Calorimetric measurement of stepwise enthalpy changes for the binding of four oxygens to adult human hemoglobin

The successive enthalpy changes for the four steps of oxygen binding by diphosphoglycerate-free adult human hemoglobin have been measured by direct calorimetry at pH 7.4 and 6°. Average results in kcal/(mole O₂) are: ΔH₁ = ?25.1 ± 2.8; ΔH₂ = ?12.6 ± 3.0, ΔH₃ = ?12.5 ± 3.0, and ΔH₄ = ?10.1 ± 1.4. These results imply a substantial temperature dependence for the cooperativity of O₂ binding by the protein and generally resemble the van't Hoff results by Roughton $\text{et al}$. [Roy. Soc. of London Proc., $\text{B 144}$, 29 (1955)] for sheep hemoglobin at pH 9.1 and a temperature range of 2° to 19°.     

Thermodynamics of equilibria of hemoglobins M Milwaukee-I and Saskatoon and isolated chains of hemoglobin a with carbon monoxide

The thermodynamic parameters of the CO-equilibria of isolated chains of hemoglobin A and of two α-chains in hemoglobins M Milwaukee-I and Saskatoon at 25°, pH 7.0 were determined. The parameters for the binding of the first CO molecule to the hemoglobins M were ΔH′=?17 and ?18 kcal/mole heme and ΔS′=?30 and ?29 e.u. for hemoglobins M Milwaukee-I and Saskatoon, respectively. In contrast to this the characteristics of the second step of the binding were ΔH′=+5.9^· and +4.3 kcal/mole and ΔS′=+51 and +49 e.u. These values for the second step were also significantly different from those of the isolated α-chain (ΔH′=?15 kcal/mole and ΔS′=?11 e.u.).     

The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate.

The reaction of β-galactosidase ($\text{E. coli}$ K12) with $\text{o}$-nitrophenyl-β-$\text{D}$-galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of $\text{o}$-nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with E_a = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on K_m but caused a small decrease in K_cat.     

Spin labels as enzyme substrates Heterogeneous lipid distribution in liver microsomal membranes

Phenobarbital-stimulated microsomal membranes of rabbit liver, containing the cytochrome P450- cytochrome P450 reductase hydroxylating enzyme system in high concentration, have been studied with a version of the spin label technique which uses nitroxide radicals as enzyme substrates. The reduction kinetics of a phosphate ester of tetramethylpiperidine nitroxide (TEMPO-phosphate) and of stearic acid nitroxide by the cytochrome P450 reductase has been studied as a function of the temperature. The Arrhenius plot of the reduction rate constants reveals a striking difference in the behaviour of the water-soluble TEMPO-phosphate label and the lipid-soluble fatty acid label: The activation energy of the fatty acid reduction decreases abruptly at about 32°C from a value of 30.8 kcal/mole to a value of 8.7 kcal/mole, whereas no such break is observed in the Arrhenius plot of the TEMPO-phosphate reduction which yields a value of the activation energy of $\text{ΔW = 13.8}$ kcal/mole in the whole temperature range investigated. Our results clearly indicate the existence of a mosaic-like structure of the membrane with the whole enzyme system being enclosed by a rather rigid phospholipid halo which is in a quasicrystalline structure below 32 °C and undergoes a crystalline-liquid crystalline phase transition at 32 °C, while the bulk lipid of the membrane is in a rather fluid state as reflected by the measured high diffusion coefficient of $\text{D}{}_{\mathrm{<mtext>diff</mtext>}}\text{= 11.0·10}{}^{\mathrm{?8}}\text{cm}{}^2\text{/s}$ at 30 °C and low activation energy of diffusion of $\text{ΔW = 3.85}\text{kcal/mole}$ of a fatty acid spin label incorporated in the membrane.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Thermal perturbation difference spectroscopy of Salmonella flagellin

The appropriateness of a two-state model for the previously reported thermal transition of $\text{Salmonella}$ flagellin and the reversibility of this process recently has been questioned by others. Spectrophotometric evidence is presented here that reveals two separate thermally-induced structural transitions in flagellin which may resolve apparent controversy. A low temperature transition (I) appears between 28 and 35°C with a midtransition temperature of 30.7°C. With increasing temperature in this transition region a progressive shift to the red of the absorbance band at 284 nm occurs. The latter, probably due to an infolding of tyrosyl residues, is paralleled by a decrease in the rate of polymerization of flagellin. The temperature profile for the spectral behavior of flagellin in transition I fulfills criteria for a two-state process and is fully reversible. A second transition, also reversible, is observed between 40 and 60°C with a midtransition temperature near 50°C. Transition II, observed as a blue spectral shift of the absorbance band at 277 nm, is better described as a multistate process. Tyrosyl residues appear to maintain the conformational integrity of the polymerizable state of flagellin.     

