Hydrophobic hydration processes: General thermodynamic model by thermal equivalent dilution determinations

The “hydrophobic hydration processes” can be satisfactorily interpreted on the basis of a common molecular model for water, consisting of two types of clusters, namely W_I and W_II accompanied by free molecules W_III. The principle of thermal equivalent dilution (TED) is the potent tool (Ergodic Hypothesis) employed to monitor the water equilibrium and to determine the number ξ_w of water molecules W_III involved in each process. The hydrophobic hydration processes can be subdivided into two Classes: Class A includes those processes for which the transformation A(−ξ_wW_I → ξ_wW_II + ξ_wW_III + cavity) takes place with the formation of a cavity, by expulsion of ξ_w water molecules W_III whereas Class B includes those processes for which the opposite transformation B(−ξ_wW_II − ξ_wW_III → ξ_wW_I − cavity) takes place with reduction of the cavity, by condensation of ξ_w water molecules W_III. The number ξ_w depends on the size of the reactants and measures the extent of the change in volume of the cavity. Disaggregating the thermodynamic functions ΔH_app and ΔS_app as the functions of T (or lnT) and ξ_w has enabled the separation of the thermodynamic functions into work and thermal components. The work functions ΔG_Work, ΔH_Work and ΔS_Work only refer specifically to the hydrophobic effects of cavity formation or cavity reduction, respectively. The constant self-consistent unitary (ξ_w = 1) work functions obtained from both large and small molecules indicate that the same unitary reaction is taking place, independent from the reactant size. The thermal functions ΔH_Th and ΔS_Th refer exclusively to the passage of state of water W_III.Essential mathematical algorithms are presented in the appendices.     

Effects of small heat shock proteins on the thermal denaturation and aggregation of F-actin

Effect of recombinant chicken small heat shock protein with molecular mass 24 kDa (Hsp24) and recombinant human small heat shock protein with molecular mass 27 kDa (Hsp27) on the heat-induced denaturation and aggregation of skeletal F-actin was analyzed by means of differential scanning calorimetry and light scattering. All small heat shock proteins did not affect thermal unfolding of F-actin measured by differential scanning calorimetry, but effectively prevented aggregation of thermally denatured actin. Small heat shock protein formed stable complexes with denatured (but not with intact) F-actin. The size of these highly soluble complexes was smaller than the size of intact F-actin filaments. It is supposed that protective effect of small heat shock proteins on the cytoskeleton is at least partly due to prevention of aggregation of denatured actin.     

Comparison of the chemical and thermal denaturation of proteins by a two-state transition model

The conformational stabilities of eight proteins in terms of the free energy differences between the native "folded" state of the protein and its "unfolded" state were determined at 298 K by two methods: chemical denaturation at 298 K and extrapolation to 298 K of the thermal denaturation results at high temperature. The proteins were expressed in Escherichia coli from the Haemophilus influenzae and E. coli genes at different levels of expression, covered a molecular mass range from 13 to 37 kg mol(-1) per monomeric unit (some exhibiting unique structural features), and were oligomeric up to four subunits. The free energy differences were determined by application of a two-state transition model to the chemical and thermal denaturation results, ranged from 9.4 to 148 kJ mol(-1) at 298 K, and were found to be within the experimental uncertainties of both methods for all of the proteins. Any contributions from intermediate states detectable from chemical and thermal denaturation differences in the unfolding free energy differences in these proteins are within the experimental uncertainties of both methods.     

Comparative studies on thermodynamic characteristics of pea legumin and legumin-T thermal denaturation

Characteristics of thermal denaturation of pea legumin and a product of its limited proteolysis with trypsin – legumin-T, in a wide range of NaCl concentrations have bean measured by means of differential scanning microcalorimetry. By the increase of NaCl concentration, the number of cooperative units (domains) increases from 1 per one polypeptide chain to 2 for legumin and 1.8 for legumin-T. Deconvolution of denaturation peaks have revealed up to three peaks, which were ascribed to the dissociation of protein macromolecules to subunits and the unfolding of - and β-polypeptide chains. The analysis of experimental data based on some assumptions showed that the splitting of C-termini of -chains, which are not constituents of cooperative domains, in the course of limited trypsinolysis results in destabilization of the quaternary structure of legumin and loosening of -chains, as well as decrease of the temperatures of their maximum stability.     

Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization

Water plays an essential role in determining the structure and function of all biological systems. Recent methodological advances allow for an accurate and efficient estimation of the thermodynamic properties of water molecules at the surface of proteins. In this work, we characterize these thermodynamic properties and relate them to various structural and functional characteristics of the protein. We find that high-energy hydration sites often exist near protein motifs typically characterized as hydrophilic, such as backbone amide groups. We also find that waters around alpha helices and beta sheets tend to be less stable than waters around loops. Furthermore, we find no significant correlation between the hydration site-free energy and the solvent accessible surface area of the site. In addition, we find that the distribution of high-energy hydration sites on the protein surface can be used to identify the location of binding sites and that binding sites of druggable targets tend to have a greater density of thermodynamically unstable hydration sites. Using this information, we characterize the FKBP12 protein and show good agreement between fragment screening hit rates from NMR spectroscopy and hydration site energetics. Finally, we show that water molecules observed in crystal structures are less stable on average than bulk water as a consequence of the high degree of spatial localization, thereby resulting in a significant loss in entropy. These findings should help to better understand the characteristics of waters at the surface of proteins and are expected to lead to insights that can guide structure-based drug design efforts.     

Differential scanning calorimetric and spectroscopic studies on the unfolding of Momordica charantia lectin. Similar modes of thermal and chemical denaturation

Thermal stability of Momordica charantia seed lectin (MCL) was investigated as a function of protein concentration, pH, scan rate, and at different ligand concentrations by using high-sensitivity differential scanning calorimetry (DSC). The DSC endotherm obtained at pH 7.4 consists of two entities with transition temperatures at ca. 333.7 K, and 338 K. The unfolding process is irreversible and could be described by a three-state model. For MCL tetramer ΔH_c/ΔH_v ratio is close to 4 for the first transition and ∼2 for the second transition, suggesting that four and two cooperative units are involved in the first and second transitions, respectively. In the presence of lactose both transitions shifted to higher temperatures, suggesting that ligand binds preferentially to the native conformation of MCL. Endotherms recorded as a function of pH indicate that MCL is more stable at lower pH. Chemical unfolding of MCL, induced by Gdn.HCl, was investigated by monitoring the intrinsic fluorescence properties of the protein. The results obtained indicate that chemical denaturation of MCL can also be described by a three-state process, involving an intermediate populated at ∼3–4 M Gdn.HCl. These observations suggest that the chemical and thermal unfolding processes are similar in that both of them proceed via an intermediate. The far UV and near UV CD spectra of MCL were nearly identical at different pH values and indicate that its secondary and tertiary structure do not change significantly with pH, suggesting that the structure of the protein is stable over a wide pH range.     

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T_m ≈ 100 °C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry–Eyring model, N ? U ? F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.     

pH dependence of the stability of barstar to chemical and thermal denaturation.

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermal and chemical denaturation of the BRCT functional module of human 53BP1

BRCTs are protein-docking modules involved in eukaryotic DNA repair. They are characterized by low sequence homology with generally well-conserved structure organization. In a considerable number of proteins, a pair of BRCT structural repeats occurs, connected with inter-BRCT linkers, variable in length, sequence and structure. Linkers may separate and control the relative position of BRCT domains as well as protect and stabilize the hydrophobic inter-BRCT interface region. Their vital role in protein function has been demonstrated by recent findings associating missense mutations in the inter-repeat linker region of the BRCT domain of BRCA1 (BRCA1-BRCT) to hereditary breast/ovarian cancer. The interaction of 53BP1 with the core domain of the p53 tumor suppressor involves the C-terminal BRCT repeat as well as the inert-BRCT linker of the tandem BRCT domain of 53BP1 (53BP1-BRCT). High-accuracy differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to characterize the heat-induced unfolding of 53BP1-BRCT domain. The calorimetric results provide evidence for unfolding to an intermediate, only partly unfolded state, which, based on the CD results, retains the secondary structural characteristics of the native protein. A direct comparison with the corresponding thermal processes for BRAC1-BRCT and BARD1-BRCT provides evidence that the observed behavior is analogous to BRCA1-BRCT even though the two domains differ substantially in the linker structure. Moreover, chemical denaturation experiments of the untagged 53BP1-BRCT and comparison with BRCA1 and BARD1 BRCTs show that no clear association can be drawn between the structural organization of the inter-BRCT linkers and the overall stability of the BRCT domains.     

Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.     

Effects of osmolytes on arginine kinase from Euphausia superba: A study on thermal denaturation and aggregation

Investigations of energy-related enzymatic properties may provide valuable information about the mechanisms that are involved in the adaptation to extreme climatic environments. The protective effects of osmolytes on the thermal denaturation and aggregation of arginine kinase from E. superba (ESAK) was investigated. When the concentration of glycine, proline and glycerol increased, the relative activation was significantly enhanced, while the aggregation of ESAK during thermal denaturation was decreased. Spectrofluorometry results showed that the presence of these three osmolytes significantly decreased the tertiary structural changes of ESAK and that thermal denaturation directly induced ESAK aggregation. The results demonstrated that glycine, proline and glycerol not only prevented ESAK from inactivation and unfolding but also inhibited aggregation by stabilizing the ESAK conformation. We measured the ORF gene sequence of ESAK by RACE, and built the 3D structure of ESAK and osmolytes by homology models. The results showed that the docking energy was relatively low and that the clustering groups were spread to the surface of ESAK, indicating that osmolytes directly protect the surface of the protein. Our study provides important insight into the protective effects of osmolytes on ESAK folding.     

