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R B Macgregor

Publications and source records attributed to R B Macgregor.

33 records · Page 2Linked to original sources
PubMed1996

Chain length and oligonucleotide stability at high pressure.

The effect of hydrostatic pressure on the helix-coil transition temperature (Tm) was measured for the DNA oligomers (dA)n(dT)n, where n = 11, 15, and 19, in 50 mM NaCl. The data were analyzed in light of previously published data for the polymer, poly(dA) center dot poly(dT) under the same conditions. As has been observed for DNA polymers, increasing the hydrostatic pressure led to an increase in the Tm of the oligomers; however, the effect of pressure diminished with decreasing chain length. The value of dTm/dP decreased linearly with the inverse of the chain length varying from 3.15 x 10(-2) degrees C MPa-1 for the polymer to 0.7 x 10(-2) degrees C MPa-1 for the 11-mer. The two-state or van't Hoff enthalpy (DeltaHvH) of the helix-coil transition was obtained by analysis of the half-width of the thermal transition. As expected, DeltaHvH decreases with decreasing chain length. In contrast to the behavior of the polymer, poly(dA) center dot poly(dT), and (dA)19(dT)19, the DeltaHvH of the two shorter duplex oligonucleotides displayed a small pressure dependence dDeltaHvH/dP approximately equal -0.4 kJ MPa-1 in both cases. The changes observed in the Tm and DeltaHvH were not sufficient to explain the magnitude of the chain-length dependence of the pressure effect. To interpret the large chain-length dependence of dTm/dP, we propose that the terminal base pairs contribute a negative volume change to the helix-coil transition. Base pairs distant from the ends exhibit behavior characterized by the polymer where end effects are assumed to be negligible, i.e., a positive volume change for the helix-coil transition. The negative volume change of separating terminal bases may originate from the imperfect interactions these base pairs form with water due to the existence of several energetically equivalent conformations. This is reminiscent of one of the mechanisms proposed to be important in the pressure-induced dissociation of multimeric proteins into their constituent subunits.

Chemical Phenomena↗
PubMed1995

Effect of cations on the volume of the helix-coil transition of poly[d(A-T)].

The pressure dependence of the helix-coil transition temperature of poly[d(A-T)] has been measured in aqueous solutions of NaCl, KCl, and CsCl at concentrations between 0.02 and 1 M. In all cases the transition temperature increases with pressure. For solutions of NaCl, KCl and dilute CsCl the change in the transition temperature is linear with pressure up to 200 MPa. In more concentrated CsCl solutions the change in Tm with pressure was hyperbolic. The molar volume change of the transition (delta Vt) was calculated using the Clapeyron equation. At the lower salt concentrations, the derived values of delta Vt increase with the radius of the cation (Na+ < K+ < Cs+). At the higher salt concentrations delta Vt of poly[d(A-T)] in Na+ and K+ became equal; however, in CsCl solutions delta Vt was approximately twice as large as delta Vt in solutions containing the other two ions. In solutions of NaCl and KCl, delta Vt increased linearly with the logarithm of the salt concentration while in aqueous CsCl the concentration dependence of delta Vt was hyperbolic. The results are interpreted in terms of the role played by the radius of the cation in deciding the strength of the interactions formed with water.

Cations↗
PubMed1995

Pressure dependence of the helix-coil transition temperature of poly[d(G-C)].

