Chemistry Faculty Scholarship

Kinetics of Cleavage of Intra- and Extracellular Simian Virus 40 DNA with the Enediyne Anticancer Drug C- 1027

Jerry Goodisman et al. — Wed, 01 May 2013 06:26:21 PDT

A kinetic analysis of cleavage of simian virus DNA (SV40 DNA) inside and outside green monkey BSC-I cells by the enediyne-protein antibiotic C-1027 and its free chromophore is described. Information on rate constants was obtained by fitting populations of forms I (closed circular DNA), II (nicked circular DNA) and III (linear DNA) of SV4f.J DNA as a function of drug concentration to a kinetic model which includes: cutting of form I to give form II with rate constant k1. cutting of form I to give form III with rate constant k4, and cutting of form II to give form III with rate constant k2. Theratio of single-strand (ss) to double-strand (ds) cutting for the holoantibiotic and the free chromophore. k1/k4, is approximately 1.8 for extracellular SV40 DNA. For intracellular DNA and extracellular DNA which has been post-treated with putrescine, ds cutting is much more probable, with k4, about four times as large as k1. This observation suggests that amine groups present in the cell are able to convert abasic sites opposite an ss break into a ds break in SV40 chkomatin. The overall rate of cleavage of form-1 DNA inside the cell is much larger than the rate outside, the sum k1 + k4 being about three times as large for intracellular DNA as for extracellular DNA.

On probing human fingertips in vivo using near-infrared light: model calculations

Jerry Goodisman et al. — Mon, 29 Apr 2013 08:01:54 PDT

We probe volar-side fingertip capillary beds with nearinfrared laser light and collect Raman, Rayleigh, and Mie scattered light and fluorescence. The results are interpreted using radiation transfer theory in the single-scattering approximation. The surface topography of the skin is modeled using the Fresnel equations. The skin is treated as a three-layer material, with a mean-field treatment of tissue composition and related optical properties. The model, with a reasonable choice of tissue parameters, gives a remarkably accurate account of the features of actual measurements. It predicts the optimal values for the incident angle of the laser beam and the distance between beam and detector. It explains the correlated temporal changes in the intensities of elastically and inelastically scattered light caused by heart-driven pulses and why they are out of phase. With appropriate boundary conditions, the model can be used to discuss the scattering from ridged skin extruded conformally into an aperture in a metal surface under constant light pressure. The probing results suggest an inherent regularity and similarity in the anatomy and composition of surface and subsurface tissues of a wide range of skin types.

Simultaneous, noninvasive observation of elastic scattering, fluorescence and inelastic scattering as a monitor of blood flow and hematocrit in human fingertip capillary beds

Joseph Chaiken et al. — Mon, 29 Apr 2013 08:01:46 PDT

We report simultaneous observation of elastic scattering, fluorescence, and inelastic scattering from in vivo near-infrared probing of human skin. Careful control of the mechanical force needed to obtain reliable registration of in vivo tissue with an appropriate optical system allows reproducible observation of blood flow in capillary beds of human volar side fingertips. The time dependence of the elastically scattered light is highly correlated with that of the combined fluorescence and Raman scattered light. We interpret this in terms of turbidity (the impeding effect of red blood cells on optical propagation to and from the scattering centers) and the changes in the volume percentages of the tissues in the irradiated volume with normal homeostatic processes. By fitting to a model, these measurements may be used to determine volume fractions of plasma and RBCs.

Quantitation of ethidium-stained closed circular DNA in agarose gels

Michael F. Shubsda et al. — Mon, 29 Apr 2013 08:01:44 PDT

The fluorescence of ethidium bromide (EB) bound to equimolar amounts of supercoiled form I and unstrained linear form III pBR322, SV40 and PM2 DNA in agarose gels has been measured by scanning a photographic negative of the gel with a microdensitometer. For SV40 and PM2 DNA, commonly used staining conditions cause both forms, i.e. linear and supercoiled, to fluoresce to the same extent. This obviates the need to use a correction factor for the fluorescence of form I DNA when measuring the amount of this form relative to the amounts of unstrained forms in agarose gels. In the case of PBR322 DNA, form I was found to fluoresce | 20% more than form III DNA.

