Equilibrium constants under physiological conditions for the reactions of choline kinase and the hydrolysis of phosphorylcholine to choline and inorganic phosphate.

The observed equilibrium constants (Kobs) of the P-choline hydrolysis reaction have been determined under physiological conditions of temperature (38 degrees) and ionic strength (0.25 M) and physiological ranges of pH and free [Mg2+]. Using sigma and square brackets to indicate total concentrations: (see article.) The value of Kobs has been found to be relatively insensitive to variations in pH and free [Mg2+]. At pH 7.0 and taking the standard state of liquid water to have unit activity ([H2O] = 1), Kobs = 26.6 M at free [Mg2+] = 0 [epsilon G0obs = -2.03 kcal/mol(-8.48 kJ/mol)], 26.8 M at free [Mg2+] = 10(-3) M, and 28.4 M at free [Mg2+] = 10(-2) M. At pH 8.0, Kobs = 18.8 M at free [Mg2+] = 0, 19.2 M at free [Mg2+] = 10(-3), and 22.2 M at free [Mg2+] = 10(-2) M. These values apply only to situations where choline and Pi concentrations are both relatively low (such as the conditions found in most tissues). At higher concentrations of phosphate and choline, the value of Kobs becomes significantly increased since HPO42- complexes choline weakly (association constant = 3.3 M-1). The value of K at 38 degrees and I = 0.25 M is calculated to be 16.4 +/- 0.3 M [epsilonG0 = 1.73 kcal/mol (-7.23 kJ/mol)]. The K for the P-choline hydrolysis reaction has been combined with the K for the ATP hydrolysis reaction determined previously under physiological conditions to calculate a value of 4.95 X 10(-3 M [deltaG0 j.28 kcal/mol (13.7 kJ/mol] for the K of the choline kinase reaction (EC 2.7.1.32), an important step in phospholipid metabolism: (see article.) Likewise, values for Kobs for the choline kinase reaction at 38 degrees, pH 7.0, and I = 0.25 M have been calculated to be 5.76 X 10(4) [deltaG0OBS = -6.77 KCAL/MOL (-28.3 KJ/mol)] at [Mg2+] = 0; 1.24 X 10(4) [deltaG0obs = -5.82 kcal/mol (-24.4 kJ/mol)] at [Mg2+] = 10(-3) M and 8.05 X 10(3) [delta G0obs = -5.56 kcal/mol (-23.3 kJ/mol)] at [Mg2+ = 10(-2) M. Attempts to determine the Kobs of the choline kinase reaction directly were unsuccessful because of the high value of the constant. The results indicate that in contrast to the high deltaG0obs for the hydrolysis of the ester bond of acetylcholine, the deltaG0obs for the hydrolysis of the ester bond of P-choline is quite low, among the lowest known for phosphate ester bonds of biological interest.     

Equilibrium constants of the reactions of choline acetyltransferase, carnitine acetyltransferase, and acetylcholinesterase under physiological conditions.

The observed equilibrium constant (Kobs) for the reaction of choline acetyltransferase (EC 2.3.1.6) has been determined under physiological conditions. Using sigma and square brackets to indicate total concentrations of all ionic species present: (see article). The value of Kobs has been determined to be 12.3 plus or minus 0.6 at 38 degrees, pH 7.0 and ionic strength 0.25 M. The value at 25 degrees is not significantly different, and the constant has been found to be insensitive to variations in ionic strength (0.03 to 0.375 M), pH (6.5 TO 7.5) OR FREE [Mg-2+] (0 to 5 mM). The Kobs of this reaction reflects the difference between the observed standard free energy change (delta G-oobs) for the hydrolysis of acetylcholine and the delta G-oobs for the hydrolysis of acetyl-CoA. Since the delta G-oobs for the hydrolysis of acetyl-CoA has been previously determined to be minus 8.54 kcal/mol (minus 35.75 kJ/mol under the same physiological conditions, the delta G-oobs for the reaction of acetylcholinesterase (EC 3.1.1.7): (SEE ARTICLE). Can be calculated to be minus 6.99 kcal/mol (minus 29.26 kJ/mol) at pH ionic strength 0.25 M and 38 degrees, taking the standard state of liquid water to have unit activity ([H2O] equals 1). The pKa for acetic acid under the same conditions, has been determined to be 4.60 plus or minus 0.01, allowing the Kobs for the pH-independent reaction (see article). To be calculated to be 3.28 times 10-2 M. Choline and carnitine are chemical analogues. The Kobs for the corresponding reaction of carnitine acetyltransferase (EC 2.3.1.7). (SEE ARTICLE). Under the same physiological conditions of pH (7.0), ionic strength (0.25 M), and temperature (38 degrees) has been determined to be 1.73 plus or minus 0.05, making the delta G-oobs for the hydrolysis of acetylcholine only 1.21 kcal/mol (5.06 kJ) less negative than that for the hydrolysis of acetylcarnitine.     