The pressure dependence of the helix-coil transition temperature (Tm) of poly[d(G-C)] was studied as a function of sodium ion concentration in phosphate buffer. The molar volume change of the transition (delta V) was calculated using the Clapeyron equation and calorimetrically determined enthalpies. The delta V of the transition increased from +4.80 (+/- 0.56) to +6.03 (+/- 0.76) mL mol-1 as the sodium ion concentration changed from 0.052 to 1.0M. The van't Hoff enthalpy of the transition calculated from the half-width of the differentiated transition displayed negligible pressure dependence; however, the value of this parameter decreased with increasing sodium ion concentration, indicating a decrease in the size of the cooperative unit. The volume change of the transition exhibits the largest magnitude of any double-stranded DNA polymer measured using this technique. For poly[d(G-C)] the magnitude of the change in delta V with sodium ion concentration (0.94 +/- 0.05 mL mol-1) is approximately one-half that observed for either poly[d(A-T)] or poly(dA).poly(dT). The delta V values are interpreted as arising from changes in the hydration of the polymer due to the release of counterions and changes in the stacking of the bases of the coil form. As a consequence of solvent electrostriction, the release of counterions makes a net negative contribution to the total delta V, implying that disruption of the stacking interactions contributes a positive volume change to the total delta V.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrostatic Pressure↗
PubMed1993

Pressure dependence of the melting temperature of dA.dT polymers.

The pressure dependence of the helix-to-coil transition temperature (Tm) of poly[d(A-T)], poly(dA).poly(dT), and poly(dA).poly(dT)2 was studied as a function of sodium chloride concentration. The molar volume change of the transition (delta V) was calculated using the Clapeyron equation and calorimetrically determined enthalpies. The delta V of the transition varied linearly with the logarithm of the salt concentration for both double-stranded polymers. The delta V of poly[d(A-T)] changed from +0.36 to +3.86 cm3 mol-1 as the sodium chloride concentration changed from 0.020 to 1.0 M. For poly(dA).poly-(dT), the delta V varied from +2.60 to +4.59 cm3 mol-1 over the range of 0.020-0.20 M NaCl. No pressure dependence of the van't Hoff enthalpy was observed for the double-helical form of either polymer. The delta V for denaturation of the triple-helical species poly(dA).poly(dT)2 was found to be +7.81 and +10.4 cm3 mol-1 at 1.0 and 3.0 M NaCl, respectively. The observed delta V values are interpreted in terms of changes in the hydration of the polymers arising from release of counterions and changes in the stacking of the bases upon denaturation. Counterion release is assumed to make a net negative contribution to the overall delta V, implying that disruption of the stacking interactions must make a positive volume change to the overall delta V. The difference in the delta V values for the two polymers remains constant as the salt concentration is changed, suggesting a difference in the partial molar volume of their single-stranded forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrostatic Pressure↗
PubMed1993

A temperature-regulated iso-hyperbaric spectrophotometer: construction and performance characteristics.

The position of an equilibrium is a function of pressure and temperature. Generally, only temperature effects are discussed for biochemical systems; however, pressure perturbations can offer interesting insights into the role of hydration in a reaction. We describe the construction and characteristics of an instrument that allows computer control of the temperature and pressure throughout the range -40 to +140 degrees C and 0.1 to 250 MPa, as well as automatic data acquisition from either a spectrophotometer or a spectrofluorimeter. To test the performance of the system the effect of pressure on the thermal denaturation of poly[d(A-T)] and poly(dA).poly(dT) has been investigated. In 20 mM NaCl, 20 mM Tris-HCl, pH 8.9, the delta V degrees for poly[d(A-T)] equals +0.30 +/- 0.09 cm3/mol (base pairs), and for poly(dA).poly(dT) delta V degrees equals +3.05 +/- 0.15 cm3/mol (base pairs). Although we focus on the use of this instrument to measure thermal denaturation curves, it can be employed to study the equilibrium of any reaction that can be followed optically in the ultraviolet or visible.

DNA↗
PubMed1992

Photogeneration of hydroxyl radicals for footprinting.

