Enthalpy of the B-to-Z conformational transition of a DNA oligonucleotide determined by isothermal titration calorimetry

The influence of high concentrations of Na(+) or [Co(NH(3))(6)](3+) on the conformation of two related DNA oligomers was investigated by circular dichroism spectropolarimetry (CD), isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC). As revealed by CD, DNA oligomers, (dC-dG)(4) and (dm(5)C-dG)(4), both form right-handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na(+) at 25 degrees C. However, at 2.0 M Na(+) or 200 microM [Co(NH(3))(6)](3+), (dm(5)C-dG)(4) assumes a left-handed double helical structure (Z-DNA), whereas the unmethylated (dC-dG)(4) analog remains right-handed under those conditions. ITC was then used to determine the enthalpy change upon increasing the concentration of either Na(+) or [Co(NH(3))(6)](3+) for both DNA oligomers at 25 degrees C. The titration with Na(+) resulted in endothermic isotherms with (dm(5)C-dG)(4) being more endothermic than (dC-dG)(4) by 700 cal/mol basepair. In contrast, titration with [Co(NH(3))(6)](3+) resulted in exothermic isotherms with (dC-dG)(4) being more exothermic than (dm(5)C-dG)(4) by 720 cal/mol basepair. We attribute the enthalpy difference to the conformational transition from B-form DNA to Z-form DNA for (dm(5)C-dG)(4), a transition which does not occur for the unmethylated (dC-dG)(4). The value of approximately 700 cal/mol basepair for the enthalpy of the B-Z transition compares favorably with previously published results obtained by different techniques. DSC was used to monitor the duplex to single strand transitions for both oligomers under the different concentrations. These results indicated that methylation of the cytidine destabilizes (dm(5)C-dG)(4) relative to (dC-dG)(4). Coupling the DSC data with the ITC data allowed construction of a thermodynamic cycle which gives insight into the influence of both temperature and ionic strength on the heat content of the two DNA systems studied. Further, this study reveals the utility of using ITC for determinations of transition enthalpies with the appropriate choice of control.     

Interaction of drugs with Z-DNA: cooperative binding of actinomycin D or actinomine to the left-handed forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) reverses the conformation of the helix

The interaction of actinomycin D and actinomine with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) under B- and Z-form conditions has been investigated by optical and phase partition techniques. Circular dichroism data show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation. Actinomycin D binds in a cooperative manner to poly(dG-dC).poly(dG-dC) under both B-form and Z-form conditions. Analysis of the circular dichroism data shows that 5 +/- 1 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to a right-handed conformation for each bound actinomycin D. When the left-handed form of poly(dG-dC).poly(dG-dC) is stabilized by the presence of 40 microM [Co(NH3)6]Cl3, 25 +/- 5 base pairs switch from a left-handed to a right-handed conformation for each bound actinomycin D. Actinomine binds cooperatively to left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and to left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. Actinomine does not bind to left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl at concentrations as high as 100 microM. Each bound actinomine converts 11 +/- 3 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and 7 +/- 2 base pairs of left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. The binding isotherm data also indicate that the binding site has a right-handed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).     

Vitamin C interaction with cobalt-ammine cations. Synthesis, spectroscopic and structural characterization of cobalt-pentammine and cobalt-tetrammine sugar complexes containing L-ascorbate anion

Interaction between [Co(NH3)5Cl]Cl2, [Co(NH3)4Cl2]Cl and L-ascorbic acid has been investigated in aqueous solution and solid complexes of the type [Co(NH3)5 ascorbate]Cl2 X H2O and [Co(NH3)4 ascorbate]Cl2 X H2O have been isolated and characterized by 13C-NMR, FT-IR and electron absorption spectroscopy. Spectroscopic and other evidence suggested that the sugar anion binds monodentately in the [Co(NH3)5 ascorbate]2+ cation via the ionized O3 oxygen atom and bidentately in [Co(NH3)4 ascorbate]2+ through the O1 and O4 oxygen atoms, resulting in a six-coordinate geometry around the Co(III) ion. The intermolecular sugar hydrogen-bonding network is perturbed upon sugar metalation and the sugar moiety shows a similar conformation to that of the sodium ascorbate compound in these series of cobalt-ammine complexes.     

