The influence of the peptide bioregulator prostamax on heterochromatin of human lymphocytes in situ

It was shown that chromatin contained in human lymphocytes has two stages of denaturation: with T(d)VII = 94.4 degrees C, Q(d)VII = 50.8 J/g DNA, and T(d)VIII = 105.1 degrees C Q(d)VIII = 44.9 J/g DNA. The peptide bioregulator prostamax causes a redistribution of heat among endotherms T(d)III and T(d)IV and a shift of both endotherms to low temperatures by 2.9 and 1.0 degrees C, respectively. It was supposed that the redistribution of heat among endotherms is connected with a partial relaxation of the 30-nm-thick fiber in the 10-nm filament. A weak decrease in T(d)VIII and T(d)VII of lymphocytes treated with prostamax compared to untreated ones is connected with small structural changes of nucleosomal organization in the 10-nm filament and 30-nm-thick fiber.     

Microcalorimetric study of iodized and noniodized cells and C-phycocyanin of Spirulina platensis

It was shown that eight stages of transition are observed in the heating process of Spirulina platensis cells in temperature range 5-140 degrees C. The first stage covers the temperature range 5-53 degrees C with maximum approximately 45 degrees C. The heat evolved in this temperature range is equal to 380 +/- 20 J/g of dry biomass, it does not change at scanning rate lower than 0.083 degrees C/min and belongs, mainly, to cell respiration in a stationary regime, in the dark. It was shown that endotherm approximately 66 degrees C belongs to denaturation of C-phycocyanin which denaturates in solutions with Td = 64.2 degrees C, deltaHd = 34.7 +/- 2.1 J/g and for it deltaHd(cal)/deltaH(V.H) is equal to 10.8 +/- 1.2. The endotherms with Td equal to 58 and 88 degrees C are connected with denaturation of phycobilisome proteins and endotherm with Td = 48 degrees C and deltaHd = 4.2J/g of dry biomass-with denaturation of protein which, apparently, is connected with cell respiration.     

Microcalorimetric study of human serum

It was established that albumin of donor blood serum denatures in two temperature ranges. It is shown that the first stage of denaturation with Td = 61.5 degrees C is dominant and corresponds to melting of regions not bound to fatty acids. The second stage with Td = 80 degrees C corresponds to melting of regions bound to fatty acids. Serum denaturation heat is equal to 20.2 J/g dry protein. A change in denaturation heat capacity is 0.21 J/(g.K). Analysis of thermal parameters of deconvolution peaks showed that albumin of donor blood serum is in a fatless state and its multiple binding centers are essentially free as compared with freshly isolated albumin and may play an important role in binding of ligands in vivo. The thermal parameters of denaturation of some important human blood serum proteins including gamma-globulins, transferrin ceruloplasmin and protease inhibitors were also determined.     

Differential scanning calorimetry studies of rabbit cardiac sarcolemma

Thermal transitions were measured by differential scanning calorimetry for rabbit cardiac sarcolemma in 3-(N-morpholino)propanesulfonic acid buffer at pH 7.5, in glycerol-buffer and dimethyl sulfoxide - buffer mixtures, after heat denaturation, and after enzymatic degradation of the proteins. Specific solvent effects on the protein transitions were observed. Glycerol stabilized some of the four protein transitions, while dimethyl sulfoxide destabilized all protein transitions. The thermal transitions in the lower temperature range were studied for both the membranes and the lipid extracted from the membranes. A very small endotherm was observed for both the lipid extracted from the sarcolemma and the intact membrane (0.1-0.2 cal/g; 1 cal = 4.1868 J). A larger endotherm was observed in both the glycerol-buffer and dimethyl sulfoxide - buffer mixtures. Major perturbation of the protein by enzymatic degradation (papain or trypsin digestion), by heat denaturation, or by reaction with excess N-ethylmaleimide all produced larger endotherms near 20 degrees C. The very small magnitude of the endotherm near 20 degrees C suggests that it is not a typical gel - liquid crystalline transition of the bilayer. However, the occurrence of an endotherm in the extracted lipid suggests that some reorientation of lipid is involved.     

Conformational properties of streptokinase--differential scanning calorimetric investigations.