Further studies on the temperature dependence of the binding of testosterone to human pregnancy plasma proteins

The binding of testosterone by pregnancy plasma proteins has been studied by a new equilibrium dialysis system. The temperature dependence on the association constant has been investigated and the enthalpy change ΔH and entropy change ΔS have been calculated.By a computer optimization program, the binding constant of the high affinity testosterone binding protein has been estimated from Scatchard plots. The binding reactions were carried out at 5°, 25° and 37° C. The corresponding values were 3.1.10 1.2.10⁹ and 7.2.10⁸ liter/mole. The resulting enthalpy and entropy changes were ?2.0 kcal/mole and 35.0 cal/(mole.degree) respectively.It can be concluded that the binding of testosterone to the specific binding protein is an exothermic reaction and is stabilized by hydrophobic binding forces.     

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373–377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues ($\text{n = 3}$ through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by ²²Na permeability measurements, differential scanning calorimetry, fluorescence polarization, ³¹P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with $\text{n = 2}$ or 3 exhibit a sharp transition at 46°C or 49°C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with $\text{n > 3}$ exhibit a main transition between 38 and 43°C with enthalpies $\text{< 10}\text{kcal/mol}$ and after prolonged cooling (more than 3 days at 4°C) a broad endotherm at about $\text{20 ± 3°}\text{C}$ with enthalpies $\text{> 4}\text{kcal/mol}$. These same dispersions display a permeability peak at 20–25°C and a second increase in ²²Na efflux in the temperature range 30–40°C. The results of ³¹P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentanol-phosphocholine ($\text{n = 2}$) bilayers have restricted rotation below the main phase transition temperature.     

A comparison of van't Hoff and calorimetric heats of binding to DNA using an ethidium selective electrode

A liquid membrane electrode selective for ethidium ion was used to measure free ethidium in mixtures with calf thymus DNA. Electrode response was unaffected by variation in ionic strength from 1 mm to 0.5 m, and was not degraded over the temperature range studied. DNA-ethidium binding isotherms obtained with the electrode at 17.4, 25.4, 30.1, and 40.6 °C were fitted to a single class of excluded sites model for $\text{}\text{?}$gn ranging from 0.01 to 0.16. van't Hoff analysis of these data yielded ΔH = ?8300 cal/mol ethidium bound (in 0.5 m KCl, 10 mm Tris buffer, pH 10, 1 mm EDTA). Direct calorimetric measurements of the heat of complex formation led to a value of ?7600 cal/mol at 25 °C in the same medium; the two results were not significantly different at the 95% confidence level. The agreement supports the validity of the ethidium selective electrode, and illustrates its utility in the study of ligand binding to nucleic acids and related materials.     