Studies on activities, cell up take and DNA binding of four multinuclear complexes of the form: [[trans-PtCl(NH(3))(2)](2)mu-[trans-Pd(NH(3))(2)-(H(2)N(CH(2))(n)NH(2))(2)]]Cl(4) where n=4-7

The activity against human cancer cell lines including ovarian: A2780, A2780(cisR), cell up take, DNA-binding and nature of interaction with pBR322 plasmid DNA have been studied for four multinuclear complexes code named DH4Cl, DH5Cl, DH6Cl and DH7Cl, having the general formula: [[trans-PtCl(NH(3))(2)](2)mu-[trans-Pd(NH(3))(2)-(H(2)N(CH(2))(n)NH(2))(2)]]Cl(4) where n=4, 5, 6 and 7 for DH4Cl, DH5Cl, DH6Cl and DH7Cl, respectively. The compounds are found to exhibit significant anticancer activity against ovarian cancer cell lines: A2780, A2780(cisR) and A2780(ZD0473R). DH6Cl in which the linking diamine has six carbon atoms is found to be the most active compound. As the number of carbon atoms in the linking diamine is decreased below six and increased above six, the activity is found to decrease, illustrating structure-activity relationship. All the multinuclear compounds are believed to form a plethora of long-range interstrand GG adducts with DNA dictated by the sequence of bases in the DNA strands. Increasing prevention of BamH1 digestion with the increase in concentration of the compounds is due to global changes in DNA conformation brought about by interstrand long-range binding of the compounds with DNA.     

Reversal of the streptomycin injury of Escherichia coli

The number of viable Escherichia coli in a young, actively growing culture is decreased approximately 99.9 per cent by a 30 second exposure to 25 phig. streptomycin/ml. The injury induced by the antibiotic is only potentially lethal, however, and may be reversed by subculture within 5 minutes into fresh culture medium, NH(4)NO(3), NH(4)Cl, (NH(4))(2)HPO(4), NH(4) citrate, and NH(4) tartrate. Subculturing into water, glucose, or MgSO(4) results in a more marked decrease in the number of viable organisms. In KNO(3), NaNO(3), K(2)HPO(4), and Na(2)SO(4) solutions reversal occurs first, followed by a rapid decrease in viability. True reversal of the streptomycin injury takes place, as demonstrated by the rapid rate of recovery to the viable count of the original culture. Development of resistance has been eliminated as the cause of regrowth since the streptomycin sensitivity of recovered cultures remained the same as that of the original culture. The use of water as diluent for viability determinations potentiates the lethal effect of streptomycin activity. Several compounds, at various dilutions, substituted for water as the diluent gave rise to four types of responses, group I, NH(4)NO(3), NH(4)Cl, KNO(3), NaNO(3), Ca(NO(3))(2), showed complete reversal of the streptomycin injury at all levels of the salts tested, from 0.01 to 0.5 M concentrations. Group II, NaCl and K(2)HPO(4) showed complete reversal at 0.03 and 0.1 M. Group III, glucose and urea allowed complete reversal at 0.5 M. Group IV, glycerol and glycerine showed no reversal at 0.5 M concentration. The reversal of the streptomycin injury to young actively growing bacteria is suggested as a tool for studying the pathology of the injury to the cells.     

Pendrin in the mouse kidney is primarily regulated by Cl- excretion but also by systemic metabolic acidosis

The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.     

Condensation of DNA by multivalent cations: experimental studies of condensation kinetics

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH(3))(3+)(6)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH(3))(3+)(6)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH(3))(3+)(6)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius of the condensates first decreases, then increases with increasing [Co(NH(3))(3+)(6)] as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. However, is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with time. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH(3))(3+)(6)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH(3))(3+)(6)](xs) = [Co(NH(3))(3+)(6)] - [Co(NH(3))(3+)(6)](crit). (5) Data for low [DNA] and low [Co(NH(3))(3+)(6)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.     

Cation radius effects on cell-free translation in rabbit reticulocyte lysate

The effect of monovalent cation concentrations on the translation was examined in the rabbit reticulocyte cell-free system. The translation of standard reporter gene luciferase was studied using different concentrations of LiCl, NaCl, KCl, RbCl, CsCl, NH(4)Cl, and (CH(3))(4)NCl and the acetates of Na(+), K(+), and NH4(+). Only the salts of K(+), Rb(+), and NH4(+) and to some minor extent of Cs(+) significantly supported translation. Optimum concentrations were dependent on the cation used. Optimum concentrations ranged between 40 mM (NH(4)Ac), 80 mM (KCl, NH(4)Cl), and 100 mM (RbCl, KAc). The maximum efficiency of translation depends on the ionic radius of the cation used. KCl and RbCl were superior to all other salts tested in stimulating in vitro translation. The results were confirmed, using a second reporter system, M-hirudin. Here, however, broad optima were observed with RbCl being slightly superior to KCl in supporting translation.     

Solubilized DNA-dependent RNA polymerase from human placenta: A Mn(2+)-dependent enzyme

The 100-fold purified RNA polymerase activity from human placenta is completely dependent upon added DNA. The enzyme is most active at 3 mM Mn(2+) in the presence of 100 mM (NH(4))(2)SO(4). Denatured DNA is a better template than native DNA. alpha-Amanitin completely inhibits the incorporation of 3H-UMP, while rifampicin has no influence upon the enzymatic activity.     

