Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2-

Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2- have been made. The reaction was found to follow a second-order rate law: k 4.2 x 10(3) M(-1) s(-1) [25 degrees, micro0.1 M, pH 7.0 (phosphate)]; deltaH+/+ 3.2 kcal/ mol; AS+/+ -30 cal/mol-deg. The electrostatics-corrected self-exchange rate constant (k11 corr) calculated for cytochrome c551 based on the Fe(EDTA)2- cross reaction is 2 M(-1) s(-1), as compared to a value of 6 M(-1) s(-1) for horse heart cytochrome c. The close correspondence of the two k11 corr values is taken as an indication that the two proteins employ very similar electron transfer mechanisms in their reactions with Fe(EDTA)(2-). It is proposed that this mechanism involves reagent contact, but little protein conformational change, at the partially exposed heme edge.     

Isokinetic Relationship in the Oxidation of Cuprous Stellacyanin by Cobalt(III) Complexes

Rate parameters have been obtained for the oxidation of cuprous stellacyanin by cobalt(III) ions of the form cis(N)-[CoN₂O₄]^?, including cis(N)-[Co(NTA)(gly)]^?, cis(N)-[Co(IDA)₂]^?, [Co(en)(ox)₂]^?(μ 0.5 M(phosphate), pH 7.0), and Co(EDTA)^?(μ 0.1 M(NaCl), pH 7.2, 0.001 M phosphate). An excellent isokinetic correlation between the activation parameters ΔH^≠ and ΔS^≠ exists for the reactions of aminopolycarboxylatocobalt(III) ions with reduced stellacyanin (β = 300 ± 12 K; correlation coefficient = 0.995). It is concluded that enthalpy-entropy compensation in these reactions may be understood in terms of differing orientations preferred by the various oxidants in forming precursor complexes with the reduced blue protein. While ΔH^≠ and ΔS^≠ values for electron transfer from stellacyanin to cis(N)-[CoN₂O₄]^? ions vary over ranges of 10.7 kcal/mol and 34 cal/mol-deg, respectively, room temperature rate constants are relatively constant (3.6–34.5 M^?1 sec^?1), as expected from Marcus theory for outer sphere electron transfer.     

Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.     

Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium deltaG degrees, deltaH degrees, deltaS degrees, deltaC degrees(p), and activation deltaG++ of S45A at 37 degrees C are 13.7+/-0.1 kcal/mol, -21.1+/-0.5 kcal/mol, -23.7+/-1.8 cal/mol K, -223+/-12 cal/mol K, and 20.0+/-2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the deltaH++ and deltaS++ of dissociation, 25.8+/-1.2 kcal/mol and 18.7+/-4.3 cal/mol K. The large increases of deltaH++ and deltaS++ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.     

Kinetics of the hemerythrin-oxygen interaction.

The kinetics of the reaction of Golfingia gouldii hemerythrin with O2 have been studied by stopped flow spectrophotometry. For the second order oxygenation process, k1 = 7.4 X 10(6) M-1 s-1, deltaH1++ = 8.2 kcal-mol-1 and deltaS1++ = +1 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The rate constant is unchanged when protein concentration is varied from 3 to 25 muM, the ionic strength is increased to 0.07 M, and the pH moved to 6.8. The deoxygenation of oxyhemerythrin is studied with stopped flow by scavenging liberated O2 with S2O4(2-). For the first order dissociation, k-1 = 51 s-1, deltaH-1++ = 20.6 kcal-mol-1 and deltaS-1++ = +19 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The value of k-1 is independent of [protein] = 50 to 200 muM, [S2O4(2-)] = 5 to 100 mM I = 0.015 to 0.30 M and pH 6.8 to 9.0. Using myoglobin instead of S2O4(2-) as scavenger gives similar results. Combination of activation parameters for the oxygenation and deoxygenation processes gives K1 = 1.5 X 10(5) M-1, deltaH = -12.4 kcal-mol-1, and deltaS = -18 e.u., values in good agreement with independent thermodynamic data. Perchlorate ion (0.05 M) enhances k-1 about 3-fold and hardly effects k1. There is no sign of other than a single reaction in either direction, and octameric hemerythrin apparently behaves kinetically as eight single units.     

Thermodynamics of glucagon aggregation.