Effect of temperature on the in vitro assembly of bacterial flagella

The temperature dependence for the rate of reconstitution or polymerization (k₊) at neutral pH of the protein, flagellin, to flagella was measured using Ostwald-type viscometers. Similarly, the kinetics for the reverse process, the thermally-induced depolymerization of flagella filaments to the flagellin monomer (k_?) was measured. The temperature at which k_? equals zero was used to define the thermal dissociation temperature or melting point of flagella filaments. The remarkable similarity of melting points obtained (36.8 ± 0.2 deg. C) for flagella isolated from three Salmonella strains (SJ670, SJ25 and SJ30 bearing H-antigen types i, 1.2 and e, n, x, respectively) suggests that the structural stability of these different protein filaments is also similar.On increasing the temperature between 12 and 28°C, k₊ increased smoothly and had a Q₁₀ of 1.8. Above 28.0, k₊ decreased rapidly and fell to zero at a temperature near 37°C, its precise value varying with the bacterial strain. This result supports the prior hypothesis (Gerber &; Noguchi, 1967) that on heating, a reversible co-operative transconformation occurs between different states of the protein; in one state, flagellin (M) can polymerize to flagella, whereas its conformational isomer(s) may do so with difficulty or not at all.For strains SJ25 and SJ30 the rates of polymerization and depolymerization both fall to zero near 37°C. Therefore, mixtures of monomer and flagella fragments (short polymers or “seeds”), in all ratios, appear to be in equilibrium at temperatures near this critical temperature, and neither polymerization of flagellin to flagella nor melting of polymers is apparent.Measurements made on flagella from strain SJ670 showed that k₊ and k_? approached zero at 45 and 37°C, respectively. Within this temperature range the conc entration of monomer in equilibrium with filaments was determined. By a null -point type experiment, solutions of monomer and seed were mixed to find the ratio that showed neither increases (polymerization) nor decreases (depolyme rization) in viscosity with time. An unexpected finding was that the temperature defines a critical monomer concentration, which exists in equilibrium with any concentration of filaments (and not the ratio of monomer-to-filament concentrations). Thus, the polymerization of fiagellin to flagella corresponds to a phase change akin to either crystallization or condensation.Application, of the Clapeyron-Clausius equation to the results obtained yields a heat of condensation of 70 kcal/mol of monomeric protein. The enthalpy change associated with M ? M_i is estimated as 110 kcal/mol of protein. Since the heat content of these various forms of flagella protein lies in the order M_i > F > M, by difference we estimate the enthalpy change for the conversion of monomers to polymers to be 40 kcal/mol of monomer.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Analysis of protein self-association by combined calorimetric and molecular sieve studies: application to beta-lactoglobulin A

The application of isothermal calorimetry to the study of self-association reactions between identical protein subunits has been explored to assess the types of information obtainable from heat of dilution curves (i.e., the molar heat of dilution as a function of total solute concentration). Relationships between the heat of dilution, subunit association constants, and enthalpies of formation for the various association complexes in a self-associating system have been formulated. A method is described for constructing heat of dilution curves from sequential step-wise dilution experiments in a batch-type calorimeter. The relationship between calorimetric and van't Hoff enthalpies is formulated for systems undergoing self-association reactions. Comparison between the two is shown by numerical simulation to provide a very sensitive test for the presence of intermediate species.Calorimetric measurements were made on the self-association of β?lactoglobulin-A at 5.0 °C, pH 4.65, in 0.1 m NaCl and in 0.1 m acetate. Heat of dilution curves constructed from these data were used to estimate the equilibrium constant and enthalpy of formation, assuming a monomer-tetramer association process. Values of 31 ± 4 kcal/mole tetramer for the enthalpy and 1.3 ± 1.0 × 10¹¹ liter³/mole³ for the constant of tetramerization were determined from the calorimetric measurements in NaCl. The corresponding values for calorimetric measurements in acetate were 33 ± 4 kcal/mole and 1.6 ± 1.0 × 10¹¹ liter³/mole³.The calorimetric results were compared with thermodynamic information obtained from association data between 5 and 25 °C in 0.1 m acetate using molecular sieve chromatography. Within experimental error, the molecular sieve data at 5 °C could be fit to a monomer-tetramer association reaction with a monomer molecular weight of 36,000. From these studies a van't Hoff enthalpy of 38 ± 4 kcal/mole tetramer and an equilibrium constant of 4.6 ± 1.0 × 10¹¹ liter³ mole³ at 5 °C were obtained. Comparison between calorimetric and van't Hoff enthalpies indicates that the self-association of β-lactoglobulin-A under these conditions can be adequately described by a monomer-tetramer reaction. The results suggest that, a small fraction (e.g., 5–10%) of species having intermediate states of aggregation may be present, but preclude the presence of large fractions of intermediate species having appreciable enthalpies of association.     

Cytochrome c interaction with membranes. The enthalpy of oxidation of cytochromes c, c2, and a1,2.

The enthalpy of oxidation of horse-heart cytochrome c bound to phospholipid vesicles was found to be 14.6 ± 0.3 kcal/mole at 25 °C, pH 7.0, equal to the value for oxidation of the free form of the cytochrome. The affinity constants for binding of the reduced and oxidized forms of cytochrome c were the same at 4 °C and 30 °C, indicating that ΔH ° of binding contributes negligibly to the overall enthalpy of oxidation of the bound cytochrome c. The free energy (ΔG °′) of oxidation of the bound cytochrome c was 1.3 kcal/mole smaller than that for the free form, the difference being due to the change in entropy favoring the oxidized state of the cytochrome in the bound state. Measurement of the ΔH °′ for the oxidation of cytochrome a relative to the ferri/ferrocyanide couple shows it to be the same, within the limits of experimental error to that for the oxidation of cytochrome c.     