The two-domain structure of streptokinase (Sk) was demonstrated by scanning calorimetric investigations at neutral pH and low ionic strength. The melting pattern of the protein is composed of two two-state transitions at TtrS1 = 45.9 +/- 0.4 degrees C with delta H1 = 431 +/- 18 kJ/mol, and TtrS2 = 60.1 +/- 1.3 degrees C with delta H2 = 306 +/- 16 kJ/mol. The partial specific heat capacity of native Sk was determined to be Cp = 1.42 +/- 0.17 J/K/g and the denaturational heat capacity change associated with the two transitions, delta Cp1 = 0.21 J/K/g and delta Cp2 = 0.38 J/K/g, respectively. The overall melting pattern of Sk remains almost unchanged at a variety of tested solvent compositions, except at pH 4 (and below) and in the presence of denaturants. The two domains show different susceptibility to urea. It is proposed that the less thermostable domain is located within the N-terminal part (residues 1-230), and the more thermostable one, within the C-terminal region.     

Single-strand nuclease action on heat-denatured spermiogenic chromatin.

The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.     

Evaluation of a temperate-environment test of heat tolerance in prior heatstroke patients and controls

It has been reported that scores from a temperate-environment step test describe the heat-tolerance status of prior heatstroke patients (HP). This investigation evaluated the ability of this temperate-environment heat-tolerance test (HTT) to indicate altered heart rate (HR) and rectal temperature (Tre) responses of HP, after 7 days of heat acclimation. On day 1, ten male HP (61 +/- 7 days post-heatstroke) and five control subjects (C) bench-stepped (0.30 m high, 27 steps.min-1) for 15 min (25.8 degrees C dry bulb, 16.2 degrees C wet bulb). On days 2-8, subjects underwent heat acclimation (40.1 degrees C dry bulb, 23.8 degrees C wet bulb; treadmill, 90 min.day-1). Heat acclimation resulted in significant decreases in final HR (152 +/- 5 vs 130 +/- 3 beats.min-1, P less than 0.025) and final Tre (38.62 +/- 0.11 vs 38.13 +/- 0.07 degrees C, p less than 0.01) in HP. One HP but no C was defined heat intolerant, exhibiting inability to adapt to daily exercise in the heat. On day 9, HP repeated HTT, exactly as performed on day 1; mean group HTT scores did not change (day 1 = 39 +/- 6; day 9 = 48 +/- 6, P greater than 0.05). All physical characteristics and physiological responses of HP (days 1, 2, 7, 9) were statistically similar (P greater than 0.05) to those of C. In contrast to heat-acclimation data, HTT scores (score less than or equal to 30) indicated that four HP were heat intolerant on day 1 and two HP were heat intolerant on day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

A calorimetric study of human CuZn superoxide dismutase.

Structural alterations, as manifested by thermal transitions, caused by removal or binding of metal ions to human and bovine CuZn superoxide dismutases (SODs) were investigated by differential scanning calorimetry. Although holo forms of the two mammalian enzymes exhibited irreversible thermal transitions (delta Hcal. = 27.7 J/g and Td = 104 degrees C for bovine SOD; delta Hcal. = 23.6 J/g and Td = 101 degrees C for human SOD), only the bovine apoenzyme showed the presence of a less thermostable form (delta Hcal. = 10.7 J/g and Td = 63 degrees C). These observations suggested that human apo-SOD had considerably less conformational order than bovine apo-SOD. Reconstitution of human and bovine apoenzymes with Cu2+ and Zn2+ resulted in recovery of thermodynamic parameters and specific activity. Binding of Zn2+ alone to human apo-SOD resulted in the formation of two distinct structural units, detectable by differential scanning calorimetry, which underwent conformational disorder at 82 and 101 degrees C respectively. Saturation of binding sites with both Zn2+ and Cu2+ appeared to stabilize the enzyme structure further as shown by elimination of the low-temperature transition and the appearance of another thermal transition at a higher temperature.     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

A calorimetric analysis of human plasma fibronectin: effects of heparin binding on domain structure