Heats of dilution of concentrated glucagon solutions have been measured calorimetrically at 10 and 25 degrees C in 0.2 M potassium phosphate buffer of pH 10.6. Analysis of the data in terms of a monomer-trimer equilibrium gives the following thermodynamic parameters for the association reaction at 25 degrees C: delta G degrees = 7.34 kcal/mol of trimer, delta H degrees = -31.2 kcal/mol, deltaS degrees = -80 cal/(K mol), deltaCp = 430 cal/(K mol). The sensitivity of heat of dilution data to the association constant and stoichiometry of the reaction is discussed.     

Thermal and urea-induced unfolding of the marginally stable lac repressor DNA-binding domain: a model system for analysis of solute effects on protein processes

Thermodynamic and structural evidence indicates that the DNA binding domains of lac repressor (lacI) exhibit significant conformational adaptability in operator binding, and that the marginally stable helix-turn-helix (HTH) recognition element is greatly stabilized by operator binding. Here we use circular dichroism at 222 nm to quantify the thermodynamics of the urea- and thermally induced unfolding of the marginally stable lacI HTH. Van't Hoff analysis of the two-state unfolding data, highly accurate because of the large transition breadth and experimental access to the temperature of maximum stability (T(S); 6-10 degrees C), yields standard-state thermodynamic functions (deltaG(o)(obs), deltaH(o)(obs), deltaS(o)(obs), deltaC(o)(P,obs)) over the temperature range 4-40 degrees C and urea concentration range 0 相似文献   

Reactivity of cuprous stellacyanin as a quinone and semiquinone reductase

The reactivity of cuprous stellacyanin as a quinone and semiquinone reductase has been examined. Rate constants (25.0 degrees C) measured for the oxidation of stellacyanin by 1,4-benzoquinone and benzosemiquinone are 2.3 X 10(4) M-1 s-1 (delta H not equal to = 4.4 kcal/mol, delta S not equal to = -24 eu) and 5.1 X 10(6) M-1 s-1, respectively [pH 7.0, I = 0.1 M (phosphate)]. The agreement of these rate constants with those calculated on the basis of relative Marcus theory is discussed. Stellacyanin is more effective than laccase in quenching benzosemiquinone, suggesting that the physiological role of this metalloprotein is to regulate the concentration of free radicals generated through the laccase-catalyzed oxidation of phenols.     

Energetics of the equilibrium between two nucleotide-free myosin subfragment 1 states using fluorine-19 nuclear magnetic resonance

A new fluorine-containing reagent has been synthesized and used to specifically label the reactive sulfhydryl [sulfhydryl-1 (SH1)] of myosin subfragment 1 (S-1). The labeled S-1 (S-1-CF3) demonstrates activated calcium and magnesium adenosinetriphosphatase (ATPase) activities relative to S-1 and a lower potassium ethylenediaminetetraacetate (EDTA) ATPase activity. Maximal effect is obtained with the modification of one thiol per S-1. The 19F NMR spectrum of S-1 CF3 contains only one resonance with a line width of 110 Hz, which implies a rotational correlation time of 2.3 X 10(-7) s. The chemical shift of this resonance is sensitive to temperature, PH, ionic strength, and nucleotides bound in the active site. The temperature dependence of the chemical shift clearly indicates two limiting states for the S-1-CF3 with a highly temperature-dependent equilibrium between 5 and 40 degrees C. The low-temperature state appears to be identical with the state resulting from the binding of Mg.ADP or Mg.AMPPNP at 25 degree C. The energetics of the conformational change have been studied under various conditions. At pH 7 in 25 mM cacodylate, 0.1 M KCl, and 1 mM EDTA, delta H degree = 30 kcal/mol and delta S degree = 105 cal deg-1 mol-1. A decrease in pH to 6.5 results in an increased population of the low-temperature state with delta H degree = 31 kcal/mol and delta S degree = 107 cal deg-1 mol-1. Similarly, the low-temperature state is favored by low ionic strength. In 5.8 mM piperazine-N,N'bis(2-ethanesulfonic acid) and 1 mM EDTA (pH 7), delta H degree = 8 kcal/mol and delta S degree = 27 cal deg-1 mol-1. We have also obtained 19F NMR spectra of S-1-CF3 in D2O solution with 30% ethylene glycol at pH 7.1. Increasing concentrations of ethylene glycol progressively stabilize the high-temperature states.     

The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits. Kinetic and thermodynamic aspects.

