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.     

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)     

Conformational changes required for pyruvate kinase activity as modulated by monovalent cations.

The interaction of a series of alkylamines with muscle pyruvate kinase was investigated by kinetic and physical studies in order to understand the mechanisms by which certain monovalent cations can activate the enzyme and to define several of the important conformational changes necessary for catalytic activity. Monomethylammonium ion interacts with pyruvate kinase to activate the enzyme. Dimethyland trimethylammonium ions do not activate, but are competitive inhibitors against activating cations. Tetramethylammonium ion neither activates nor inhibits pyruvate kinase activity. When the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy. This conformational change is similar to that observed with other activating cations and appears to be a necessary but no sufficient conformational change in the formation of an active complex. The interaction of the substrate phosphoenolpyruvate with the pyruvate kinase-Mn2+ complex in the presence of these cations was studied by water proton relaxation rate measurements. The affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate is decreased by a factor of 5 in the presence of any of the alkylamines compared to the affinity measured in the presence of K+ or NH4+. No change in the Km of phosphoenolpyruvate is observed however when it is measured in the presence of monomethylammonium ion, suggesting that the decrease in affinity for the substrate is not the reason for lack of enzymic activity. The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex about the bound Mn2+, as reflected by the enhancement values (epsilont) measured, differs depending upon the nature of the monovalent cation. The epsilon t values measured in the presence of the alkylamines are larger (epsilont - 5.7 +/- 0.2) than those measured in the presence of K+ or NH4+ (epsilont = 1.9 +/- 0.1).     

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.     

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.     

Temperature dependence of the dissociation rate constants for the nonactivated (molybdate-stabilized) and activated progesterone receptor-hormone complexes from the chick oviduct

Both the nonactivated and activated forms of the chick oviduct cytosol progesterone receptor-hormone complexes displayed first-order dissociation kinetics at temperatures between 0 and 25 degrees C. The rate constant was always 2-3-times greater for the nonactivated than for the activated complex. The thermodynamic parameters calculated from the Eyring plot for the nonactivated and activated forms, respectively, were: delta H+ = 28.6 +/- 0.2 and 29.9 +/- 1.5 kcal/mol; -T delta S+ = 7.4 +/- 0.6 and 7.7 +/- 1.6 kcal/mol; and delta G+ = 21.3 +/- 0.5 and 22.1 +/- 0.1 kcal/mol. These values suggest that activation results in an increase in enthalpy of the ligand-receptor interaction, thus stabilizing the complex. The dissociation rate constants for the native complex obtained by two different experimental approaches, namely, isotope dilution ('chase') and dissociation against charcoal, indicated the absence of cooperativity in the receptor-ligand binding.     

Phosphorus-31 nuclear relaxation rate studies of the nucleotides on phosphoenolpyruvate carboxykinase

The interactions of nucleotide substrates with the enzyme phosphoenolpyruvate carboxykinase and its Mn2+ complex were investigated by several methods. Direct binding shows the formation of stoichiometric complexes. The presence of Mn2+ increases the affinity of the enzyme for nucleotide. A higher affinity for GTP (Kd less than 2 microM) than for GDP (Kd = 15 microM) was determined. Solvent proton relaxation rate studies indicate no substantial difference in titration curves for free nucleotide or for Mg-nucleotide to the enzyme-Mn complex. The effect of Mn2+ on the 31P relaxation rates of IDP and of ITP in the binary Mn-nucleotide complex indicates the formation of direct coordination complexes. The distances of the alpha- and beta-31P of IDP to Mn2+ are identical (3.5 A). The Mn2+ distance to the beta- and gamma-31P of ITP is also identical (3.7 A) and is 0.2 A further from the alpha-phosphorus. In the presence of P-enolpyruvate carboxykinase, the effect of Mn2+ on the 31P relaxation rates was measured at 40.5 MHz and at 121.5 MHz. The dipolar correlation time was calculated to be 0.6-5.4 ns, depending upon assumptions made. The Mn2+ to phosphorus distances indicate the nucleotide substrates form a second sphere complex to the bound Mn2+. From 1/T2 measurements, electron delocalization from Mn2+ to the phosphorus atoms is indicated; this effect occurs although direct coordination does not take place. The exchange rate of GTP from the enzyme-Mn complex (koff = 4 X 10(4) s-1) is rapid compared to kcat with a lower energy of activation (9.2 kcal/mol) than for catalytic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Biochemical characterization of positive cooperativity in the binding of 1 alpha, 25-dihydroxyvitamin D3 to its chick intestinal chromatin receptor

