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W E Antholine

Publications and source records attributed to W E Antholine.

At least 37 records · Page 2Linked to original sources
PubMed1997

Fe- and Co-bleomycins bound to site specific and nonspecific DNA decamers: comparative binding and reactivity of their metal centers.

Co- and Fe-bleomycins (Blms) have been reacted with DNAa, d(GGAAGCTTCC)2, containing a specific site for cleavage, and DNAb, d(GGAAATTTCC)2, a closely related nonspecific 10-mer, to survey whether features of structure and reactivity of these adducts vary systematically as a function of the base sequence of the DNA oligomer. The ESR spectrum of NO-Fe(II)BlmDNAa is rhombically perturbed in comparison with that of NO-Fe(II)BlmDNAb, which is nearly identical to the spectrum of NO-Fe(II)Blm. The ESR spectrum of Fe(III)BlmDNAa in phosphate buffer is low-spin; that of Fe(III)BlmDNAb is high-spin as seen with Fe(III)Blm alone. According to absorbance spectroscopy, O2-Fe(II)BlmDNAa is stabilized in comparison with the DNAb adduct. Similar stabilization of O2-Co(II)Blm bound to DNAa but not to DNAb was also observed by ESR spectroscopy. HO2(-)-Co(III)Blm A2 binds in slow exchange on the NMR time scale to DNAa at its 5'-G-pyrimidine-3' site of cleavage. In contrast, fluorescence and NMR spectroscopy demonstrate that most of HO2(-)-Co(III)Blm A2 binds stoichiometrically in fast exchange to DNAb. The reactions of Fe(III)BlmDNAa and Fe(III)BlmDNAb with ascorbate and O2 reveal that the latter becomes activated and cleaves its 10-mer, producing base propenals, at a faster initial rate. Thus, in two series of metallobleomycins, (A) NO-Fe(II)Blm, O2-Fe(II)Blm, Fe(III)Blm in phosphate buffer, and HO2(-)-Fe(III)Blm and (B) O2-Co(II)Blm and HO2(-)-Co(III)Blm, the metal domain of each species interacts differently with DNA depending upon its base sequence.

Antibiotics, Antineoplastic↗
PubMed1997

Interaction of nitrosyl Fe(II) complex of bleomycin with DNA.

ESR spectra of Fe(III)BLM and NO-Fe(II)BLM on highly oriented DNA-fibers indicated that not only the iron binding domain of the BLM but also the orientation of NO bond axis is fixed with respect to the DNA-double helical axis at room temperature.

Animals↗
PubMed1996

Metal coordination environment and dynamics in 113cadmium bleomycin: relationship to zinc bleomycin.

The 13C chemical shifts of Cd- and ZnBlm A2 are almost identical throughout the entire molecule, suggesting that these structures adopt similar conformations. Nuclear magnetic resonance experiments with 113Cd-bleomycin have defined part of the metal-ligand environment of the molecule. Nitrogen atoms from the primary amine, pyrimidine, and imidazole are bound to 113Cd according to 13C spectra showing 113Cd-13C spin-spin couplings. Bound and free forms of the secondary amine nitrogen may be in equilibrium, as suggested by temperature-dependent 13C studies with Cd-bleomycin. In addition, a number of other carbon resonances are in chemical exchange over the temperature range 5-54 degrees C. The temperature dependence of the line widths of carbon atoms of Zn-bleomycin strongly resembles that of Cd-bleomycin. Examination of the 113Cd resonance as a function of temperature also supports the presence of at least two differently coordinated forms of cadmium in the molecule. According to the position of the 113Cd chemical shift, at most four nitrogen atoms are bound to Cd at low temperature. Titrations of 113Cd-bleomycin with chloride or acetate demonstrate that these anions can bind to major and minor forms of the structure and that a minor species exists which does not associate with chloride.

Antibiotics, Antineoplastic↗
PubMed1996

Release of CuPTSM from human serum albumin after addition of fatty acids.

