Hemoglobins of the tadpole of the bullfrog, Rana catesbeiana. Temperature dependence of oxygen binding and pH dependence of subunit dissociation.

The temperature dependence of the oxygen equilibrium of tadpole hemoglobin has been determined between 0 degrees and 32 degrees for the unfractionated but phosphate-free lysate and between 12 degrees and 32 degrees for each of the four isolated components between pH 6 and 10 in 0.05 M cacodylate, Tris, or glycine buffers containing 0.1 M NaCl and 1 mM EDTA. Under these conditions the Bohr effect (defined as deltalog p50/deltapH) of the unfractionated lysate is positive at low temperatures between pH 6 and 8.5 and is negative above pH 8.5 to 8.8 at any temperature. As the temperature rises the Bohr effect below pH 8.5 changes greatly. In the interval pH 7.0 to 7.5, the magnitude of the Bohr effect decreases from + 0.28 at 0 degrees to zero at about 24 degrees and becomes negative, as in mammalian hemoglobins, above this temperature. Measurements with the isolated components show that the temperature dependence of oxygen binding for Components I and II and for Components III and IV is very similar. For both sets of components the apparent overall enthalpy of oxygenation at pH 7.5 is about -16.4 kcal/mol and -12.6 kcal/mol at pH 9.5. The measured enthalpies include contributions from the active Bohr groups, the buffer ions themselves, the hemoglobin groups contributing buffering, and any pH-dependent, oxygenation-dependent binding of ions such as chloride by the hemoglobin. The apportioning of the total enthalpy among these various processes remains to be determined. Between pH 8 and 10.5 tadpole oxyhemoglobin undergoes a pH-dependent dissociation from tetramer to dimer. The pH dependence of the apparent tetramer-dimer dissociation constant indicates that at pH 9.5 the dissociation of each tetramer is accompanied by the release of approximately 2 protons. In this pH range the oxygen equilibrium measurements indicate that about 0.5 proton is released for each oxygen molecule bound. The results are consistent with the conclusion that one acid group per alphabeta dimer changes its pK from about 10 to 8 or below upon dissociation of the tetramer.     

Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy (T delta S degrees = +10.32 kcal/mol) and no change in enthalpy. Binding to albumin is driven by enthalpy (delta H degrees = -8.34 kcal/mol) and is accompanied by a decrease in entropy (T delta S degrees = -2.88 kcal/mol). Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic (delta H degrees was -3.3 and -5.5 kcal/mol, respectively) and by entropic (T delta S degrees was +4.44 and +2.91 kcal/mol, respectively) components. The implications of these finding are discussed.     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Duplex formation of a nonionic oligo(deoxythymidylate) analogue (heptadeoxythymidylyl-(3'-5')-deoxythymidine heptaethyl ester (d-(Tp(Et))7T)) with poly(deoxyadenylate). Evaluation of the electrostatic interaction.

The heptaethyl ester of heptadeoxythymidylyl-(3'-5')-deoxythymidine (d-[Tp(Et)]7T or d-T8-Et) has been prepared by chemical methods. The material, consisting of a mixture of diastereoisomers, forms a 1:1 complex with (dA)n in neutral aqueous buffer; this interaction is virtually independent of ionic strength. The octamer triester does not bind to (dA)n-(dT)n, and it interacts with (rA)n only at low temperatures. By cochromatography with (dA)n on Sephadex G-50, d-T8-Et fractions with different binding affinities for the polyadenylates were obtained. This heterogeneity in binding affinity is ascribed to the diastereoisomerism of d-T8-Et. Enthalpies of dupoex formation were determined by the concentration variation method. At 0.1 M sodium ion concentration, the enthalpy of binding of the various d-T8-Et fractions to (dA)n is essentially invariant (-8.1 kcal/mol of base pairs at 0 degrees C to -8.6 kcal at 25 degrees C) and 1.6 kcal/mol of base pairs more negative than the enthalpy of binding of the phosphodiester analogue, d-(Tp)7T, to (dA)n (-6.8 kcal/mol of base pairs at 11 degrees C). This difference is the electrostatic contribution to the enthalpy of duplex formation, arising from the interstrand electrostatic repulsion and the intrastrand repulsion in d-(Tp)7T. The entropy of binding to (dA)n is more negative for the octamer triesters than for the diester analogue, and is different for the various d-T8-Et fractions. This is interpreted in terms of varying degrees of restriction of rotational freedom for the ethyl substituents upon double helix formation.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