Fibronectin domain structure, as influenced by interaction with heparin, calcium, or chondroitin sulfate C, was analyzed by differential scanning calorimetry. A complex thermal denaturation transition was observed with a large sharp endotherm at 63 degrees C, a broad endotherm between 70 and 80 degrees C, and an exotherm at 80-90 degrees C. Analysis of the denaturation profiles revealed the existence of four thermal transitions, 59.1, 62.2, 67.3, and 74.3 degrees C, and an exotherm at 83.9 degrees C. The calorimetric enthalpies of the four endotherms are 1146 +/- 259, 866 +/- 175, 1010 +/- 361, and 676 +/- 200 kcal/mol, respectively. In all cases, the calorimetric to van't Hoff enthalpy ratio was greater than 1.0. Computer analysis of the primary structure of fibronectin revealed 29 +/- 8% homology among the type I homology units and 28 +/- 7% homology among type III homology units, suggesting that different structural domains could arise from the same homology type. This may explain why more thermal transitions are observed for fibronectin than there are homology types. Addition of heparin to fibronectin in varying molar ratios, i.e., 10:1 to 30:1, resulted in a larger calorimetric enthalpy for the first type of structural domain (Tm = 59.1 degrees C) of fibronectin. At higher heparin to fibronectin ratios (40:1 or 75:1), the enthalpy of this domain decreased, while the others remained unchanged. In the presence of 5 mM calcium chloride, fibronectin thermal denaturation occurred at lower temperatures and was associated with precipitation of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factor VIII A1 domain residues 97-105 represent a light chain-interactive site

Results from a recent study on subunit association in factor VIIIa indicated that the A1 and A3C1C2 domains contribute approximately 90% of the interchain binding energy in factor VIII and that A3 domain residues 1954-1961 participate in the interaction with A1 domain (Ansong, C., and Fay, P. J. (2005) Biochemistry 44, 8850-8857). Enhanced trypsin-accessibility at four sites within residues 89-142 in free A1 compared with that of A3C1C2-bound A1, as determined by mass spectrometry, suggested that residues within this region are interactive with the A3C1C2 domains. A synthetic peptide to A1 domain residues 97-105, predicted to be A3 domain-interactive from molecular modeling, inhibited the formation of a functional A1/A3C1C2 dimer (apparent K(i) = 0.64 +/- 0.21 microM) and reduced the efficiency of energy transfer between a fluorescein-labeled A1 subunit and an acrylodan-labeled A3C1C2 subunit. B-domainless factor VIII point mutants, His99Ala, Val101Ala, and Gly102Ser, exhibited reduced specific activity (32%, 51%, and 45%, respectively) compared with that of factor VIII wild type. Furthermore, the activity of factor VIII His99Ala was less stable (t(1/2) = 2.3 +/- 0.2 min) compared with that of factor VIII wild type (t(1/)(2) = 6.2 +/- 0.7 min) following heat denaturation analysis. This reduced stability appeared to result from an approximately 40% increase in the dissociation rate for the mutant factor VIII heterodimer as judged by solid-phase binding assays. These results indicate that residues within segment 97-105 of the A1 domain interact with residues within the A3C1C2 domains of the light chain and contribute to the interface in the factor VIII heterodimer.     

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.     

The nuclear matrix is a thermolabile cellular structure

Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at Tm = 48 degrees C and 55 degrees C with an onset temperature of approximately 40 degrees C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with Tm = 48 degrees C, indicating increased exposure of hydrophobic residues at this transition. The Tm = 48 degrees C transition has a similar Tm to those predicted for the critical targets for heat-induced clonogenic killing (Tm = 46 degrees C) and thermal radiosensitization (Tm = 47 degrees C), suggesting that denaturation of nuclear matrix proteins with Tm = 48 degrees C contribute to these forms of nuclear damage. Following heating at 43 degrees C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei Isolated from heat-shocked cells and is a form of indirect thermal damage.     

High-resolution differential scanning calorimetric study of myosin, functional domains, and supramolecular structures

High-resolution differential scanning calorimetry (DSC) has been employed to study the thermal stability of myosin, its major constitutive fragments (S-1, light chains, and rod), and also reconstituted thick filaments. The thermal denaturation of soluble myosin was complex and was characterized by a multistep endothermic process for the temperature range from 41 to 60 degrees C. The shape of the endotherm was highly dependent on the pH and the ionic strength of the solution, although the delta Hcal (calorimetric enthalpy) of denaturation (1715 +/- 75 kcal/mol) was insensitive to these changes for the solvent conditions used in this study. This value also agrees, within experimental error, with the sum of the denaturation enthalpies obtained for isolated fragments (1724 +/- 79 kcal/mol). In identical conditions of ionic strength, pH, and heating rate, the computer-calculated differential endotherms of domains belonging to S-1 and light chains were superimposable with those of the isolated fragments. Their responses to changes in the solvent condition were also similar. We suggest that the observed functional independence of the major domains in myosin reflects also the independence of their structural stability. The thermal unfolding of the isolated rod was multiphasic and readily reversible (95%). It occurred between 41 and 60 degrees C, with an delta Hcal of 1058 +/- 59 kcal/mol. The melting of S-1 showed a single peak at 46.3 +/- 0.1 degrees C with an delta Hcal of 255 +/- 12 kcal/mol. Light chains melted at 51.0 +/- 0.2 degrees C with an delta Hcal of 85 +/- 15 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat debt as an index for cold adaptation in men