Copper-62 labeled pyruvaldehyde bis (N4-methyl-thiosemicarbazonato) copper(II), CuPTSM, has been used to probe tissue perfusion by means of positron emission tomography. Despite promising results from animals, problems have been encountered in the use of 62CuPTSM to quantitate regional myocardial blood flow in humans. Ultrafiltration and plasma/erythrocyte partitioning studies with radiotracer have previously shown that CuPTSM is bound much more strongly by human serum albumin (HSA) than by dog serum albumin (DSA), limiting its ability to freely diffuse from blood into tissue. In this study, it is confirmed by electron spin resonance (ESR) that CuPTSM strongly binds to HSA with an apparent gparallel value of 2.12 and an apparent Aparallel value of 186 G. It is also shown that both spin-labeled stearic acid (5-SASL) and nonspin-labeled stearate inhibit CuPTSM binding to HSA. CuPTSM is completely released from HSA when the ratio of 5-SASL to HSA is 5:1. When pure sodium stearate is used, the binding of CuPTSM significantly decreased, about 73% of CuPTSM is released with a ratio of 4:1 stearate to HSA. These results highlight a means of liberating CuPTSM from HSA.

Electron Spin Resonance Spectroscopy↗
PubMed1995

An electron paramagnetic resonance method for quantification of copper isotopes in proteins: application to a mixed-isotope derivative of laccase.

We have developed a method of denaturing a copper-containing protein and sequestering the copper in the form of the bis(acetylacetonato)copper(II) complex. Because of the excellent resolution of the hyperfine structure in the resulting EPR spectrum, the method is useful for determining the 65Cu/63Cu isotope ratio in proteins such as tree laccase. Here we analyzed a mixed isotope sample prepared by reconstituting 63Cu-enriched type 2-depleted laccase with 65Cu(I) and found that it contained only 25% 65Cu. This proves that reconstitution of the type 2 copper is possible under conditions that permit little or no exchange between resident copper and the external pool. However, exchange does occur under reducing conditions when we expose the holoprotein to a large excess of exogenous copper.

Binding Sites↗
PubMed1995

Electron spin-lattice relaxation of the [Cu(1.5) ... Cu(1.5)] dinuclear copper center in nitrous oxide reductase.

Relaxation times have been obtained with time-domain EPR for the dinuclear mixed valence [CuA(1.5) ... CuA(1.5)[ S = 1/2 center in nitrous oxide reductase, N2OR, from Pseudomonas stutzeri, in the TN5 mutant defective in copper chromophore biosynthesis, in a synthetic mixed valence complex, and in type 1 and 2 copper complexes. Data confirmed that the intrinsic electron spin-lattice relaxation time, T1, for N2OR in the temperature range of 6-25 K is unusually short for copper centers. At best, a twofold increase of T1 from g perpendicular to g parallel was measured. Optimized fits of the saturation-recovery data were obtained using both double-exponential and stretched-exponential functions. The temperature dependence of the spin-lattice relaxation rate of mutant N2OR is about T5.0 with the stretched-exponential model or T3.3 and T3.9 for the model using the sum of two exponentials. These T1s are intrinsic to the mixed valence [CuA(1.5) ... CuA(1.5)] center, and no interaction of the second copper center in wild-type N2OR with the [CuA(1.5) ... CuA(1.5)] center has been observed. The T1 of the mixed valence center of N2OR is not only shorter than for monomeric square planar Cu(II) complexes, but also shorter than for a synthetic mixed valence complex, Cu2(N[CH2CH2NHCH2CH2NHCH2CH2]3N). The short T1 is attributed to the vibrational modes of type 1 copper and/or the metal-metal interaction in [CuA(1.5) ... CuA(1.5)].

Amino Acid Sequence↗
PubMed1994

Iron requirement for cellular DNA damage and growth inhibition by hydrogen peroxide and bleomycin.