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.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Thermal inactivation and stabilization of lysozyme substrate-- Micrococcus lysodeicticus cells

Heat inactivation of the acetonic powder of Micrococcus lysodeicticus cells suspended in phosphate buffer pH 6.2 was quantitatively characterized in the temperature range from 34 to 52 degrees. The total value of the rate constant for heat inactivation of the cells equals 2.88 X 10(8) exp(-18360/RT) sec-1. The activation parameters of the process at 34 degrees are the following: delta H* = 17.7 kcal/mole; delta S* = 21.8 E. U.; delta F* = 24.4 kcal/mole. The effect of ethylene glycol, mannitol, dextran, polyvinyl alcohol (PVA) and polyethylene glycols with different molecular weights on the lysis rate and cell stability was studied. Polyvinyl alcohol was found to be the most effective stabilizer. At concentrations of about 10(-5) it enhances the thermostability of the cells threefold.     

Inhibition of lactate and malate dehydrogenase by dyes containing vinylsulfone and chlorotriazine groups

The difference spectra of lactate and malate dehydrogenase complexes with four native dyes containing vinylsulfonic and triazinic groups (light-resistant yellow 2KT, red-violet 2KT, etc.) were monitored in 0.1 M phosphate buffer pH 8.2 at 20 degrees C. The dissociation constants were calculated from the spectral data. The most stable complexes were lactate dehydrogenase--light-resistant yellow 2KT and malate dehydrogenase--light-resistant yellow 2KT ones. The values of delta H degree = 5.75 kcal/mole and standard thermodynamic parameters, delta G degree = -6.5 kcal/mole and delta S degree = 41.2 e. u., were calculated from the values of association constants for temperature dependence. The thermodynamic characteristics confirmed the key role of hydrophobic interactions in lactate dehydrogenase--reactive dye complex formation. All the dyes under study competitively inhibit lactate and malate oxidation by the corresponding dehydrogenases. The inhibition constants of both enzymes by the four dyes were determined at 20 degrees C in 0.1 M phosphate buffer pH 8.2. Light-resistant yellow 2KT appeared to be the most effective inhibitor of the enzymes.     

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.     

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.     

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.     

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.     

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.     

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.     

Thermodynamics of complex formation between nicotinamide adenine dinucleotide and pig skeletal muscle lactate dehydrogenase.

Formation of the binary complex between the reduced coenzyme nicotinamide adenine dinucleotide (NADH) and pig skeletal muscle lactate dehydrogenase (LDH, EC 1.1.1.27) has been investigated by calorimetric and equilibrium dialysis techniques in 0.2 M potassium phosphate buffer (pH 7.0) at various temperatures. Analysis of thermal titration curves at two temperatures (25 and 31.5 degrees) shows that the experimental enthalpy data can be rationalized assuming four independent and equivalent binding sites for the tetrameric enzyme. Binary complex formation is characterized by a negative temperature coefficient, delta cp, of the binding enthalpy, which amounts to -1300 plus or minus 53 cal/(deg mol of LDH) in the temperature range of 5-31.5 degrees. Despite the slightly smaller standard deviation resulting when polynomial regression analysis of the second degree is applied to the temperature dependence of the enthalpy values, binding enthalpies seem to be adequately represented in the temperature range studied by the equation delta H = -1.3T + 2.3, kcal/mol of LDH, T referring to the temperature in degrees C. By combination of the results obtained from equilibrium dialysis and calorimetric studies a set of apparent thermodynamic parameters for binding of NADH to LDH in 0.2 M potassium phosphate buffer at pH 7 has been established.     

Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction

A filter retention assay is used to examine the kinetic and equilibrium properties of the interaction between phage R17 coat protein and its 21-nucleotide RNA binding site. The kinetics of the reaction are consistent with the equilibrium association constant and indicate a diffusion-controlled reaction. The temperature dependence of Ka gives delta H = -19 kcal/mol. This large favorable delta H is partially offset by a delta S = -30 cal mol-1 deg-1 to give a delta G = -11 kcal/mol at 2 degrees C in 0.19 M salt. The binding reaction has a pH optimum centered around pH 8.5, but pH has no effect on delta H. While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions. The ionic strength dependence of Ka reveals that ionic contacts contribute to the interaction. Most of the binding free energy, however, is a result of nonelectrostatic interactions.     