We have characterized a positive cooperativity mechanism in the binding of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to its chick duodenum chromatin receptor. The Hill plot which can take account of the possibility of cooperativity resulted in a much better fitting of the experimental data than the Scatchard model (r = +0.998 versus r = -0.94). Concentrating the chromatin receptor preparation from 10 to 40% resulted in an increase of the Hill coefficient (nH) from 1.09 +/- 0.08 to 1.46 +/- 0.08 (S.D.). Increasing the temperature of incubation from 1 degree C to 40 degrees C resulted in a decrease of nH from 1.46 +/- 0.08 to 1.10 +/- 0.02 (S.D.). The calculation of the thermodynamics of the interaction of 1,25-(OH)2D3 with the second binding site of the receptor (from a Van't Hoff plot) showed that this process occurred spontaneously (delta G0 = -11.6 kcal X mol-1 at 1 degree C), was entropy-driven (delta S0 = +26 cal degree-1 mol-1), and was energy-requiring (delta H0 = -4.37 kcal X mol-1). The temperature controlled reversibility of the cooperativity demonstrates that this phenomenon is not an artifact. Finally, in a study of the rate of dissociation of [3H]1,25-(OH)2D3 from the duodenal receptor preparation, we have found two slopes (k-1 = 32 X 10(-3) min-1; k-2 = 3.2 X 10(-3) min-1); this suggests the existence of two species of receptor. These receptor species could result possibly from either a monomer-dimer system or from a conformational change of a monomer via site-site interactions. In conclusion, the positive cooperativity in the binding of 1,25-(OH)2D3 to the two binding sites of its intestinal receptor is an entropy-driven process and requires energy, is reversible with temperature, and has been shown to take place in concentrated chromatin aggregates.     

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.     

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.     

Thermodynamic analysis of inducer binding to the lactose repressor protein: contributions of galactosyl hydroxyl groups and beta substituents

Kinetic and equilibrium studies of the binding of modified beta-D-galactoside sugars to the lac repressor were carried out to generate thermodynamic data for protein-inducer interactions. The energetic contributions of the galactosyl hydroxyl groups to binding were assessed by using a series of methyl deoxyfluoro-beta-D-galactosides. The C-3 and C-6 hydroxyls contributed less than or equal to -2.3 and -1.7 +/- 0.3 kcal/mol to the binding free energy change, respectively, whereas the C-4 hydroxyl provided only a nominal contribution (-0.1 +/- 0.2 kcal/mol). Favorable contributions to the total binding free energy change were observed for replacement of O-methyl by S-methyl at the beta-anomeric position and for S-methyl by S-isopropyl. Negative delta H degrees values characteristic of protein-sugar complexes [Quiocho, F. A. (1986) Annu. Rev. Biochem. 55, 287-315] were observed for a series of beta-D-galactosides differing at the beta-glycosidic position. A decrease in delta H degrees of approximately 6 kcal/mol upon replacement of the O-methyl substituent by S-methyl indicates a substantial increase in van der Waals' interactions and/or hydrogen bonding in this region of the ligand binding site. The more favorable free energy change for the binding of the S-isopropyl vs S-methyl compound is due mainly to more positive entropic contributions, consistent with an increase in apolar interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselectivity of interaction of phosphoenolpyruvate analogues with various phosphoenolpyruvate-utilizing enzymes

The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.     

Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

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.     

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.     

Cyanate binding to the ferric heme octapeptide from cytochrome c. A model for anion binding to high spin ferric hemoproteins

Equilibrium constants for the binding of cyanate to the ferric heme c octapeptide in 50% ethylene glycol, 50% aqueous buffer were measured spectrophotometrically. Equilibrium constants measured at several temperatures from -20 degrees C to 0 degrees C exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta Ho = -1.3 X 10(3) +/- 0.9 X 10(3) J/mol (-3.1 X 10(2) +/- 2 X 10(2) cal/mol), delta So = -3 +/- 3 J/K X mol (-0.6 +/- 0.8 cal/K X mol). The equilibrium constant for cyanate binding at 25 degrees C and pH 7.4 is 1.21 which is approximately 2 to 3 orders of magnitude lower than that observed for cyanate binding to methemoglobin and metmyoglobin. Krel, the ratio of the hemoprotein to model heme octapeptide binding constants, for NCO- is smaller than Krel for N3- suggesting that hydrogen bonding between the terminal ligand atoms and the distal histidine in hemoglobin and myoglobin does not contribute to the increased protein ligand stabilization observed for these anions relative to the model. A donor-acceptor interaction between the distal histidine and the electrophilic middle atoms of these bound ligands is proposed.     

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues.     

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.     

Flanking DNA-sequences contribute to the specific binding of cI-repressor and OR1.

The binding of cI-repressor to a series of mutant operators containing OR1 of the right operator of bacteriophage lambda was investigated. Sites OR2 and/or OR3 were inactivated by either point or deletion mutations. The free energy of binding repressor to OR1 in the wildtype operator, delta G1, is -13.7 +/- 0.3 kcal/mol. delta G1 determined for an OR2- operator created by a single point mutation in OR2 is -13.6 +/- 0.2 kcal/mol. In contrast, delta G1 for the binding of repressor to a cloned synthetic OR1 operator containing only 24 bp of lambda sequence is -12.2 +/- 0.1 kcal/mol. When sequence 5' to OR1 is present, the binding affinity increases to -13.0 +/- 0.1 kcal/mol. In addition, the proximity of OR1 to a fragment-end decreases delta G1 from -13.7 to -12.3 +/- 0.1 kcal/mol. These results suggest that the DNA sequence outside the 17 bp OR1 binding-site contributes to the specific binding of cI-repressor.