Use of fractals and kinetic equations to model thermally induced hillock formation and growth in thin metal films

Joseph Chaiken et al. — Mon, 29 Apr 2013 08:01:42 PDT

We investigated the applicability of a model based on fractals and the Smoluchowski kinetic equations to describe hillock formation in thin metal films. We have previously used this model to analyze cluster and ultrafine particle production. We show how to extract two parameters from measured hillock size distributions which may reveal the scaling of the mobility of clusters and vacancies in films with varying hillock size. On the basis of our application of this model to certain data taken from the literature, the model shows considerable potential for being able to provide an internally consistent quantitative basis for monitoring thermally driven mass redistribution processes in metal films.

Understanding how the platinum anticancer drug carboplatin works: From the bottle to the cell

Jerry Goodisman et al. — Mon, 29 Apr 2013 08:01:40 PDT

Carboplatin, a platinum anticancer drug used to treat many types of human cancer, contains a bidentate dicarboxylate chelate leaving ligand, a structural feature that makes it much less chemically reactive than the first-generation platinum anticancer drug cisplatin, which contains two monodentate chloride leaving ligands. In water, carboplatin exists in a monomer–dimer equilibrium with an association constant of K (M⁻¹) ≈ 391, a property that accounts for the long-term stability of its ready-to-use infusion solution. When administered in the clinic, carboplatin is believed to exert its biological effects by interacting with genomic DNA and proteins. The slower substitution kinetics of carboplatin, compared to cisplatin, has prompted investigators to focus on mechanisms by which the compound can be activated in vivo. Carbonate, which is in equilibrium with hydrogen carbonate, carbonic acid, and dissolved carbon dioxide, is ubiquitous in biological systems, and is found in high concentrations in the blood, the interstitial fluid, and the cytosol. Activation of carboplatin by carbonate, CO₃²⁻ (k₁ = 2.04 ± 0.81 × 10⁻⁶ in 24 mM carbonate buffer, pH 7.5 at 37 °C), for example, leads to the formation of platinum species that are more cytotoxic than the parent drug. This short review focuses on the reason for the unusual stability of carboplatin in its aqueous ready-to-use infusion solution, describes the reactions of the drug with biologically common nucleophiles and summarizes the activation chemistry that make the drug more reactive toward substances present in the biological system.

Modification of carboplatin by Jurkat cells

Anthony J. Di Pasqua et al. — Mon, 29 Apr 2013 08:01:38 PDT

Using [¹H,¹⁵N] heteronuclear single quantum coherance (HSQC) NMR and¹⁵N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt²⁺ anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k1=kc[CO32-]+km+kuN, where k_c is the rate constant for reaction of 1 with carbonate in the medium, k_m is the rate constant for reaction of 1 with all other components of the medium, and k_u is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium (<1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by the cells.

Formation of monofunctional cisplatin-DNA adducts in carbonate buffer

Alexandra Binter et al. — Mon, 29 Apr 2013 08:01:37 PDT

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768–12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8 mM HEPES, N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid, 5 mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8 mM carbonate buffer, 5 mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

General Correction for Electrode Sphericity in Voltammetry of Nernstian Systems

Jerry Goodisman — Thu, 25 Apr 2013 07:49:31 PDT

The current is considered at a stationary reversible spherical electrode whose potential E(t) as a function of time is given, such that E(t) determines the ratio of oxidized to reduced species at the electrode surface. Writing the current as that for planar geometry, I0, plus corrections for sphericity, we derive formulae for the corrections. The first two are expressed as integrals over I0, with no explicit dependence on the potential, for any form of E(t), and whether the reduced species diffuses into the electrode or into the solution. If the ratio of diffusion constants for oxidized and reduced species is taken as unity, and if oxidized and reduced species are both in the solution, the corrections become extremely simple (no integrations or differentiations), regardless of the form of E(t). The first correction is evaluated in several cases where this does not obtain.