Laser-induced time-resolved photoacoustic calorimetry study on photo-dissociation of human and bovine oxyhemoglobin

The dynamics of the enthalpy and volume changes related to the photo-dissociation of oxygen from human and bovine oxyhemoglobin are investigated by nanosecond time-resolved photoacoustic calorimetry (PAC). The values of enthalpy and volume change associated with the above process are deltaH = 37.8 +/- 3 kcal/mol, deltaV = 5.0 +/- 1 ml/mol for human HbO(2); and deltaH = 35.7 +/- 3.5 kcal/mol, deltaV = 4.8 +/- 1 ml/mol for bovine HbO(2), respectively. A possible explanation for the similar values between both human and bovine oxyhemoglobin is proposed. In addition, the PAC results for human HbO(2) and HbCO are compared and discussed.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.     

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Studies on the heme environment of horse heart ferric cytochrome c. Azide and imidazole complexes of ferric cytochrome c.

Horse heart ferric cytochrome c was investigated by the following three methods: (I) Light absorption spectrophotometry at 23 degrees C and 77 degrees K; (II) Electron paramagnetic resonance (EPR) spectroscopy at 20 degrees K; (III) Precise equilibrium measurements of ferric cytochrome c with azide and imidazole between 14.43 and 30.90 degrees C. I and II have demonstrated that: (1) Ferric cytochrome c azide and imidazole complexes were in the purely low spin state between 20 degrees K and 23 degrees C; (2) The energy for the three t2g orbitals calculated in one hole formalism shows that azide or imidazole bind to the heme iron in a similar manner to met-hemoglobin azide or imidazole complexes, respectively. III has demonstrated that: (1) The change of standard enthalpy and that of standard entropy were -2.3 kcal/mol and -1.6 cal/mol per degree for the azide complex formation, and -1.4 kcal/mol and 2.9 cal/mol per degree for the imidazole complex formation. (2) A linear relationship between the change of entropy and that of enthalpy was observed for the above data for the cyanide complex formation. The complex formation of ferric cytochrome c was discussed based on the results of X-ray crystallographic studies compared with hemoglobin and myoglobin.     

New approach to the study of hormone-protein interaction using the microcalorimetric method.

Binding enthalpies of various hormones to bovine serum albumin (BSA) and human serum albumin (HSA) in 50 mM phosphate buffer, pH 7.4, at 37 degrees C have been determined by direct microcalorimetry. The observed enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to BSA were found to be -13.24 plus or minus 0.11 -10.31 plus or minus 0.02, -2.37 plus or minus 0.46, -17.64 plus or minus 0.32 and -17.14 plus or minus 0.36 kcal/mol of hormone, respectively. under the same experimental conditions the enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to HSA were found to be -23.94 plus or minus 0.32, -18.88 plus or minus 0.49, -11.14 plus or minus 0.02, -9.88 plus or minus 0.14 and -20.85 plus or minus 0.39 kcal/mol of hormone, respectively.     