Formyltetrahydrofolate synthetase monomers are converted to catalytically active tetramers in the presence of monovalent cations. The stoichiometry of the reaction is 4M + 2C+ in equilibrium M4C2(2+). A positive deltaS compensates for an unfavorable positive deltaH so that the overall reaction is exergonic. Both deltaH and deltaS become more positive as the temperature is increased. Association of subunits of the enzyme prepared from Clostridium cylindrosporum is second order with respect to monomer concentration, consistent with a rate-determining dimerization step. Activation parameters for this step at 20 degrees are: deltaG, 12.6 kcal mol-1; deltaH, 12.5 kcal mol-1; deltaS, -05 e.u. The rate-limiting step for the cation-dependent association of Clostridium acidi-urici monomers is believed to be a conformational alteration since first order kinetics is observed. The Eyring plot of the kinetic data obtained for the C. acidi-urici system has a sharp break at 15 degrees. Activation parameters for cation-induced association at 20 degrees are: deltaG, 21.5 kcal mol-1; deltaH, 14.0 kcal mol-1; deltaS, -26.6 e.u.     

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.     

Kinetic and spectral parameters of the amines oxidative N-dealkylation with participation of the liver microsomal cytochrome P-450. Amines oxidation with organic hydroperoxides

Kinetics of demethylation of a number of amines involving hepatic microsomal cytochrome P-450 and organic hydroperoxides (tret-butyl- and cumylhydroperoxide) have been investigated. Decomposition rate constants for the substrate-cytochrome P-450-ROOH complexes have been determined in a generalized form. Activation parameters, deltaH* and deltaS*, are calculated for decomposition of the complexes. There is a linear relation between deltaH* and deltaS*: deltaH*=18.7 kcal + 333 degrees K deltaS*. Compensation relationship is characterized by the value of alpha=333 degrees K/Taverage=1.11. The nature of the limiting step in the cytochrome P-450-NADPH-O2-system and the cytochrome P-450-ROOH-system is discussed.     

Temperature dependence of cholesterol binding to cytochrome P-450scc of the rat adrenal. Effect of adrenocorticotropic hormone and cycloheximide.

A type I absorbance change is observed in suspensions of adrenal cortical mitochondria as the temperature is increased from 0-22 degrees. This "heat-generated" type I absorbance change is similar in magnitude to the pregnenolone-induced type II absorbance change of these mitochondria. Studies with inhibitors of cholesterol side chain cleavage indicate that the heat-generated type I absorbance change represents the specific interaction of cytochrome P-450scc with endogenous cholesterol in the mitochondria. This finding is confirmed by low temperature EPR spectroscopy on temperature-equilibrated, quick frozen adrenal mitochondrial samples. The EPR resonance at g = 8.2, which is that of the high spin cholesterol-bound cytochrome P-450scc, is absent in the samples incubated at 0 degrees and increases in magnitude with increasing temperature of incubation. Studies of the pH dependence of the heat-generated type I and pregnenolone-induced type II absorbance changes reveal that both are diminished by increasing pH over the range 6 to 8. Adrenocorticotropic hormone (ACTH) treatment of rats results in adrenal mitochondria which show a greatly increased heat-generated type I absorbance change. The latter correlates with an increased pregnenolone-induced type II absorbance change and increased EPR g = 8.2 signal. Prior treatment of animals with cycloheximide eliminated the ACTH-induced increase in the heat-generated type I absorbance change, the pregnenolone-induced type II absorbance change and the EPR g = 8.2 signal. We estimate that the hydrophobic bonding of cholesterol to cytochrome P-450scc occurs with a deltaH0' of approximately +15 kcal/mol and a deltaS0' of approximately +55 cal/mol deg. Our data support the concept of a labile protein which participates directly in this process.     

Reduction of methemoglobin by cobaltocytochrome c catalyzed by mediators.

The reduction of methemoglobin by cobaltocytochrome c (Cocyt c) has been measured using nine mediators of different half-reduction potentials, Em, 7. The rate increases with the increase of Em, 7 for the mediator but dropped precipitously when it becomes more positive than the Em, 7 for the methemoglobin/hemoglobin couple. The reaction is most efficient with phenzaine methosulfate, therefore it was studied in detail. The reaction is first order in the concentrations of Cocyt c and phenazine methosulfate. The average second-order rate constant for Cocyt c + phenazine methosulfate (M) k1 leads to Cocyt c+ M-. is 2.9 x 10(4) M-1 s-1 at 25 degrees C, 0.1 M phosphate pH 7.0. There is a slight negative temperature dependence of k1 at low temperature; at higher temperatures the process has deltaH not equal to approximately 27 kJ mol-1 and deltaS not equal to approxmately - 75 J mol-1 K-1. The effect of anions reflects the dependence of Em, 7 for the methemoglobin/hemoglobin couple with various anions. There is no significant effect on k1 by the addition of inositol hexakisphosphate. The variation of k1 with pH is complicated. The experimental rate constants are compared with values calculated with the theory of nonadiabatic multiphonon process of electron tunneling.     