The Metal in the Polarisable Interface Coupling With the Solvent Phase

Jerry Goodisman et al. — Thu, 25 Apr 2013 07:49:30 PDT

A quantum mechanical treatment of the conduction electrons of a metal in a polarisable interface shows that they can make an appreciable contribution to the electrical capacitance. Results for six metals are given, showing how differences in metal properties account qualitatively for experimentally observed differences in interfacial capacities, when the solvent is replaced by a dielectric film. To justify the neglect of solvent structure when metal properties are treated, the coupling between metal and solvent is discussed for orientable point solvent dipoles, and for a representation of the solvent polarisation by a pair of charged planes. The electron profile affects the polarisation of the solvent near the point of zero charge, but the solvent structure has an almost negligible effect on the metal contribution to the capacity. One parameter in our model, the distance from metal ions to the first solvent layer, can be expected to vary as the interface is charged, due to changed bonding. Coupling by such an effect can be quite important, and severely decreases the variation of metal capacity with charge.

Contribution of the Metal to the Differential Capacity of an Ideally Polarisable Electrode

Jerry Goodisman et al. — Thu, 25 Apr 2013 07:18:10 PDT

We consider the response of the metal in the ideally polarisable electrode to charging of the electrode, using a model for the metal surface in contact with the solvent of the electrolyte phase previously presented by us in this journal. We show that the effect of the solvent on the electrons of the metal cannot be considered to be simply that of a repulsive barrier. When the electrode charge varies, qM(dip), the metal contribution to the double layer, is modified, implying a contribution which varies along the electrocapillary curve. By considering an electrostatic interaction between metal and solvent, we find an acceptable value for the contribution of the metal to the double-layer capacity. Furthermore, the introduction of appropriate parameters for the metals shows that one should expect CGa > CHg at the potential of zero charge, in accord with experimental observations. The influence of the choice of parameters, particularly those which express the interpenetration of metal and solvent in our model, is discussed as well as other possible models. The different contributions of electrons of different metals probably need to be considered in evaluating models for the inner-layer contribution to the capacity.

Characterization of Hairpin-Duplex Interconversion of DNA Using Polyacrylamide Gel Electrophoresis

Michael Shubsda et al. — Mon, 15 Apr 2013 10:36:31 PDT

We show how polyacrylamide gel electrophoresis of radiolabeled DNA can be used to measure the hairpin-duplex equilibrium constant for DNA in solution. As an aid to the interpretation of the experiments, the differential equations associated with diffusion, migration and chemical reaction of the DNA forms are solved and intensity patterns generated. Two kinds of experiments were performed on several DNA 12-mers: in the first, electrophoresis time was constant while DNA concentration varied; in the other, concentration was constant while time varied (a 'load-and-run' gel). The observed patterns depended on the gel temperature and not the temperature at which the DNA was equilibrated before loading in the well, because reequilibration occurs before the DNA leaves the well to enter the gel proper. During this time, mixing also occurs, changing the concentration and ionic strength of the sample. A method of calculating the true DNA concentration, including the unmeasured concentration added with the radiolabel, is given. When the intensity pattern consists mainly of monomer and dimer peaks, the equilibrium constant K is easily calculated from peak intensities. However, when there is significant intensity between the peaks (which the calculations show results from monomer-dimer interconversion in the gel), K will be inaccurate. An accurate value of K may be determined from a load-and-run gel by extrapolating back to time 0. When the intensity pattern consists of a single broad peak (from rapid monomer-dimer interconversion in the gel), K cannot be calculated without additional information. The rate of interconversion increases with temperature. Estimated rates in the gel are more than an order of magnitude smaller than in bulk solution at the same temperature. Derived values of K for several DNAs are compared with literature values.