Hydroxyl radicals yield footprints of DNA-ligand interactions that are uniform in intensity and display single base pair resolution. It is shown here that brief illumination of dilute aqueous solutions of hydrogen peroxide with a standard uv transilluminator can be used to generate hydroxyl radicals for footprinting studies. Photogenerated hydroxyl radicals are used to footprint netropsin, a drug that interacts with the minor groove of DNA. The method presented eliminates two of the reagents used in conventional Fenton-reaction-based hydroxyl radical footprinting. It has the further advantage that the extent of cleavage of the DNA can be precisely regulated by controlling the illumination time. Because light is used to drive the reaction, photogenerated hydroxyl radicals can be used to footprint DNA-ligand interactions under experimental conditions of temperature and pressure inaccessible to Fenton-reaction chemistry.

Base Sequence↗
PubMed1992

Footprinting of EcoRI endonuclease at high pressure.

Hydroxyl radicals generated by irradiation with gamma rays have been used to footprint EcoRI endonuclease with single base pair resolution at pressures up to 144 MPa. At atmospheric pressure (0.1 MPa) a 10 base pair footprint was found. With increasing pressure three types of responses were observed: (1) bases distant from the recognition sequence showed a moderate increase in solvent exposure; (2) the bases at the point of enzymatic activity showed a large increase in cleavage by the hydroxyl radicals; and (3) the two center-most bases exhibited no pressure-induced change in solvent accessibility. The results are interpreted in terms of localized conformational changes of EcoRI.

Autoradiography↗
PubMed1991

Gd3+ vibronic side band spectroscopy. New optical probe of Ca2+ binding sites applied to biological macromolecules.

A new spectroscopic technique is presented for obtaining infraredlike spectra of the binding sites of Ca2+ and other metals in biological macromolecules. The technique, based on the Ca(2+)-like binding properties of Gd3+, utilizes vibronic side bands (VSB) that appear in Gd3+ fluorescence. In the fluorescence spectrum of Gd3+, the separation in photon frequency between a VSB and its electronic origin at approximately 32,150 cm-1 (approximately 311 nm) is a direct measure of the vibrational frequency of a ligand coordinated to Gd3+ ion. As a consequence, the VSB are uncomplicated by molecular vibrations distant from the Gd3+ binding site. The vibrational spectra resulting from the VSB of Gd3+ coordinated to a Ca2+ binding protein, a phospholipid, and DNA are presented.

Binding Sites↗
PubMed1990

Reversible inhibition of EcoRI with elevated pressure.

The endonuclease activity of EcoRI is completely inhibited at 200 MPa, 37 degrees C using the plasmid pBR 322 as a substrate. When assayed at 133 MPa approximately half the activity at atmospheric pressure was observed; from these data the standard molar volume change is estimated to be -80 cm3mol-1. Upon return to atmospheric pressure the enzyme reacted in a standard manner with its restriction site in pBR 322. Pressurization did not decrease the specificity of the endonuclease activity of the enzyme for its canonical site. These results are discussed in terms of the role of ionic interactions in protein-DNA interactions.

Atmospheric Pressure↗
PubMed1990

Hydration of dA.dT polymers: role of water in the thermodynamics of ethidium and propidium intercalation.

We report differences in the interaction of two structurally similar phenanthroline intercalators, ethidium and propidium, with poly(dA).poly(dT) and poly[d(A-T)] as a function of ionic strength based on titration microcalorimetry, fluorescence titration, and hydrostatic pressure measurements. Both ethidium and propidium bind more strongly to poly[d(A-T)].poly[d(A-T)] than to poly(dA).poly(dT). Ethidium intercalation into the latter polymer displays titrations with positive cooperativity; this is not found with propidium. The enthalpy of intercalation (delta H degrees) is exothermic for both dyes with poly[d(A-T)].poly[d(A-T)]; however, the value of this parameter is nearly zero in the case of poly(dA).poly(dT). The molar volume change (delta V degrees) accompanying dye intercalation is negative under all conditions for poly[d(A-T)].poly[d(A-T)] whereas it is positive for poly(dA).poly(dT). The changes observed in delta V degrees correlate well with the entropy changes derived from the titration and calorimetric data for this reaction. The results, interpreted in terms of the relative hydration of these two polymers, are consistent with a higher extent of hydration of poly(dA).poly(dT) relative to poly[d(A-T)].poly[d(A-T)].

