to their measured values. No significant difference was found between predicted and measured values in SMR nor in DEE. We observed a significant relationship between the severity of nocturnal desaturation and the difference between predicted and measured DEE in apneic patients (r = -0.74, P < 0.05) and a similar negative trend with SMR (r = -0.65, P = 0.08). These preliminary data suggest that a nocturnal hypoxia may influence adaptive thermogenesis in apneic patients and complicate their body weight regulation.

Adult↗
PubMed2002

Characterization of Streptococcus pneumoniae thymidylate kinase: steady-state kinetics of the forward reaction and isothermal titration calorimetry.

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis. The tmk gene from the bacterial pathogen Streptococcus pneumoniae was identified. The gene, encoding a 212-amino-acid polypeptide (23352 Da), was cloned and overexpressed in Escherichia coli with an N-terminal hexahistidine tag. The enzyme was purified to homogeneity, and characterized in the forward reaction. The pH profile of TMK indicates that its activity is optimal at pH 8.5. The substrate specificity of the enzyme was examined; it was found that not only ATP, but also dATP and to a lesser extent CTP, could act as phosphate donors, and dTMP and dUMP could serve as phosphate acceptors. Furthermore, AZT-MP (3'-azido-3'-deoxythymidine 5'-monophosphate) was shown not to be a substrate for S. pneumoniae TMK. Steady-state kinetics and inhibition studies with adenosine 5'-[beta-thio]diphosphate and dTDP in addition to isothermal titration calorimetry were performed. The data showed that binding follows an ordered pathway, in which ATP binds first with a K(m) of 235 +/- 46 microM and a K(d) of 116 +/- 3 microM, and dTMP binds secondly with a K(m) of 66 +/- 12 microM and a K(d) of 53 +/- 2 microM.

Adenosine Triphosphate↗
PubMed2005

Characterization of different strains of poliovirus and influenza virus by differential scanning calorimetry.

Vaccines against poliomyelitis and influenza contain inactivated forms of poliovirus and influenza virus. These antigens are generated on an industrial scale from the purified active viruses that have been analysed in this study by DSC (differential scanning calorimetry). Multiple unfolding transitions are seen for influenza virus A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) and B/Shangdong/7/97. These data, combined with previously reported data on other influenza viruses, indicates that each influenza virus strain has a characteristic unfolding behaviour. Only minor changes were seen in the thermogram of betaPL (beta-propiolactone)-inactivated influenza virus, which is consistent with the proposition that betaPL reacts mainly with the nucleotide fraction of the virus. We demonstrate that a peak annotation of the thermogram of the native virus is possible using bromelain-treated virus and virosomes. At pH 1.5-2.5, poliovirus of type I unfolds in a single unfolding event with respective Tm (midpoint of protein unfolding transition) values between 34 and 45 degrees C. At pH 2, polioviruses of type II unfold equally in a single event, but, compared with the type I virus, with a Tm value increased by 3.7 degrees C. At neutral pH, the DSC thermogram of type I poliovirus was very 'noisy'. Data obtained offer the possibility of precisely characterizing and identifying different viral strains.

Bromelains↗
PubMed1983

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.

Calorimetry, Differential Scanning↗
PubMed1989

A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase.

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.

Adenosine Triphosphate↗
PubMed1990

Analysis of the physical state of cholesteryl esters in arterial-smooth-muscle-derived foam cells by differential scanning calorimetry.

The physical state of cholesteryl esters (CE) in the arterial-smooth-muscle-derived foam cells may contribute to the documented reduction in CE hydrolysis. The physical state of CE may also provide a potential enhancing mechanism for increased CE accumulation. To explore these concepts, we therefore examined the influence of alterations in CE and triacylglycerol (TG) content and their fatty acid composition on the thermotropic behaviour of these lipids by differential scanning calorimetry (d.s.c.). After exposure to cationized LDL (cLDL) or after infection with herpes simplex virus type I (HSV), smooth-muscle cells accumulated significant amounts of CE. The CE/TG ratio was significantly higher in cells treated with cLDL compared with HSV infection. TG content was unaffected by either treatment. However, the fatty acid profile of both CE and TG was significantly different between treatment groups, with the polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio being significantly higher in cLDL-treated cells than in HSV-infected cells. The d.s.c.-generated thermograms of intact cells revealed that neutral lipids of both treatment groups were in the isotropic-liquid state, similar to the state of lipids derived from 'fatty streak' types of atherosclerotic lesions. Differences in the thermograms between HSV-infected and cLDL-treated cells can be ascribed to differences in the CE content and the fatty acid composition of CE and TG (PUFA/SFA ratio). Polarizing optical microscopy revealed the presence of isotropic lipids in both groups. Biochemical and physicochemical data confirm the lysosomal localization of engorged CE, and indicate that the cellular isotropic CE in these foam cells are in a physical state which favours enzymic hydrolysis.