Binding of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein to Ψ-RNA-SL3

Michael F. Shubsda et al. — Mon, 15 Apr 2013 10:36:29 PDT

The interaction of the nucleocapsid protein NCp7, from the pNL4-3 isolate of HIV-1, with Ψ-RNA-SL3, with the sequence 5’-GGACUAGCGGAGGCUAGUCC, was studied using non-denaturing gel electrophoresis. Two kinds of experiments were performed, using buffered solutions of radiolabeled RNA and unlabeled protein. In the ‘dilution’ experiments, the total RNA concentration, R_T, was varied for a series of solutions, but kept equal to the total protein concentration, P_T. In the ‘titration’ experiments, solutions having R_T constant but with varying P_T were analyzed. The solutions were electrophoresed and the autoradiographic spot intensities, proportional to the amounts of the different species present, were measured. The intensities were fit to a number of equilibrium models, differing in species stoichiometries, by finding the best values of the binding constants. It was shown that NCp7 protein and SL3 RNA combine to form at least two complexes. When P_T is below approximately 10 μM, a complex that contains two RNAs and one protein forms. Increasing P_T to approximately 100 μM causes the 2:1 complex to oligomerize, forming a species having eight RNAs and four proteins. For the dilution experiments, run at 5 ̊C at an ionic strength of 31 mM, we found K₁ for the 2:1 complex is ̴ 10¹¹ M^-2 and K₂ for the 8:4 complex is ̴ 10¹⁶ M^-3. The titration experiments returned K₁ ̴ 10⁷ M^-2 (poorly determined) and K₁ ̴ 10¹⁹ M^-3. The analysis was complicated by the loss of RNA at higher protein concentrations, due to formation of an insoluble species containing both RNA and protein, which does not enter the gel. Correcting for this changes the calculated values of equilibrium constants, but not the molecularities determined by our analysis. The observation that a small complex can oligomerize to form a larger species is consistent with the fact that NCp7 organizes and condenses the genome in the virus particle.

Constancy in Integrated Cisplatin Plasma Concentrations Among Pediatric Patients

Jerry Goodisman et al. — Mon, 15 Apr 2013 10:36:27 PDT

The authors report on the variability in the integrated quantity of free (unbound) plasma cisplatin (area under curve of plasma concentration versus time, AUC). The AUC was measured in 19 patients receiving cisplatin doses proportional to body surface areas (BSA), 30mg/m2 over 1 hour. The relative standard deviation (RSD, population standard deviation divided by mean value) for the maximum free plasma cisplatin concentration (Cmax, μM) was 0.338; for the half-life (t½, minute), 0.210; and for the AUC (μM minute), 0.320. Thus, BSA-based dosing gave significant variability in the AUC. We attempted to use (weight)a(height)b, seeking values of a and b that gave the smallest RSD in AUC, but only minimal improvement could be obtained by deviating from the BSA formula (a = b = 0.5). However, dosing proportional to (weight)d(Cmax)f (with d ≈ 3/4 and f ≈ –1) reduced the RSD in AUC from ~1/3 to ~1/10. Dosing proportional to (weight)m(Cmax)n(t½)p (with m≈ 0.7, n ≈ –1, and p ≈ –½) reduced it further, to ~1/32. In contrast, using (weight)d(Cmax)f(age)g gave no improvement over (weight)d(Cmax)f. The authors conclude that the inconsistency in AUC can be reduced 10-fold with dosing proportional to the weight and the drug pharmacokinetic parameters [(weight0.7) (Cmax t½ 0.5)].

Thermodynamics and Statistical Mechanics of the Ideally Polarisable Electrode Gibbs Isotherm and Surface Tension

Jean-Pierre Badiali et al. — Mon, 15 Apr 2013 10:36:26 PDT

A definition of the ideally polarisable electrode which allows a general statistical mechanical treatment is given, comparisons with the usual thermodynamic approach being made. It is shown that, with this definition, the system behaves as a pure capacitance to an imposed alternating potential of sufficiently low frequency. The equilibrium states of the system are described by the grand canonical distribution. Thermodynamic functions, given in terms of the grand canonical partition function, can be computed in molecular terms: In particular, general expressions for the surface tension and the pressure are obtained, and compared to those proposed by various authors. The separation of the pressure into electrical and nonelectrical parts is analyzed. Expressions are given for the electrochemical potentials of charged and of dipolar species. With these microscopic definitions, the Gibbs adsorption isotherms and the Lippmann equation are derived. For several simple models, including that of Gouy and Chapman for an ionic solution and a metal electrode, these relations are explicitly considered. The inadmissability, in a coherent model, of reducing the solvent to a medium of fixed dielectric constant, is emphasized.