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein

Despite the fact that the porcine odorant-binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 A) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (lambda(max) = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (lambda(max) = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat-induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I(320)/I(365)) upon excitation at lambda(ex) = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85 degrees C, and the calculated midpoint of transition was centered at 70 degrees C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature-induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature.     

Monolayer characterstics and thermal behaviour of phosphatidic acids

The monolayer and thermal behaviour of different phosphatidic acids are presented. At neutral pH and 22°C dilauroylphosphatidic acid and unsaturated phosphatidic acids form liquid-expanded monolayers, while dipalmitoyl- and distearoylphosphatidic acid form condensed monolayers. Dimyristoylphosphatidic acid undergoes a transition from the liquid-expanded to the condensed state. With long-chain saturated and unsaturated phosphatidic acids little change in molecular area is observed between pH 2 and 7. In contrast, the short chain saturated phosphatidic acids, dilauroyl- and dimyristoylphosphatidic acids, undergo a condensation in the pH range 2 to 7. This is so in spite of the fact that the phosphoric acid group dissociates and the phosphatidic acid molecule attains one negative charge over this pH range. This finding is interpreted to indicate that the electrostatic repulsion between negatively charged phosphatidic acid molecules is compensated for or even outweighed by other intermolecular forces. Hydrogen bonding at the lipid/water interface is supposed to play a major role. All phosphatidates studied exhibit a significant expansion in the pH range 7 to 12. The second apparent pK of the primary phosphate group of phosphatidic acids is 8.6 and the expansion observed in this pH range is therefore due to electrostatic repulsion. At neutral pH the ether analogues of saturated phosphatidic acids have monolayer properties similar to those of the ester compounds. Considering the total pH range of 2 to 12 studied the force-area curves of the ether analogues are more condensed compared to the ester compounds. Synthetic phosphatidates and their ether analogues give reversible sharp crystal(gel)-to-liquid crystal transitions while the naturally occurring egg phosphatidate gives a broad, asymmetric one. The transition temperature T_m of saturated phosphatidates increases with increasing hydrocarbon chain length and at a given chain length T_m decreases markedly with unsaturation. The T_m values of the ether analogues are about 10°C higher and the ΔH values are 10–15% lower than those of the corresponding esters.     

Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation

A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn-HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a deltaG(H(2)O) of 1.741 kcal/mol, D1/2 of 2.3M, and m value of 0.755kcal/molM with urea and a deltaG(H(2)O) of 1.927kcal/mol, D1/2 of 1.52M, and m value of 1.27 kcal/molM with Gdn-HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 degrees C for 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a deltaCp of 0.864kcal/(molK). The deltaG(25 degrees C) at, pH 8.5, was 2.06kcal/mol and was in good agreement with the deltaG(H(2)O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.     

Thermodynamic features of the chemical and thermal denaturations of human serum albumin

The unfolding process of human serum albumin between pH 5.4 and 9.9 was studied by chemical and thermal denaturations. The experimental results showed that there is no correlation between the stability of albumin at different pH values determined by both methods. The free energy change of unfolding versus concentration of guanidine showed a close dependence on the pH, suggesting that the variation of the electrical charge of albumin influences the final state of the unfolded form of the protein. Spectroscopic techniques, such as native fluorescence of the protein and circular dichroism, demonstrated that the unfolded state of the protein obtained from both methods possesses a different helical content. The solvophobic effect and the entropy of the chains have no influence on the final unfolding state when the protein is unfolded by thermal treatment, while, when the protein is unfolded by chemical denaturants, both effects depend on the medium pH. The results indicate that guanidine and urea interact with albumin by electrostatic forces, yielding a randomly coiled conformation in its unfolded state, while thermal denaturation produces a molten globule state and the aggregation of the protein; therefore, both methods yield different structurally unfolded states of the albumin.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Effects of thermal stratification and mixing on reservoir water quality