Cobalt hexammine inhibition of the hammerhead ribozyme

The effects of Co(NH(3))(6)(3+) on the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH(3))(6)(3+) efficiently displaces Mn(2+) bound to the ribozyme with an apparent dissociation constant of K(d app) = 22 +/- 4.2 microM in 500 microM Mn(2+) (0.1 M NaCl). Displacement of Mn(2+) coincides with Co(NH(3))(6)(3+) inhibition of hammerhead activity in 500 microM Mn(2+), reducing the activity of the WT hammerhead by approximately 15-fold with an inhibition constant of K(i) = 30.9 +/- 2.3 microM. A residual 'slow' activity is observed in the presence of Co(NH(3))(6)(3+) and low concentrations of Mn(2+). Under these conditions, a single Mn(2+) ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn(2+) site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R. , Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn(2+) displacement induced by addition of Co(NH(3))(6)(3+). Taken together, the data indicate that a high-affinity Co(NH(3))(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.     

Ethidium binding to left-handed (Z) DNAs results in regions of right-handed DNA at the intercalation site

The equilibrium binding of ethidium to the right-handed (B) and left-handed (Z) forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) was investigated by optical and phase partition techniques. Ethidium binds to the polynucleotides in a noncooperative manner under B-form conditions, in sharp contrast to highly cooperative binding under Z-form conditions. Correlation of binding isotherms with circular dichroism (CD) data indicates that the cooperative binding of ethidium under Z-form conditions is associated with a sequential conversion of the polymer from a left-handed to a right-handed conformation. Determination of bound drug concentrations by various titration techniques and the measurement of circular dichroism spectra have enabled us to calculate the number of base pairs of left-handed DNA that adopt a right-handed conformation for each bound drug; 3-4 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to the right-handed form for each bound ethidium, while approximately 25 and 7 base pairs switch conformations for each bound ethidium in complexes with poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2, respectively. The induced ellipticity at 320 nm for the ethidium-poly(dG-dC).poly(dG-dC) complex in 4.4 M NaCl indicates that the right-handed regions are nearly saturated with ethidium even though the overall level of saturation is very low. The circular dichroism data indicate that ethidium intercalates to form a right-handed-bound drug region, even at low r values where the CD spectra show that the majority of the polymer is in a left-handed conformation.     

Flexibility of DNA and RNA upon binding to different metal cations. An investigation of the B to A to Z conformational transition by Fourier transform infrared spectroscopy

The interaction of DNA and RNA with Cu(II), Mg(II), [Co(NH3)6]3+ [Co(NH3)5Cl]2+ chlorides and, cis- and trans-Pt(NH3)2Cl2 (CIS-DDP, trans-DDP) has been studied by Fourier Transform Infrared (FT-IR) spectroscopy and a correlation between metal-base binding and conformational transitions in the sugar pucker has been established. It has been found that RNA did not change from A-form on complexation with metals, whereas DNA exhibited a B to Z transition. The marker bands for the A-form (C3'-endo-anti conformation) were found to be near 810-816 cm-1, while the bands at 825 and 690 cm-1 are marker bands for the B-conformation (C2'-endo, anti). The B to Z (C3'-endo. syn conformation) transition is characterized by the shift of the band at 825 cm-1 to 810-816 cm-1 and the shift of the guanine band at 690 cm-1 to about 600-624 cm-1.     

Circular dichroism study of the irreversibility of conformational changes induced by polyamine-linked dinuclear platinum compounds

In this work, the reversibility of both the B-->Z and B-->A conformational change in polymer DNA induced by polynuclear platinum compounds was studied. The compounds examined were: [[trans-PtCl(NH(3))(2)](2)[NH(2) (CH(2))(6)NH(2)]](2+) (BBR3005); [[trans-PtCl(NH(3))(2)](2)[mu-spermine-N1,N12]](4+) (BBR3535); [[trans-PtCl(NH(3))(2)](2)[mu-spermidine-N1,N8]](3+) (BBR3571); [[trans-PtCl(NH(3))(2)](2)[mu-BOC-spermidine]](2+) (BBR3537); and [[trans-PtCl(NH(3))(2)](2)[mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]](4+) (BBR3464). The conformational changes were assessed by circular dichroism and the reversibility of the transitions was tested by subsequent titration with the DNA intercalator ethidium bromide (EtBr). Fluorescent quenching was also used to assess the ability of ethidium bromide to intercalate into A and/or Z-DNA induced by the compounds. The results were compared with those produced by the simple hexamminecobalt cation [Co(NH(3))(6)](3+). The data suggest that while conformational changes induced by electrostatic interactions are confirmed to be reversible, covalent binding induces irreversible changes in both the A and Z conformation. The relevance of these changes to the novel biological action of polynuclear platinum compounds is discussed.     