Animals↗
PubMed1994

Studies on cytochrome c-heparin interactions by differential scanning calorimetry.

The effects of heparin on the thermotropic properties of ferricytochrome c have been studied using high-sensitivity differential scanning calorimetry. Saturating concentrations of heparin at low ionic strength induced an important shift of the transition temperature Tm from 84.1 degrees C to 59.8 degrees C. This was accompanied by unusually large cooperativity of thermal denaturation of this complex, indicating strong intermolecular interactions between protein molecules. The destabilization of cytochrome c when mixed with heparin was not observed at high ionic strength, under which conditions complex was not formed.

Calorimetry, Differential Scanning↗
PubMed1986

Glucose oxidation rates in newborn infants measured with indirect calorimetry and [U-13C]glucose.

Indirect calorimetry and primed constant infusion of [U-13C]glucose were combined in 16 appropriate-for-gestational age newborn, parenterally fed infants, in order to measure glucose utilization and glucose oxidation respectively. Glucose intake ranged between 10.0 and 24.1 g day-1 kg-1 and energy intake between 156.9 and 439.3 kJ day-1 kg-1. Glucose utilization (P less than 0.001), glucose oxidation (P less than 0.001) and metabolic rate (P less than 0.005) increased significantly with rising glucose intake. The significant difference between glucose utilization and oxidation (P less than 0.001) can be accounted for by an increasing storage as fat. As lipogenesis from glucose consumes 15-24% of the original glucose energy, the increasing metabolic rate accompanying rising glucose intake is probably due to increasing lipogenesis.

Calorimetry, Indirect↗
PubMed1991

Energy expenditure and substrate metabolism measured by 24 h whole-body calorimetry in patients receiving cyclic and continuous total parenteral nutrition.

1. Twenty-four hour energy expenditure and its components, i.e. 'basal metabolic rate', activity energy expenditure and diet-induced thermogenesis were measured, using continuous whole-body indirect calorimetry, in patients receiving total parenteral nutrition while in remission from Crohn's disease (weight 51.9 +/- 9.9 kg, body mass index 19.2 +/- 2.0 kg/m2). 2. Total parenteral nutrition was infused continuously over 24 h in four subjects and cyclically, between 22.00 and 10.00 hours, in eight subjects. Twenty-four hour energy expenditure (6.83 +/- 1.10 MJ/24 h) was lower than total energy intake (10.09 +/- 1.63 MJ/24 h), resulting in a positive energy balance (3.26 +/- 1.42 MJ) in all subjects. Repeated measurements of resting energy expenditure in the continuously fed subjects (5.82 +/- 1.11 MJ/24 h) did not change significantly at different times of day (coefficient of variation 2.2-6.6%). In contrast, in cyclically fed subjects, resting energy expenditure was 24.2 +/- 9.0% higher towards the end of the 12 h feeding period than the 'basal metabolic rate', which was measured just before the start of the feeding period. 3. Diet-induced thermogenesis, calculated as the increment in resting energy expenditure above 'basal metabolic rate' over the 24 h period (adjusted for the reduction in energy expenditure during sleep), was found to be 0.60 +/- 0.29 MJ or 6.1 +/- 3.1% of the energy intake. 4. The energy cost of activity (activity energy expenditure) in the continuously fed patients, calculated as the difference between 24 h energy expenditure and the integrated 24 h measurements of resting energy expenditure, was 0.88 +/- 0.53 MJ, i.e. 12.9 +/- 5.9% of the 24 h energy expenditure. 5. The non-protein non-glycerol respiratory quotient exceeded 1.0 for varying periods of time (0.5-17 h) in 11 subjects, indicating net lipogenesis from carbohydrate. 6. The results demonstrate favourable rates of deposition, during intravenous feeding, of both energy and nitrogen over a 24 h period in patients recovering from an episode of Crohn's disease. The efficacy of these commonly used total parenteral nutrition regimens in these patients is related to three features that are absent in normal healthy individuals, namely a low basal metabolic rate, a low activity-related energy expenditure and prolonged periods of lipogenesis from carbohydrate.