Unusual enthalpy changes which accompany the titration of dimyristoylphosphatidylcholine vesicles with Triton X-100

The enthalpy changes which accompany the titration of 0.1% and 0.25% small unilamellar and multiameller vesicle samples of dimyristoylphosphatidylcholine with 2% Triton X-100 in 0.067 M phosphate buffer (pH 7.4) containing 0.15 M NaCl have been determined by titration calorimetry at 21 degrees C and 28 degrees C, the enthalpy change for both type of vesicles was zero within the limits of experimental error. At 21 degrees C, the multilamellar vesicle samples exhibited an enthalpy change of 1.35 +/- 0.48 and 2.47 +/- 0.98 kcal/mol dimyristoylphosphatidylcholine which was complete at a molar ratio of dimyristoylphosphatidylcholine to Triton of 3.21 +/- 0.84 and 5.77 +/- 1.05 for 0.1% and 0.25% dimyristoylphosphatidylcholine solutions, respectively. An exothermic transition of -2.39 +/- 0.30 and -2.05 +/- 0.69 kcal/mol phospholipid followed by an endothermic transition of 1.37 +/- 0.12 and 1.94 +/- 0.20 kcal/mol dimyristoylphosphatidylcholine was observed at 21 degrees C for 0.1% and 0.25% small unilamellar vesicle samples, respectively. In addition the nearly athermal association of the small unilemellar vesicle samples at 21 degrees C was observed, which may be an appropriate model for biological membrane fusion.     

NMR study of the alkaline isomerization of ferricytochrome c

The pH-induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D2O with a pKa measured as 9.5 +/- 0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0 +/- 0.6 s-1 at 27 degrees C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are delta H not equal to = 12.8 +/- 0.8 kcal.mol1, delta S not equal to = -12.9 +/- 2.0 e.u. for the forward reaction and delta H not equal to = 6.0 +/- 0.3 kcal.mol-1, delta S not equal to = -35.1 +/- 1.3 e.u. for the reverse reaction (pH* = 9.28). delta H degree and delta S degree for the isomerization are 6.7 +/- 0.6 kcal.mol-1 and 21.9 +/- 1.0 e.u., respectively.     

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)     

Physicochemical studies of processes involving potential photodynamic drugs on route to their targets: self-aggregation and membrane-binding of Zn-hematoporphyrin

The thermodynamics of zinc hematoporphyrin (ZnHP) dimerization and ZnHP-membrane binding were studied. The dimerization equilibrium was determined over the temperature range 19-40 degrees C, using fluorometric techniques. The dimerization constant obtained at 37 degrees C (neutral pH in phosphate-buffered saline) is 4.6 (+/- 0.6) X 10(4) M-1. The dimerization was found to decrease with temperature over the range 19-36 degrees C, the data allowing the extraction of the following thermodynamic parameters for the temperature range 19-31 degrees C: delta G0 = -9.3 kcal/mol, delta H0 = -7.4 kcal/mol, delta S0 = -6.4 eu. For temperatures above 36 degrees C the dimerization was found to be temperature independent, giving the following parameters: delta G0 = -6.6 kcal/mol, delta H0 = 0 kcal/mol, delta S0 = 21.2 eu. On the basis of the data the case is made for the existence of two types of ZnHP dimers, differing in the location of the fifth Zn2+ ligand and in the nature of the contribution of the solvent to the dimerization. For the membrane binding, large unilamellar liposomes served to model biological membranes. The binding of ZnHP to the liposomes was found to be similar, quantitatively, to the corresponding metal-free molecule, namely, fitting a case of one type of site and giving a binding constant of 1600 +/- 160 M (neutral pH and 37 degrees C) which is independent of the length of the porphyrin-liposome.     

Thermodynamic parameters of cytochrome c3-ferredoxin complex formation

The complex formation between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway was studied by microcalorimetric and pH-stat titration measurements. The stoichiometry of the complex was found to be one molecule of cytochrome c3 per monomer of ferredoxin I. The association constant determined at T = 283 K in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, 10(-2) M and pH 7.7, was KA = 1.3 X 10(6) M-1. Though the enthalpy (delta H = 19 +/- 1 kJ.mol-1) and the entropy (delta S = 183 J.K-1.mol-1) were positive and consistent with a hydrophobic process involved in the interaction, the analysis of ionic strength dependence exhibited an important electrostatic effect on the association. The use of both Tris-HCl and phosphate buffers during microcalorimetric experiments showed proton release at pH 6.6. The pH-stat study of proton release indicated that one of the charged groups involved in the interacting site underwent a pK shift from 7.35 to 6.05.     

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.