The thermodynamics of bovine and porcine insulin and proinsulin association determined by concentration difference spectroscopy

Difference spectroscopy was used to determine the equilibrium constants and thermodynamic parameters for the monomer-dimer association of bovine and porcine insulin and bovine proinsulin at pH 2.0 and 7.0. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -6.6 kcal/mol, -18 cal/mol-deg, and -12 kcal/mol, respectively. Porcine insulin behaved similarly to bovine insulin in its dimerization properties in that delta G degree 25, delta S degree, and delta H degree were found to be -6.8 kcal/mol, -14 cal/mol-deg, and -11 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -7.2 kcal/mol, -16 cal/mol/deg, and -12 kcal/mol, respectively. At pH 7.0 delta G degree 25, delta S degree, and delta H degree for dimerization of porcine insulin were -6.7 kcal/mol, -11.6 cal/mol-deg, and -10 kcal/mol, respectively. The similarity in the thermodynamic parameters of both insulin species at the different pH's suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range. The dimerization of both insulin species is under enthalpic control. This may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine proinsulin were found to be -5.3 kcal/mol, -26 cal/mol-deg, and -13 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of proinsulin were -5.9 kcal/mol, -4.2 cal/mol-deg, and -7.2 kcal/mol, respectively. Although the presence of the C-peptide on proinsulin does not drastically affect the overall free energy change of dimer formation (as compared to insulin), the other thermodynamic parameters are rather drastically altered. This may be because of electrostatic interactions of groups on the C-peptide with groups on the B-chain which are near the subunit contact site in the insulin dimer.     

Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin.

The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.     

Thermostability at ultrahigh temperatures of thermolysin and a protease from a psychrotrophic Pseudomonas.

Thermal inactivation at 110-150 degrees C of thermolysin (EC 3.4.24.4), produced by the thermophile Bacillus thermoproteolyticus, and the extracellular protease of Pseudomonas sp. MC60 a psychotroph, were investigated at 130 degrees C, both enzymes had approximately the same deltaH (22 kcal/mol) and deltaS (-13.5 cal/mol per degree) values. Both enzymes contain zinc and calcium. The amino acid compositions of the enzymes were similar except that MC60 protease exhibited a more typical tyrosine content. Comparable heat resistance at extreme temperatures of enzyme produced by psychrotrophic and thermophilic organisms emphasizes the difference between molecular properties that resist denaturation at elevated temperatures and those that allow reversible denaturation.     

Study of steroid-proteininteractions by electron spin resonance spectroscopy. Binding of a spin-labelled dihydrotestosterone to bovine serum albumin.

The interaction of bovine serum albumin with dihydrotestosterone bearing a spin label at C-3 was studied using electron spin resonance (ESR) spectroscopy. Quantitative binding parameters (Ka approximately 10(5) M-1; maximum binding capacity; two sites/mol albumin) obtained by ESR were in good agreement with those given by equilibrium dialysis. ESR study at various temperatures allowed the calculation of the thermodynamic parameters of the steroid-protein interaction: deltaG=-6.8 kcal/mol; deltaH=-7.9 kcal/mol; deltaS=-3.2 cal/mol per degree and confirmed a transition temperature of about 65 degrees C for albumin. Na, Liland Ca salts had a generally favorable effect on the interaction whereas other ions (e.g. Hg, Cu) impaired the binding process. Study of the width of the ESR spectra of the protein-bound spin-labelled steroid and extrapolation of a 2 T value to infinite viscosity (Azz coupling constant) indicated a non-polar binding site, which became increasingly hydrophobic as the temperature was raised. Since this methodology can give both pertinent quantitative and qualitative data, ESR spectroscopy should be of value in the study of steroid-protein interactions of biological significance.     

Watson-Crick base-pairing properties of bicyclo-DNA.

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.     

A calorimetric study of the CO Bohr effect of monomeric haemoglobins.

A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19 and 0.31 mol H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups. A pH-independent part of the reaction enthalpy was determined as - 19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of deltaG0 and deltaH0 values TdeltaS0 values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-1 mol-1 (8.4 JK-1 mol-1) at pH 9.5 as compared to pH 6.0.