Thermodynamics of the glutamate dehydrogenase catalytic reaction

The enthalpy change for the oxidative deamination of glutamate by NADP⁺ catalyzed by bovine liver glutamate dehydrogenase has been determined calorimetrically. The ΔH^o values are 64.6 ± 1.2 $\text{kJ}\text{mol}$ and 70.3 ± 1.2 $\text{kJ}\text{mol}$ at 25 and 35°C respectively. The equilibrium constants for the reaction at the two temperatures were determined spectrophotometrically. This enabled the determination of ΔG^o and ΔS^o of the reaction as well. ΔH^ovalues were also determined for the reaction using an alternative coenzyme and the deuterated substrate.     

The rate of γ-benzyl-L-glutamate NCA polymerizations

The primary amine initiated homopolymerization of γ-benzyl-L -glutamate NCA in dioxane at 25°C, 35°C, 50°C, and 65°C has been investigated. The reactions were virtually independent of temperature indicating an activation energy of less than 1 kcal/mole. The entropy of activation was estimated to be ?65 entropy units at 300°K. The reaction proceeded in two stages. The first stage was zero-order with respect to monomer, whereas the second was first-order with respect to monomer. Both stages were first-order with respect to initiator. These results were interpreted by assuming that the rate constant for propagation was not independent of the degree of polymerization up to the point where a conformational transition to α-helix occurred.     

Calorimetric studies on thein vitro polymerization ofPr. mirabilis flagellin

The heat effects accompanying the isothermalin vitro polymerization ofPr. mirabilis flagellin on short flagella fragments (seeds) have been measured in phosphate buffer pH 7, at various temperatures employing a batch microcalorimeter. Additionally, at 20 ?C, measurements have been performed in phosphate as well as Tris- HCl buffer at pH 7.5. The rate of both heat uptake and release during the process of polymerization was shown to be proportional to the rate of molar ellipticity changes observed by parallel circular dichroism experiments. No change in the state of protonation of flagellin occurs during the polymerization as indicated by the constancy of the enthalpy values determined in buffers with different heats of ionization. The apparent molar enthalpy of polymerization at 25 ?C, pH 7, is ?34.7±3 kcal per mole of flagellin, the relatively large error mainly resulting from uncertainties of the determination of the percentage of unpolymerized monomers after completion of the reaction. The most prominent feature of the results obtained in this study is the large temperature variation of the enthalpy, corresponding to a temperature independent heat capacity change ofδc _p =?3039±100 cal per degree per mole of flagellin, the error limits referring to the standard deviation in a linear regression analysis.     

Acid dissociation of cyclohexaamylose and cycloheptaamylose

Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pK_a's are ΔH⁰ = 8.4 ± 0.3 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?28. ± 1}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 6-Cy and ΔH⁰ = 10.0 ± 0.1 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?22.4 ±0.3}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 7-Cy. Intrinsic ¹³C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D₂O ($\text{v}\text{v}$). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 2⁰-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Calorimetric and spectroscopic investigation of the helix-to-coil transition of the self-complementary deoxyribonucleotide ATGCAT

Differential scanning calorimetry and temperature-dependent uv spectroscopy are used to thermodynamically characterize the double-strand to single-strand transition of the self-complementary deoxyribo-oligonucleotide ATGCAT. The calorimetric experiments provide a value of 33.6 kcal (mol of double strand)^?1 for the transition between 10 and 90° C. In conjunction with available temperature-dependent nmr data (which reveals terminal base pair fraying), we attempt to define specifically those interactions to which the calorimetrically measured enthalpy change refers.Values of ΔH_V.H. (van 't Hoff enthalpy change) are derived from the spectroscopic and calorimetric data and compared with the ΔH obtained directly from the calorimetric experiment. This comparison reveals that the part of the thermally-induced transition that occurs between 10 and 90°C is well represented by a two-state process. It is noted that in assessing the applicability of the two-state model it is best to compare the ΔH_cal. with ΔH_V.H. obtained from the calorimetric rather than the spectroscopic data.