Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: I. Autoassociation of 2-aminopurine

A general equation was derived, describing fluorescence quantum yield and lifetime of an autoassociating compound in liquid solutions. The autoassociation of 2-aminopurine in aqueous solution was examined within the range from 0 to 90°C. The compound seemed to associate cooperatively. The thermodynamic parameters of polymerization change with temperature, so that its free enthalpy ΔG = ?0.0797 T² + 45.4 T ?7893. The dimerization enthalpy and entropy are approximately temperature-independent (ΔH₂ = ?4.17 $\text{kcal}\text{mol}$, ΔS₂ = ?10.9 e.u.), although the function: ΔG₂ = ?0.0308 T² + 30.3 T - 7213 fits experimental points better. The observed dependences can be explained by the increasing role of the hydrophobic effect with temperature and size of the aggregates. The association rate constants were determined, and a two-step reaction mechanism was demonstrated. The first step is diffusion-controlled. The second is characterized by an activation energy of ~2 $\text{kcal}\text{mol}$ and an encounter distance of ~8.3 Å.     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: II. Heteroassociation between 2-aminopurine and thymidine

Heteroassociation between A and B compounds in liquid solution was considered. Provided that concentration of A molecules is low, a geneial equation describing fluorescence quantum yield and lifetime of compound A as a function of B molecules concentration was derived. The heteroassociation between 2-aminopurine and thymidine in aqueous solutions was examined within the range of temperatures 0 to 90° C. The equilibrium constants of the first step of association, namely heterodimer formation, were determined and its thermodynamic parameters (ΔH = ?2.76 $\text{kcal}\text{mol}$, ΔS = ?5.9 e.u.) were calculated. The observed changes of the stacking rate constants with temperature confirm the two-step mechanism of the reaction. The activation energy (~2.7 $\text{kcal}\text{mol}$) and the encounter distance (~10.7 A) are only slightly larger than in the case of 2-aminopurine autoassociation, most probably because of a stronger solvation of thymidine molecules.     

Fluctuation of the lysozyme structure

The kinetics of hydrogen-deuterium exchange in hen egg-white lysozyme (muramidase) has been followed in aqueous solutions of various pH values and in solutions with various concentrations of lithium chloride, by an infrared absorption measurement. It was found that, in every case, 34% of the total peptide hydrogen atoms exchange relatively slowly with a rate of a first-order reaction. This amount corresponds to 44 peptide groups per molecule, and this is equal to the number of peptide NH-groups which are found to be involved in hydrogen bonds with the carbonyls of other peptide groups in the lysozyme molecule in the crystalline state. Each rate constant determined is in good agreement with the value expected from two simple assumptions. (1) The scheme of the isotope exchange reaction is N ? D → D^∗ (? N^∗), where N is the native form of the molecule, D a denatured (unfolded) form, and ∗ indicates the deuterated products. (2) The N ? D fluctuation rate is much higher than the rate of the isotope exchange reaction D → D^∗. It has been shown that the N ? D transition postulated here is the same as that which can be followed by circular dichroism measurement and by some other physical measurements. The effect of lithium chloride on the exchange reaction rate is solely attributable to the change in the N ? D equilibrium caused by the salt, whereas the effect of pH (in the 5 to 8 range) is wholly ascribed to the catalytic action of the OH⁻ anion on the D → D^∗ reaction rate. From the deuterium exchange rate observed, an effective value of the mole fraction of the D form is estimated to be 3 × 10⁻⁶in the solution with no lithium chloride at 20 °C and of pH = 5 to 8.     

Comparison of the structures of the native form of rat liver 5S rRNA and yeast tRNAphe: small-angle and wide-angle X-ray scattering study

The small-angle and wide-angle X-ray scattering of tRNA^phe (yeast) and ribosomal 5S RNA (rat liver) in solution have been analysed and compared. tRNA^phe in solution is folded into a compact L-shaped structure similar to its structure in crystals. The geometry of the secondary structure of the double helical regions is also equivalent to the A-form in the crystalline state. Despite differences between the molar mosses of 5S rRNA (40 000 g mol^?1) and tRNA^phe (25 000 g mol^?1), and the fact that the 5S rRNA molecule is more anisometric than the tRNA^phe molecule, there are many structural similarities. The geometrical parameters of the secondary structure of double helical regions in both RNA molecules are almost identical; the mean rise per base pair is about 0.253–0.28 nm and the mean turn angle is about 32.5–33.5. Identical cross-sectional radii of gyration, R_sq,1 ≈ 1.16 nm and R_sq,2 = 0.92 nm, identical molar mass per unit length, , and a mean thickness of the molecules D ≈ 1.65 nm suggest a similar, nearly coplanar organization of isolated, double helical arms. Furthermore, there are compact regions in the central parts of both molecules, which are the sites of tertiary interactions in the tRNA^phe molecule and are a potential site of tertiary interactions in the SS rRNA molecule for stabilization of the complicated L-shape of the two molecules. Both molecules have a pseudo-twofold axis,w hich may play a role in recognition for binding of specific proteins.     