Studies with Euglena gracilis and HL-60 cells have assessed the need for intracellular iron in the mechanisms of inhibition of cell growth and DNA damage by H2O2 and bleomycin. Cell culture media were directly depleted of iron in order to deprive cells of nutrient iron. Major pools of cellular iron were reduced in both cell types. Nevertheless, iron bound in e.s.r.-observable haem protein and ribonucleotide diphosphate reductase in HL-60 cells was not decreased. In both control cell populations, there was a concentration-dependent reduction in proliferation and cell survival caused by H2O2. In comparison, the proliferation rates of both iron-deficient cell types were significantly less sensitive to H2O2. H2O2 caused concentration-dependent single-strand breakage in DNA in control HL-60 and Euglena gracilis cells. Iron deficiency reduced the amount of strand breaks in HL-60 cells at each concentration of H2O2 used. Single-strand breakage caused by H2O2 in Euglena gracilis was a direct function of the concentration of iron in which the cells had been grown. Growth inhibition and both single- and double-strand DNA damage caused by bleomycin were substantially reduced or eliminated in iron-deficient cells. Copper bleomycin behaved like metal-free bleomycin when assayed for the capacity to cause DNA damage in iron-normal and iron-deficient HL-60 cells. In contrast, iron bleomycin was equally active under the two conditions in these cells.

Animals↗
PubMed1994

P. mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: electron paramagnetic resonance investigation of the copper binding site.

The copper binding site in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase has been investigated by multifrequency electron spin resonance spectroscopy. Methodology has been developed to introduce copper in vitro into the isolated apoenzyme. The X-band EPR of Cu2+ (mixed isotopes or 63Cu2+) introduced in this way is very similar to the EPR spectra of samples in which copper is introduced during protein expression. g parallel and A parallel values in the X-band spectra support the prediction of nitrogen ligands to the tightly bound copper. In the g parallel region of S-band EPR spectra (mI = -1/2) of lyase-bound 63Cu2+, superhyperfine interactions due to nitrogen ligands are observed. Computer simulation with appropriate g values and A values and strain parameters was used to satisfactorily model both the X-band and the S-band spectra. The g parallel value of 2.282 and the A parallel value of 470 MHz are consistent with two nitrogen and two oxygen donor atoms for a square planar type 2 copper center. By simulating the mI = -1/2 line of the S-band spectrum, the superhyperfine features could be well modeled. This simulation approach was also used to distinguish between two and three nitrogen donor atoms. Based on the intensity patterns of the superhyperfine lines and the estimated coupling constants, it is concluded that at least two (and probably only two) nitrogen donor atoms are liganded to the tightly bound copper in HMG-CoA lyase. Additionally, kinetic experiments demonstrate that a spin-labeled substrate analog (R.CoA) is a competitive inhibitor of HMG-CoA lyase (KI = 98 microM). ESR titration experiments indicate that R.CoA binds to lyase with an equilibrium dissociation constant of 103 microM. Bound spin label exhibits a rotational correlation time, tau c, of 20 ns, in agreement with the value predicted for immobilization on a protein composed of two 32-kDa subunits.

Acyl Coenzyme A↗
PubMed1993

Metallothionein reacts with Fe2+ and NO to form products with A g = 2.039 ESR signal.

The interaction of metallothionein, Fe2+, and nitric oxide has been examined by electron spin resonance spectroscopy. When either apoMT 2 or Zn-MT 2 from rabbit liver was mixed with Fe2+ and NO, paramagnetic products with g values of 2.013 and 2.039 were found. These are characteristic of iron nitrosyl thiolates with stoichiometry, Fe(NO)2(SR)2. This product was not generated when beef heart mitochondrial aconitase was used as the source of iron.

Animals↗
PubMed1993

Low frequency EPR of Pseudomonas aeruginosa azurin. Analysis of ligand superhyperfine structure from a type 1 copper site.

The type 1 copper in Pseudomonas aeruginosa azurin was studied by electron paramagnetic resonance (EPR) spectroscopy at low microwave frequencies. Partially resolved ligand hyperfine structure was observed in the perpendicular region of the spectra at both S-band (2.4 GHz) and L-band (1.1 GHz). A trial and error method, requiring several hundred simulations, has been used to simulate the low frequency EPR data and yield an optimum value of 30 MHz for ACUx, more than one half that previously reported. The fit between the simulated and experimental data is sensitive to changes in the Euler angles and, in particular, to the angle alpha which rotates the Cu A-tensor about the z-axis. Thus, the A- and g-tensors for copper in P. aeruginosa azurin do not appear to be coincident. A value for the Euler angle beta of at least 10 degrees does not disturb the fit between the simulated and experimental data. These studies demonstrate the advantage of evaluating EPR parameters from simulations at more than one frequency, especially at low frequencies where ligand superhyperfine structure may be resolved for type 1 copper.