Discontinuous release of heat at successive steps of oxygenation in human and bovine hemoglobin at pH 9.0

We have measured the temperature dependence of the oxygen-binding isotherms of human and bovine hemoglobin at pH 9.0 in 0.1 M borate buffer. In both hemoglobins the ionization of the Bohr protons is finished at this pH; therefore, their heat does not interfere with the measurements. Two sets of curves have been obtained, which have been analyzed by either singular or global procedures for estimating the enthalpy changes of subsequent steps of oxygenation. The data indicate that in human hemoglobin the reaction with oxygen is enthalpy driven for steps 1, 2, and 4 while it is entropy driven for step 3. In bovine hemoglobin this phenomenon is even more evident: steps 2 and 4 are enthalpy driven while steps 1 and 3 are entropy driven. The discontinuous distribution of heat at subsequent steps of oxygenation suggests that the T to R transition in hemoglobin is not a monotonic process and involves conformations with novel characteristics.     

Oxygenation properties of hemoglobin from the earthworm, Lumbricus terrestris. Effects of pH, salts, and temperature

Oxygen equilibrium curves of the extracellular hemoglobin from Lumbricus terrestris were determined under a variety of conditions. These data were characterized by (i) a rather small free energy of cooperativity (1.6-2.8 kcal/mol), (ii) a large and strongly pH-dependent Hill coefficient with a maximum value of 7.9, (iii) a high sensitivity of the upper asymptote of the Hill plot to pH, and (iv) a maximum association constant as large as that of the free beta subunit of human hemoglobin A. The effects of LiCl, KCl, NaCl, BaCl2, CaCl2, SrCl2, and MgCl2 on the oxygen equilibrium were measured. Cations, not Cl-, were found to control oxygen binding. Divalent cations have a larger effect on oxygen affinity than monovalent cations, and their effectiveness decreased in the order listed above within each valence class. These specific effects depend in part on ionic radius and cannot be explained in terms of ionic strength. The data indicate that the oxygenation-linked binding of a Ca2+ ion is accompanied by the release of two protons; the binding of a Na+ ion is associated with the release of one proton. These findings indicate that the oxygenation-linked cation-binding site contains two acid groups that do not readily dissociate their protons except when replaced by cations. Incubation at either pH 6.2 or 8.9 had no effect on subsequent measurements of oxygen equilibria at pH 7.8. The apparent heat of oxygenation was found to be -11.8, -7.3, and -9.3 kcal/mol at pH 9.0, 7.4, and 6.6, respectively. These differences indicate that proton-binding processes contribute to the heat of oxygenation.     

Calorimetric and spectroscopic investigation of drug-DNA interactions. I. The binding of netropsin to poly d(AT)

We report the first calorimetric investigation of netropsin binding to poly d(AT). Temperature-dependent uv absorption, circular dichroism (CD), batch calorimetry, and differential scanning calorimetry (DSC) were used to detect, monitor, and thermodynamically characterize the binding process. The following results have been obtained: 1) Netropsin groove binding is accompanied by a large exothermic enthalpy of 9.2 kcal/mol of drug bound at 25 degrees C. This indicates that a large negative binding enthalpy may be a necessary but not a sufficient criterion for drug intercalation. We suggest that the exothermic binding might be correlated with specific H-bonding interactions. 2) From the difference in DSC transition enthalpies in the presence and absence of netropsin, we calculate a binding enthalpy of -10.7 kcal/mol of netropsin at 88 degrees C. 3) We calculate a positive delta S for netropsin binding to poly d(AT) at 25 degrees C. This positive entropy change may reflect netropsin-induced release of condensed cations and/or bound water. 4) The netropsin-saturated duplex monophasically melts 46 degrees C higher than the free duplex. The unsaturated duplex melts through two thermally-resolved transitions that correspond to netropsin-free and netropsin-bound regions. These two regions interact dynamically with no substantial influence on the thermal stabilities of the separate domains. 5) Netropsin binding decreases the cooperativity of the duplex to single strand transition.     