Adult↗
PubMed2001

The thermal unfolding and domain structure of Na+/K+-exchanging ATPase. A scanning calorimetry study.

The thermal unfolding and domain structure of Na+/K+-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry (HS-DSC). The excess heat capacity function of Na+/K+-ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Td = 327.5 K was identified as corresponding to the beta subunit, while two other domains belong to the alpha subunit. The thermal unfolding of the low-temperature domain leads to large changes in the amplitude of the short-circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2-mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (approximately 12 J.g-1), suggesting that thermal denaturation of Na+/K+-ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.

Animals↗
PubMed2001

Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.

The binding of two mono-acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C-terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1-myristoylglycerophosphatidylcholine (MyrGro-PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 microM and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro-PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen-addad, C. & Pebay-Peyroula, E. (1999) Eur. J. Biochem. 264, 562-568.]. Results from isothermal titration calorimetry experiments indicated non-classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two omega-hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.

Calorimetry↗
PubMed2002

Characterization of heparin binding by a peptide from amyloid P component using capillary electrophoresis, surface plasmon resonance and isothermal titration calorimetry.

Synthetic peptides based on amino-acid residues 27-38 of human serum amyloid P component represent a novel type of heparin binders as they do not contain clusters of basic amino acids or other known features associated with protein or peptide heparin binding. Here, we characterize the binding using capillary electrophoresis (CE), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). By CE, heparin-binding activity was readily apparent for both a regular peptide and a slightly N-terminally modified form, while a sequence-scrambled peptide had no measurable binding. Dissociation constants in the 1-15 microm range were estimated, but only a minor part of the binding isotherm was covered by the experiments. SPR measurements using immobilized peptides verified heparin binding, the range of the binding constants, and the reduced binding of the sequence-scrambled peptide. Structurally defined heparin oligosaccharides were used to establish that while the tetrasaccharide is too small to exhibit strong binding, little difference in binding strength is observed between hexa- and tetradeca-saccharides. These experiments also confirmed the almost complete lack of activity of the sequence-scrambled peptide. The amino-acid sequence-dependent binding and the importance of a disulfide bond in the peptide were verified by ITC, but the experimental conditions had to be modified because of peptide precipitation and ITC yielded significantly weaker binding constants than the other methods. While the precise function of the peptide in the intact protein remains unclear, the results confirm the specificity of the glycosaminoglycan interaction with regard to peptide sequence by applying two additional biophysical techniques and showing that the N-terminal part of the peptide may be modified without changing the heparin binding capabilities.

Binding Sites↗
PubMed2003

A differential scanning calorimetry study of tetracycline repressor.

Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix-turn-helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: N(2)--->D(2), where k is a first-order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two-state model: N(2)<--->U(2)--->D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DeltaH(cal) = 1067 kJ x mol(-1)) and an entropy change (DeltaS = 3.1 kJ x mol(-1)).

Calorimetry, Differential Scanning↗
PubMed1999

Apolipoprotein A-I(Milano) unfolds via an intermediate state as studied by differential scanning calorimetry and circular dichroism.

In this study the thermal and denaturant induced denaturation behaviors of apolipoprotein A-I(Milano) (apo A-IM) have been studied by differential scanning calorimetry and circular dichroism spectroscopy, as well as solution properties by analytical ultracentrifugation. Thermal denaturation is dependent on pH, sodium phosphate concentration and NaCl concentration. The protein is highly self-associated at the protein concentrations used in this study. Denaturation of apo A-IM at pH 7.4 and 8.0 occurs in two steps. The midpoint between the transition is at 37 degrees C. The first step at 31 degrees C involves melting of tertiary structure and rearrangement of protein association complexes, i.e. a transition into an intermediate molten globular-like state. Subsequent melting of this intermediate state into an unfolded state occurs at 52 degrees C. At pH 2.8 the protein lacks all tertiary structure and denaturation occurs over a large temperature interval, indicating the induction of a molten globular-like state at low pH.

Apolipoprotein A-I↗
PubMed2000

Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry.

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin, which denatures with a maximum at 64 degrees C in the absence and at 78 degrees C in the presence of a twofold molar excess of phalloidin. After the F-actin-tropomyosin complex had been heated to 90 degrees C and then cooled (i.e. after complete irreversible denaturation of F-actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F-actin to be probed very precisely.

Actins↗