Several types of cold adaptation in men have been described in the literature (metabolic, insulative, hypothermic). The aim of this study is to show that the decrease of heat debt can be considered as a new index for cold adaptation. Ten male subjects were acclimated by water immersions (temperature 10-15 degrees C, 4 immersions/wk over 2 mo). Thermoregulatory responses before and after acclimation were tested by a standard cold test in a climatic chamber for 2 h at rest [dry bulb temperature (Tdb): 10 degrees C; relative humidity (rh): 25%]. After adaptation, four thermoregulatory modifications were observed: an increase in the delay for the onset of shivering (32.7 +/- 7.99 instead of 14.1 +/- 5.25 min); a decrease of body temperature levels for the onset of shivering [rectal temperature (Tre): 37.06 +/- 0.08 instead of 37.31 +/- 0.06 degrees C; mean skin temperature (Tsk): 24.83 +/- 0.56 instead of 26.86 +/- 0.46 degrees C; mean body temperature (Tb): 33.03 +/- 0.20 instead of 34.16 +/- 0.37 degrees C); a lower level of body temperatures in thermoneutrality (Tre = 37.16 +/- 0.08 instead of 37.39 +/- 0.06 degrees C; Tsk = 31.29 +/- 0.21 instead of 32.01 +/- 0.22 degrees C; Tb = 35.92 +/- 0.08 instead of 36.22 +/- 0.05 degrees C); a decrease of heat debt calculated from the difference between heat gains and heat losses (5.66 +/- 0.08 instead of 8.33 +/- 0.38 kJ/kg). The different types of cold adaptation observed are related to the physical characteristics of the subjects (percent body fat content) and the level of physical fitness (VO2max).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.     

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

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

Factor VIII A3 domain residues 1954-1961 represent an A1 domain-interactive site

Factor VIIIa consists of subunits designated A1, A2, and A3C1C2. Reassociation of the A1 and A3C1C2 subunits monitored by the factor Xa generation assay and fluorescence resonance energy transfer yielded intersubunit affinity values (K(d)) of 58.0 +/- 12.5 and 58.8 +/- 16.8 nM, respectively. These affinity values were equivalent to that previously determined for factor VIII heavy and light chains [Wakabayashi, H., et al. (2001) Biochemistry 40, 10293-10300], suggesting that the A2 domain makes a minimal contribution to the interchain affinity in factor VIII. Ca(2+) showed no effect on A1/A3C1C2 intersubunit affinity (K(d) = 51.6 +/- 16.6 nM), while Cu(2+) enhanced the A1/A3C1C2 intersubunit affinity approximately 5-fold (K(d) = 12.5 +/- 2.3 nM). A synthetic peptide to A3 domain residues 1954-1961 inhibited association of the A1 and A3C1C2 subunits (K(i) = 65.8 +/- 11.9 microM). Three factor VIII point mutants, His1957Ala, Gly1960Val, and His1961Asp, were stably expressed in BHK cells and purified. All mutants exhibited reduced specific activity (39, 42, and 4%, respectively) compared with that of wild-type factor VIII, and their activity was less stable following heat denaturation analysis (t(1/2) values of 13.3 +/- 0.7, 8.7 +/- 0.3, and 8.1 +/- 0.1 min, respectively) compared with that of the wild type (18.8 +/- 0.8 min). This reduced stability appeared to result from an approximately 2-fold increased dissociation rate for the Gly1960Val and His1961Asp dimers as judged by solid-phase binding assays. We propose that residues 1954-1961 of the A3 domain contribute to interactions with the A1 domain, facilitating their association in factor VIII.     

Thermodynamics of condensation of nuclear chromatin. A differential scanning calorimetry study of the salt-dependent structural transitions

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.