Thermodynamics of the binding of human apolipoprotein A-I to dimyristoylphosphatidylglycerol

The interaction of human serum apolipoprotein A-I with dimyristoylphosphatidylglycerol was analyzed by isothermal titration calorimetry. Binding of the apolipoprotein A-I to large unilamellar vesicles of dimyristoylphosphatidylglycerol, a negatively charged phospholipid, is characterized by thermodynamic parameters which are invariant over the 30-40 degrees C temperature range. The enthalpy change resulting from the first additions of lipid are positive and decline in magnitude with subsequent additions of lipid. After several additions of lipid, the sign of the enthalpy changes to negative and then reaches a constant value/injection. This exothermic process is larger and opposite in sign to the heat of dilution. Similar behavior is also observed when the lipid is in the form of a dispersion in distilled water. Only a non-saturable exothermic process is observed at 30 degrees C with large unilamellar vesicles of the zwitterionic lipid, dimyristoylphosphatidylcholine. The beginning of an exothermic process can also be observed prior to the larger endotherm in the first injections of large unilamellar vesicles of dimyristoylphosphatidylglycerol into the protein. We analyze the enthalpy changes for the reaction of dimyristoylphosphatidylglycerol with the protein as arising from two distinct processes, one endothermic and the other exothermic. The binding isotherms for the high affinity binding of the apolipoprotein A-I to large unilammelar vesicles of dimyristoylphosphatidylglycerol, over the temperature range 30-40 degrees C, gave an enthalpy change of 1.43 +/- 0.07 kcal/mol of protein and a free energy change of -5.91 +/- 0.04 kcal/mol of protein for the binding of the protein to a cluster of 25 +/- 2 lipid molecules. Thus this reaction is entropically driven.     

Arctic life adaptation--III. The function of whale (Balaenoptera acutorostrata) hemoglobin

1. The oxygen binding properties of the hemoglobin from the Lesser Rorqual, Balaenoptera acutorostrata, has been investigated with respect to the possible effects of organic phosphates on gas transport in arctic environments. 2. The intrinsic oxygen affinity of the hemoglobin is high and strongly modulated by the effects of organic phosphates. 3. In the absence of organic phosphates, the temperature sensitivity of oxygen binding expressed by the heat of oxygenation, delta H, is -16.2 kcal/mol when corrected for the heat of oxygen in solution. 4. In the presence of organic phosphates there is a marked decrease in the temperature sensitivity delta H approximately -5 kcal/mol). 5. This feature is of great importance for oxygen unloading in the flippers and the tail, where the temperature is lower than the trunk of the whale. 6. Furthermore the organic phosphates strongly increase the Bohr coefficient, delta log P50/delta pH, from less than -0.3 in stripped hemoglobin to about -1.5 when the hemoglobin is saturated with P6-inositol. 7. This feature may be of great physiological importance by reducing the CO2 tension and acidosis after a prolonged dive.     

Water-phase palmitate concentrations in equilibrium with albumin-bound palmitate in a biological system.

The palmitate (PA) binding and transport capacity of human and bovine red cell membranes enables us to establish, in a biological system, the existence of a well-defined monomer concentration in equilibrium with PA bound to bovine serum albumin (BSA, 30 microM) inside the resealed red cell ghosts. Supernatants of suspensions of the [3H]PA-labeled ghosts contain a tiny quantity of dissolved binding capacities besides the monomer PA. This is demonstrated by linear regression of supernatant tracer concentrations versus ghost concentrations in a dilution series. The extrapolated value, corresponding to zero ghost concentration, is the monomer PA concentration in equilibrium with PA bound to BSA within the ghosts in molar ratio (nu). Measurements have been carried out for nu between 0.1 and 1.5 and at 0 degrees C, 10 degrees C, 23 degrees C and 38 degrees C. The important nu-dependent binding of PA to the ghost membrane itself enables us to use preparations of BSA-free ghosts in the same way, whereas this is impossible in the case of arachidonic acid. Within the physiological range of nu the PA monomer concentrations are accounted for by an apparent dissociation equilibrium constant (Kd) 3.4 10(-8) M at 38 degrees C calculated on basis of three equivalent binding sites per mol BSA. Kd depends on temperature with a well-defined enthalpy of 38.4 kJ/mol.