Thermal behavior of bovine β-trypsin at physiological temperature range

The effects of temperature on the steady-state kinetics of β-trypsin hydrolysis of α-Ntosyl-l-arginine methyl ester (k_cat, K_m) and its inhibition by phenylguanidinium ion (K_i) were studied in the temperature range 27–37 °C, at 1 °C intervals, pH 8.0. Within this temperature range inhibition of β-trypsin by phenylguanidine was strictly competitive. The Eyring and van't Hoff plots were nonlinear; interpretation of the data was based on two possible alternatives: in the first, there occurs a thermal transition centered at 31 °C, characterized by ΔH° = 42.2 ± 8.7 kcal/mol and ΔS ° = 138 ± 29 e.u. According to the second interpretation the phenomenon would be determined by a large value of Δ Cp; its value was estimated to be ΔCp = ?7192 cal/deg · mol. A decision as to what interpretation is more adequate must wait until further experimental information is obtained.     

Increase in temperature induces the Z to B transition of poly[d(G-C)] in water-ethanol solution

An increase in temperature from 20 to 50° C results in the complete transition from the Z to B form of poly(d(G-C)], dissolved in a 55% ethanol-water solution. The transition is fully reversible and displays a slow kinetics. The transition profiles for the free polynucleotide and for that in the presence of ethidium bromide, which is known to stabilize the B form, are obtained by circular dichroism. Based on these data the enthalpy value for the B-Z transition in our conditions is estimated to be ΔH_BZ = ?0.7 $\text{kcal}\text{mol}$.     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

Dissociation of pig liver carboxylesterase measured by quantitative micro-complement fixation

Rate and apparent equilibrium constants for the dissociation of pig liver carboxylesterase into three subunit molecules have been determined by complement fixation. The dependence of the dissociation equilibria on pH are consistent with dissociation reactions involving the addition of two protons per subunit, a pH-independent dissociation, and a dissociation upon the loss of one proton per subunit. The rate constants for dissociation are consistent with terms first order in hydrogen and hydroxide ions and a pH-independent path. The equilibrium constants in the range 3–35 °C at pH 7.2 exhibit no dependence on temperature; the association reaction is entropy driven with $\text{ΔS = 68}\text{cal mol}{}^{\mathrm{?1}}{}^{\mathrm{°}}\text{K}{}^{\mathrm{?1}}$. The rate constants for the pH-independent dissociation follow ΔH^≠ ? 6 kcal mol^?1. The order of effectiveness of concentrated salts in promoting denaturation is correlated with their effect on the activity coefficient of acetyltetraglycine ethyl ester and suggests that peptide groups become more exposed upon dissociation. The increased dissociation in the presence of urea derivatives containing alkyl substituents suggests exposure of hydrophobic regions upon dissociation; this is also consistent with ΔH = 0 for dissociation. It is likely that hydrophobic interactions contribute to the stability of the trimeric whole molecule.     

Interfacial adsorption of an inhalation anesthetic onto ionic surfactant micelles and its desorption by high pressure

The effects of pressure and temperature on the critical micelle concentration (CMC) of sodium dodecylsulfate (SDS) were measured in the presence of various concentrations of an inhalation anesthetic, methoxyflurane. The change in the partial molal volume of SDS on micellization, $\text{Δ}\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, increased with the increase in the concentration of methoxyflurane. The CMC-decreasing power, which is defined as the slope of the linear plot between ln(CMC) vs. mole fraction of anesthetic, was determined as a function of pressure and temperature. Since the CMC-decreasing power is correlated to the micelle/water partition coefficient of anesthetic, the volume change of the transfer (ΔV_p^o) of methoxyflurane from water to the micelle can be determined from the pressure dependence of the CMC-decreasing power. The value of ΔV_p^o amounts 6.5±1.8 cm³·mol^?1, which is in reasonable agreement with the volume change determined directly from the density data, 5.5±0.6 cm³ · mol^?1. Under the convention of thermodynamics, this indicates that the application of pressure squeezes out anesthetic molecules from the micelle. The transfer enthalpy of anesthetic from water to the micelle is slightly endothermic. The partial molal volume of methoxyflurane in the micelle (112.0 cm³·mol^?1) is smaller than that in decane (120.5 cm³·mol^?1) and is larger than that in water (108.0 cm³·mol^?1). This indicates that the anesthetic molecules are incorporated into the micellar surface region, i.e., the palisade layer of the micelle in contact with water molecules, rather than into the micelle core.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide: A7U7