Competitive substitution of hexammine cobalt(III) for Na+ and K+ ions in oriented DNA fibers

Competition of the trivalent cation, Co(NH3)(3+)(6), with K+ and Na+ ions in binding to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH), containing different combinations and concentrations of KCl and NaCl and constant concentration (0.8 mM) of Co(NH3)(6)Cl(3). The degree of Co(NH3)(3+)(6) binding to DNA does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na+ or K+). The ion exchange selectivity coefficient of monovalent-trivalent ion competition, D(1)(c3), increases with the concentration of Me+, C(o)(+), and the monotonic dependence of log D(1)(c3) vs log C(o)(+) has an inflection between 100 and 300 mM that is caused by a structural transformation of DNA from A- to B-form. The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, discretely charged polyions with density and spatial distribution of the charged groups modeling B- and A-forms of DNA. The GCMC method for discretely charged models of the DNA polyion produces a quantitative agreement with experimental data on trivalent-monovalent ion competition in dependence on DNA structural state and salt concentration. Based on this and previous studies it is concluded that the affinity of DNA for the cations decreases in the order Co(NH3)(3+)(6) > Ca2+ > Mg2+ > Na+ approximately K+ > Li+. DNA does not exhibit selectivity for Na+ or K+ in ethanol/water solutions either in the absence or in the presence of Co(NH3)(3+)(6), Ca2+, and Mg2+.     

Synthesis, DNA binding, and oxidative cleavage studies of Fe(II) and Co(III) complexes containing bioactive ligands

The two complexes containing bioactive ligands of the type and [Fe(L)] (PF(6))(2) (1) (where L = [1-{[2-{[2-hydroxynaphthalen-1-yl)methylidine]amino}phenyl)imino] methyl}naphthalene-2-ol]) and [Co(L(1)L(2))] (PF(6))(3) (2) (where L(1)L(2) = mixed ligand of 2-seleno-4-methylquinoline and 1,10-phenanthroline in the ratio 1:2, respectively) were synthesized and structurally characterized. The DNA binding property of the complexes with calf thymus DNA has been investigated using absorption spectra, viscosity measurements, and thermal denaturation experiments. Intrinsic binding constant K(b) has been estimated at room temperature. The absorption spectral studies indicate that the complexes intercalate between the base pairs of the CT-DNA tightly with intrinsic DNA binding constant of 2.8 × 10(5) M(-1) for (1) and 4.8 × 10(5) M(-1) for (2) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2, respectively. The oxidative cleavage activity of (1) and (2) were studied by using gel electrophoresis and the results show that complexes have potent nuclease activity.     

Halophilic Properties of Metal Binding Protein Characterized by High Histidine Content from Chromohalobacter salexigens DSM3043

Periplasmic metal binding protein characterized by high histidine content was cloned from moderate halophile, Chromohalobacter salexigens. The protein, termed histidine-rich metal binding protein (HP), was expressed in and purified from E. coli as a native form. HP bound to Ni- and Cu-loaded chelate columns with high affinity, and Co- and Zn-columns with moderate affinity. Although the secondary structure was not grossly altered by the addition of 0.2–2.0 M NaCl, the thermal transition pattern was considerably shifted to higher temperature with increasing salt concentration: melting temperature was raised by ~20 °C at 2.0 M NaCl over the melting temperature at 0.2 M NaCl. HP showed reversible refolding from thermal melting in 0.2–1.15 M NaCl, while it formed irreversible aggregates upon thermal melting at 2 M NaCl. Addition of 0.01–0.1 mM NiSO₄ stabilized HP against thermal melting with high reversibility, while addition above 0.5 mM resulted in irreversible melting due to aggregation.     

Helix-coil transition of the self-complementary dG-dG-dA-dA-dT-dT-dC-dC duplex.

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-dA-dA-dT-dT-dC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters delta H degree = -216.7 kJ/mol and delta S degree (25 degrees C) = -0.632 KJ mol-1 K-1 in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG.dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x 10(3) s-1 at 32 degrees C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.     

The effects of elevated pH and high salt concentrations on tubulin

The effects of incubating phosphocellulose-purified bovine tubulin at 4 degrees C in nucleotide-free buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH4)2SO4, or NH4Cl have been studied. At pH greater than or equal to 7.5 or at NaCl concentrations greater than or equal to 0.7 M, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 M 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 M NH4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 M (NH4)2SO4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220-260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 mM guanosine 5'-triphosphate (GTP) or 4 M glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 M NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 M NaCl is decreased in the presence of 1 mM GTP, and tubulin is protected almost completely from 1 M NaCl-induced loss of GTP (and retains the ability to exchange [3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.