Binding Sites↗
PubMed1987

Viscosity dependence of ethidium-DNA intercalation kinetics.

The kinetics of ethidium intercalation into double-stranded poly[d(G-C)] were investigated by use of repetitive pressure-jump chemical relaxation at 20 degrees C in low ionic strength (0.1 M NaCl) aqueous buffers containing either glycerol or methanol. The viscosity of the various solvents differed by more than an order of magnitude while other physical properties (e.g., dielectric constant) remained approximately constant. The single-reciprocal kinetic relaxation time (tau -1) increases linearly with DNA concentration. The observed association rate constant is lower in all organic-aqueous mixtures than in water and is inversely proportional to the viscosity. These results provide evidence for an additional step in the intercalation mechanism which is identified as an obligatory DNA conformational change preceding ethidium intercalation. From the data presented, the equilibrium constant of this local conformational change is approximately 10(-3), i.e., greatly favoring the structure incapable of intercalation. The corresponding kinetics were not directly determined; however, in order to be consistent with all of the data the forward and/or reverse rate constants of the conformational change must be larger than the rate of the intercalation reaction. Thus, it is proposed that the rate of the conformational change back to the nonintercalating B-DNA structure is greater than approximately 500 s-1, implying a rate of opening greater than approximately 0.5 s-1, in agreement with other hydrogen exchange and NMR data. The observed overall rate constant for the dissociation of ethidium is inversely proportional to the solvent density, possibly reflecting a dependence on the solvent free volume. The overall volume change of intercalation is less negative in the organic-aqueous solvent mixtures than in water.

DNA↗
PubMed1985

Pressure-jump study of the kinetics of ethidium bromide binding to DNA.

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1985

Binding of N-dansylgalactosamine to the lectin from Erythrina cristagalli as followed by stopped-flow and pressure-jump relaxation kinetics.

The binding kinetics of N-dansylgalactosamine to the lectin from Erythrina cristagalli have been studied using stopped-flow and pressure-jump chemical relaxation by monitoring ligand fluorescence. Both methods gave results which are consistent with a simple bimolecular association reaction. The association rate constant, k + 1 = 4.8 X 10(4) M-1 s-1, is far too low to be controlled by diffusion; the dissociation rate is 0.4-0.66 s-1, depending upon the method of determination and the experimental conditions. Identical reaction-rate parameters were obtained at pH 7.3, where soluble aggregates can be present in the lectin solution and at pH 4.7 where such aggregates are absent. The slow rates of carbohydrate binding seem to be characteristic for most lectins and lend support to the idea that they are evolutionary related and have structurally similar binding sites. Analysis of the relaxation amplitudes of the pressure-jump experiments yielded a molar reaction volume change, delta V0, upon binding of +7 ml/mol. This volume change can be caused by desolvation of the ligand upon binding.

Binding Sites↗
PubMedDate not supplied

Estimation of the polarity of the protein interior by optical spectroscopy.

Crystallographic studies of myoglobin have shown that the haem pocket is lined with nonpolar amino-acid residues. In order to estimate the true polarity of the interior of proteins, certain studies have used bound fluorophores, the spectroscopic properties of which reflect the polarity of their environment. These studies have most often used 1-amino-8-naphthalene sulphonate (ANS) as a probe, but a more suitable probe, in principle, is 6-propionyl 2-(N,N-dimethyl)aminonaphthalene (PRODAN). We have synthesized a molecule with the advantageous spectroscopic properties of PRODAN but with a higher affinity for apomyogloblin: 2'-(N,N-dimethyl)amino-6-naphthopyl-4-trans-cyclohexanoic acid (DANCA), and report here its use to determine the polarity of the myoglobin haem pocket. Our results show that the pocket is actually a polar environment, and the polarity can be accounted for by peptide amide dipoles.

Animals↗