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A₇U₇ are reported over a concentration range of 6.9 μM to 19.6 μM in single strand in 1 M NaCl. The rate constants for helix formation are about 2 × 10⁶ M^?1 s^?1 and decrease with increasing temperature yielding an activation enthalpy of ?6 $\text{kcal}\text{mole}$. The rate constants for helix dissociation range from 3 to 250 s^?1 and increase with increasing temperature yielding an activation enthalpy of +45 $\text{kcal}\text{mole}$. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.     

Structural dynamics of bacterial ribosomes: V. Magnesium-dependent dissociation of tight couples into subunits: Measurements of dissociation constants and exchange rates

The magnesium ion-dependent equilibrium of vacant ribosome couples with their subunits

$\text{70}\text{S}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{50}\text{S}\text{+30}\text{S}$

has been studied quantitatively with a novel equilibrium displacement labeling method which is more sensitive and precise than light-scattering. At a concentration of 10^?7m, tight couples (ribosomes most active in protein synthesis) dissociate between 1 and 3 mm-Mg²⁺ at 37 °C with a 50% point at 1.9 mm. The corresponding association constants K_a′ are 5.1 × 10⁵m^?1 (1 mm-Mg²⁺), 3.5 × 10⁷m^?1 (2 mm), and 1.2 × 10⁹m^?1 (3 mm), about five orders of magnitude higher than the K_a′ value of loose couples studied by Spirin et al. (1971) and Zitomer & Flaks (1972).In this range of Mg²⁺ concentrations ($\text{37 °C, 50 mm-}\text{NH}{}_4{}^{\mathrm{+}}$) the rate constants depend exponentially and in opposite ways on the Mg²⁺ concentration: k₁ = 2.2 × 10^?3s^?1, k_?1 = 7.7 × 10⁴m^?1s^?1 (2mm-Mg²⁺); k₁ = 1.5 × 10^?4s^?1, k_?1 = 1.7 × 10⁷m^?1s^?1 (5 mm-Mg²⁺). Under physiological conditions (Mg²⁺ ～- 4 mm, ribosome concn ～- 10^?7m), the equilibrium strongly favors association and the rate of exchange is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10}\text{min}$). In the range of dissociation (2 mm-Mg²⁺), association of subunits proceeds without measurable entropy change and hence ΔG^O = ΔH^O. The negative enthalpy change of ΔH^O = ? 10 kcal suggests that association of subunits involves a shape change.Below a critical Mg²⁺ concentration (～- 2 mm), the 50 S subunits are converted irreversibly into the b-form responsible for the transition to loose couples. The results are compatible with two classes of binding sites, one class binding Mg²⁺ non-co-operatively and contributing to the free energy of association by reduction of electrostatic repulsion, and another class probably consisting of hydrogen bonds between components at opposite interfaces whose critical spatial alignment rapidly denatures in the absence of stabilizing magnesium ions.     

Osmotic pressure method to measure salt induced folding/unfolding of bovine serum albumin

A new approach has been developed to monitor protein folding by utilizing osmotic pressure and a range of salt concentrations in a well characterized protein, bovine serum albumin (BSA). It is hypothesized that both the ‘effective’ osmotic molecular weight, A_e, and the solute/solvent interaction parameter, I, in the empirical relation $\text{M}{}_{\mathrm{solvent}}\text{M}{}_{\mathrm{solute}}\text{= (}\text{RTϱ}\text{A}{}_{\mathrm{e}}\text{)}\text{1}\text{gp}\text{+ I}$ [1] can be used as measures of protein folding. I is a measure of solvent perturbed by the solute and is thought to depend directly upon the solvent accessible surface area (ASA). It is reasoned that larger solvent accessible surface area of an unfolded or denatured protein should perturb more water and produce larger I-values. Thus I-values allow calculation of a unfolded protein fraction, f_u^a, due to changes in relative solvent accessible surface area. It has been observed that A_e decreases for filamentous, denatured proteins due to segmental motion of the molecule [2]. This allows calculation of unfolded protein fraction from the effective molecular weight, f_u^m. Colloid osmotic pressure of BSA was measured in a range of salt concentrations at 25°C, and pH = 7 (above the isoelectric point of BSA at pH = 5.4). Both S and I were used to monitor protein folding as the salt concentration was varied. In general, larger and variable I-values and smaller A_e were observed at salt concentrations less than 50 mmolal NaCl (I_max = 8.9), while constant I = 4.1 and A_e = 66,500 were observed above 50 mmolal NaCl. The two expressions for fractional unfolding (f_u^a and f_u^m) are in general agreement. Small differences in the parameters below 50 mmolal salt concentration are explained with well known shifts in the relative amounts of α-helix, β-sheet and random coil in denatured BSA. The relative amounts of these shifts agree with predictions in the literature attributed to continuous BSA expansion rather than an ‘all-or-none’ conversion.     