Effect of Solvent on Properties of the Liquid Metal Surface

J. P. Badiali et al. — Mon, 15 Apr 2013 10:36:24 PDT

We calculate the difference in the surface potentials between the free surface of a liquid metal and the same metal in an ideally polarizable interface at the point of zero charge. This difference, δXm, is due to the deformation of the electronic cloud of the metal by the solvent molecules. The simple model used for the free (metal-metal vapor) surface yields qualitatively correct work functions for a number of metals (Hg, Cd, In, Zn, Pb, Ga, A1). Two simple ways to model the metal-solvent interaction are proposed and calculations of δXm made for each. One, the dielectric film model, considers only an electrostatic interaction between metal electrons and solvent, while the other, the repulsive core model, considers only the exchange repulsion between metal electrons and the cores of solvent molecules. For Zn, Cd and Hg the dielectric film model, with parameters chosen according to conventional electrochemical wisdom, gives values for δXm which are close to those estimated in the literature. For Ga and A1, the effect of the solvent is much greater because of the larger electron density and smaller ion size. The repulsive core model can give similar results, but there is an arbitrariness in the choice of the barrier strength parameter. Again, Ga is more sensitive to the presence of solvent. The effect of changing certain parameters in both models, and of combining the two, is considered.

Site-Specific Binding Constants for Actinomycin D on DNA Determined from Footprinting Studies

Jerry Goodisman et al. — Wed, 27 Mar 2013 09:44:28 PDT

We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at -7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3’ end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3’ end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about - 120 base pairs of the restriction fragment (- 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5’ - 3’) GC, with TGCT having the highest binding constant, 6.4 X lo6 M-l. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.

Quantitative Measure of Cytotoxicity of Anticancer Drugs and Other Agents.

Zhimin Tao et al. — Wed, 27 Mar 2013 09:44:26 PDT

Many anticancer drugs act on cancer cells to promote apoptosis, which includes impairment of cellular respiration (mitochondrial O(2) consumption). Other agents also inhibit cellular respiration, sometimes irreversibly. To investigate the sensitivity of cancer cells to cytotoxins, including anticancer drugs, we compare the profiles of cellular O(2) consumption in the absence and presence of these agents. Oxygen measurements are made at 37 degrees C, using glucose as a substrate, with [O(2)] obtained from the phosphorescence decay rate of a palladium phosphor. The rate of respiration k is defined as -d[O(2)]/dt in a sealed container. Different toxins produce different profiles of impaired respiration, implying different mechanisms for the drug-induced mitochondrial dysfunction. The decrease in the average value of k over a fixed time period, I, is proposed as a characteristic value to assess mitochondrial injury. The value of I depends on the nature of the toxin, its concentration, and the exposure time as well as on the cell type. Results for several cell types and 10 cytotoxins are presented here.

Surface Tensions of Molten Salt Mixtures

Jerry Goodisman — Wed, 27 Mar 2013 09:44:24 PDT

It is always found that the surface tension of a mixture of molten salts lies below the mean of the surface tensions of the pure components, weighted by mole fractions. Part of this effect is certainly due to the well-known fact that the composition near the surface is enriched in the component of lower surface tension. We show that an effect of the long-range forces, present for fluids involving Coulombic interactions, is of greater importance. The effect is due to the electroneutrality constraint on the distribution functions. As we show by separate calculations, both effects must be considered to obtain good agreement with experimental results for alkali halide melts.

Methyl Proton NMR Shifts of Coordinated Methanol in a Series of Co(CH,0H),X2+ Compounds

J.R Vriesenga et al. — Wed, 27 Mar 2013 09:44:22 PDT

The isotropic contact NMR shifts of the coordinated cis and tram methanol methyl proton resonances of a series of Co(CH30H)5X2+ complexes were measured, where the ligand X was pyridine, /I- and a-picoline, DMF, DMSO, acetonitrile, and water. The question of how to separate the observed shifts into their respective Fermi contact and dipolar (pseudocontact) contributions was examined in detail with the result that a new procedure for carrying out the separation was proposed. The dipolar shifts for these complexes were found to vary widely, with the acetonitrile complex exhibiting the largest dipolar shift. For the DMSO and water complexes the sign of the dipolar shift was reversed relative to the others. Qualitatively this sign reversal may be related to the difference in ligand field strengths of methanol and X relative to the spin orbit soupling constant for cobalt (II). It was also concluded that the Fermi contact shift varied between complexes.