In this study, the effect of thermal stratification on water quality in a reservoir has been investigated by field observations and statistical analysis. During the summer period, when stratification is evident, field observations indicate that the observed dissolved oxygen concentrations drop well below the standard limit of 5 mg l⁻¹ at the thermocline, leading to the development of anoxia. The reasons for variations in the dissolved oxygen concentrations were investigated. Variations of air temperature and other meteorological factors and lateral flows from side arms of the lake were found to be responsible for the increase of dissolved oxygen concentrations. It was also observed that turbidity peaked mostly in the thermocline region, closely related to the location of the maximum density gradient and thus low turbulence stabilizing the sediments in the vertical water column. Relatively cold sediment-laden water flowing into the lake after rain events also resulted in increased turbidity at the bottom of the lake. Nondimensional analysis widely used in the literature was used to identify the strength of the stratification, but this analysis alone was found insufficient to describe the evolution of dissolved oxygen and turbidity in the water column. Thus correlation of these parameters was investigated by multivariate analysis. Fall (partial mixing), summer (no mixing), and winter (well mixed) models describe the correlation structures between the independent variables (meteorological parameters) and the dependent variables (water-quality parameters). Statistical analysis results indicate that air temperature, one day lagged wind speed, and low humidity affected variation of water-quality parameters.     

The role of water in the reversibility of thermal denaturation of lysozyme in solid and liquid states

Although unfolding of protein in the liquid state is relatively well studied, its mechanisms in the solid state, are much less understood. We evaluated the reversibility of thermal unfolding of lysozyme with respect to the water content using a combination of thermodynamic and structural techniques such as differential scanning calorimetry, synchrotron small and wide-angle X-ray scattering (SWAXS) and Raman spectroscopy. Analysis of the endothermic thermal transition obtained by DSC scans showed three distinct unfolding behaviors at different water contents. Using SWAXS and Raman spectroscopy, we investigated reversibility of the unfolding for each hydration regime for various structural levels including overall molecular shape, secondary structure, hydrophobic and hydrogen bonding interactions. In the substantially dehydrated state below 37 wt% of water the unfolding is an irreversible process and can be described by a kinetic approach; above 60 wt% the process is reversible, and the thermodynamic equilibrium approach is applied. In the intermediate range of water contents between 37 wt% and 60 wt%, the system is phase separated and the thermal denaturation involves two processes: melting of protein crystals and unfolding of protein molecules. A phase diagram of thermal unfolding/denaturation in lysozyme - water system was constructed based on the experimental data.     

Calculation of thermodynamic properties of protein in Escherichia coli K-12 grown on succinic acid, energy changes accompanying protein anabolism, and energetic role of ATP in protein synthesis

Using an average of the results from three methods of calculation, estimations are made of the thermodynamic properties of a unit carbon formula weight (UCFW) of Escherichia coli K-12 protein. These resulted in values fro DeltaG(f) of -38.09 kJ (-9.10 kcal)/ UCFW, for DeltaH(f) of -68.18 kJ (-16.29 kcal)/UCFW, and for DeltaS(f) of -94.2 J (-22.5 cal)/UCFW deg. The absolute entropy of one UCFW of E. coli K-12 protein is calculated to be 73.8 J/UCFW deg. Using these values, the corresponding changes in thermodynamic properties accompanying the anabolism of protein by this microorganism to from one UCFW of protein by this microorganism to from one UCFW of protein are calculated to be 1.97 kJ (0.47 kcal)/UCFW for DeltaG, 0.75 kJ (0.18 kcal)/UCFW for DeltaH, and -4.09 J (-0.98 cal)/UCFW deg for DeltaS. All these values are sufficiently close to zero that they may be considered to be so. The question is raised as to the quantity of ATP energy conserved within the substance of the protein as it is synthesized from succinic acid. It is calculated that only 3.8% of the total free energy available from ATP that is required during protein anabolism can have been conserved within the substance of the protein, there being a net conversion of the remaninder into heat and entropy.     

Controlled morphologies of copper oxide single crystalline nanostructures by wet chemistry and thermal decomposition processes

A wet-chemical method to synthesize CuO nanocubes (0D), nanorods (1D) and nanosheets (2D) is demonstrated with using Cu(OAc)₂ as an inorganic precursor and triethylammine as a basic source in the presence of tetraoctylammonium bromide in a H₂O-C₂H₅OH mixed-solvent. We found that the nanocubes spontaneously self-assembled into nanorods that could further self-assemble into nanosheets via oriented attachment of the nanorods. Based on the structural characterization, we found that the CuO nanosheets grow primarily along the [0 1 0] direction. A single phase of CuO nanostructures was observed. Another simple thermal decomposition method was also developed to prepare monodispersed single crystalline CuO nanocubes controlled by the decomposition of the inorganic Cu(OAc)₂ precursor with triethylammine and TOAB in H₂O-C₂H₅OH solution on a Si wafer and indium tin oxide conducting glass. The composition of the CuO was further confirmed by XRD and XPS analyses.