Calorimetric investigations of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid

The enthalpy of the helix-coil conversion of phenylalaninespecific transfer ribonucleic acid from brewer's yeast (tRNA^Phe_{brewer's yeast}) has been measured using both an LKB 10700-2 batch miciocalorimeter and an adiabatic differential scanning calorimeter. In the mixing calorimeter the conversion from coil to helix was induced by mixing a tRNA^Phe solution with a solution containing an excess of MgSO₄. We measured the enthalpy of this reaction stepwise in the temperature range from +9 to +60° C. For the enthalpy of folding of tRNA^Phe from coil to helix this method yielded the remarkably high value of ?310 $\text{kcal}\text{mole}$ of tRNA^Phe. With the differential scanning calorimeter in which the helix-coil conversion is simply induced by raising the temperature we found a value of +240 $\text{kcal}\text{mole}$ of tRNA^Phe at a Tm value of 76° C and a value of +200 $\text{kcal}\text{mole}$ of tRNA^Phe at a T_m value of 50° C. A comparison of the apparent van't Hoff enthalpies with the calorimetrically measured enthalpies shows, that the cooperativity of the system increases continually with rising melting temperatures - which are achieved by increasing Mg²⁺ concentrations - reaching a constant value at about 57° C. Above this temperature value the thermodynamic behaviour of the helix-coil conversion of tRNA^Phe may be approximately described by the model of an all-or-none process.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: III. Intramolecular association of 9,9'-[1,3-propylene]-bis-2-aminopurine

General equations relating fluorescence quantum yield and lifetime of a compound with its intramolecular stacking equilibrium and kinetics were derived. Intramolecular stacking association of 9,9'-[1,3-propylene]-bis-2-aminopurine in aqueous solution was examined within the range of temperatures from 0 to 90°C. A two-state thermodynamic model of the association was verified. The stacking enthalpy and entropy can be taken, with a good approximation, as temperature-independent (δH = ?2.0 $\text{kcal}\text{mol}$, ΔS = ?3.25 e.u.) although the function ΔG = ?0.00886 T² + 8.847 T ?2876 describes more precisely the observed changes of stacking free enthalpy with temperature. The association rate constants were determined. Activation energy of the reaction (2 $\text{kcal}\text{mol}$) is the same as in the case of association between free 2-aminopurine molecules. It confirms a two-step mechanism of the process. The advantages and shortcomings of the fluorescence quenching method are discussed.     

Synthesis and bioassay of 3-deoxy-1α-hydroxyvitamin D3, an active analog of 1α,25-dihydroxyvitamin D3

Two synthetic routes to 3-deoxy-1α-hydroxyvitamin D₃, an analog of 1α,25-dihydroxyvitamin D₃, are described. One involved the six-step conversion of 1α,2α-epoxy-6,6-ethylenedioxy-5α-cholestan-3- one to 1α-acetoxycholest-5-ene, whereas, in the second, the same intermediate was prepared from 1α-hydroxycholesterol. Conversion of the Δ⁵-sterol to the required 5,7-diene was accomplished most efficiently via 7-keto and 7-tosylhydrazone intermediates. Bioassay of 3-deoxy-1α-hydroxyvitamin D₃ in the rat establishes that the analog can fulfill all common vitamin D functions including stimulation of intestinal calcium transport, mobilization of calcium and phosphate from bone, stimulation of growth, and calcification of bone. Direct comparison indicates the compound to have $\text{1}\text{20}$ to $\text{1}\text{50}$ of the activity of 1α-hydroxyvitamin D₃, but it acts with a time course indistinguishable from the latter.     

Thermodynamics of the interaction of daunomycin with DNA

The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [³H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 × 10⁶ M^?1 (4°C) and 3.9 × 10⁵ M^?1 (65°C). The free energy change was ?8.2 to ?8.8 $\text{kcal}\text{mol}$, the enthalpy change ?5.3 $\text{kcal}\text{mol}$ and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appeals to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.