Azurin↗
PubMed1993

Multiquantum EPR of the mixed valence copper site in nitrous oxide reductase.

This work demonstrates the use of multiquantum EPR to study the magnetic properties of copper complexes and copper proteins. Pure absorption spectra are obtained because of the absence of field modulation. The signal intensity of 3-quantum spectra is proportional to the spin lattice relaxation time T1, while its linewidth in a frequency difference sweep is T1(-1). A change in lineshape for the EPR detectable mixed value [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase is attributed to suppression of the forbidden transitions. The data confirm the unusually fast relaxation time for this site, which requires temperatures of less than 100 K to resolve hyperfine structure. The T1's for the mixed valence [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase are very similar to T1's for the Cua site in cytochrome c oxidase. The similar relaxation properties, together with previous multifrequency EPR results, support the hypothesis that the EPR detectable sites in cytochrome c oxidase and nitrous oxide reductase are mixed valence [Cu(1.5) . . . Cu(1.5)] configurations.

Binding Sites↗
PubMed1993

Low dietary iron prevents free radical formation and heart pathology of copper-deficient rats fed fructose.

Two studies were conducted to determine whether hepatic iron overload in rats fed fructose plays a role in the exacerbation of the signs associated with copper deficiency. When fed the adequate iron diet (50 micrograms Fe/g), copper-deficient rats fed either fructose or starch exhibited hepatic iron overload of similar magnitude. However, only livers of copper-deficient rats fed fructose exhibited the presence of high peaks associated with an iron compound detected by electron spin resonance. In addition, only copper-deficient rats fed fructose developed anemia, pancreatic atrophy, and heart hypertrophy with histopathologic changes, and they died prematurely of heart-related abnormalities. Lowering dietary iron from 50 micrograms/g to 30 micrograms/g was not sufficient to protect the animals against the pathologic consequences of copper deficiency. In contrast, the consumption of a fructose diet inadequate in both copper (0.6 micrograms/g) and iron (17 micrograms/g) resulted in the reduction of hepatic iron, which in turn caused the amelioration of the deficiency, compared with rats fed the adequate iron (50 micrograms/g) diet. None of these rats developed pancreatic atrophy, none exhibited myocardial lesions, and none died of the deficiency. Electron spin resonance spectra of their livers did not show the presence of free radicals. The data suggest that hepatic iron overload plays a role in the exacerbation of copper deficiency only when fructose diets are consumed.

Animals↗
PubMed1992

Effect of gallium on the tyrosyl radical of the iron-dependent M2 subunit of ribonucleotide reductase.

Gallium, a pharmacologically important metal which resembles iron, was shown in previous studies to inhibit ribonucleotide reductase. To better understand its mechanism of action, we have examined the interaction of gallium with the iron-dependent M2 subunit of ribonucleotide reductase. In its active form, M2 contains an iron center and a tyrosyl free radical which is detectable by ESR spectroscopy. In the present study, cytoplasmic extracts prepared from murine leukemic L1210 cells after an 18-hr incubation with 960 microM gallium nitrate displayed a > 60% inhibition in their M2 tyrosyl radical ESR signal. However, this signal was restored within 15 min to levels greater than that of controls by the addition of increasing concentrations of ferrous ammonium sulfate. Gallium citrate added directly to cytoplasmic extracts from control cells also decreased the tyrosyl radical signal, an effect which could be reversed by iron. Immunoblot analysis revealed that incubation with gallium did not diminish the amount of M2 protein in cells, thus indicating that the decrease in the tyrosyl radical signal was not due to a decrease in cellular M2 content. In immunoprecipitation studies of 59Fe-labeled M2, gallium displaced 55-60% of the 59Fe incorporated into M2. Our studies suggest that gallium displaces iron from the M2 subunit of ribonucleotide reductase, resulting in a loss of the tyrosyl radical and an accumulation of inactive M2 within the cell.

Animals↗
PubMed1992

A comparative EPR investigation of the multicopper proteins nitrous-oxide reductase and cytochrome c oxidase.

The multicopper proteins, nitrous-oxide reductase (N2OR) and cytochrome c oxidase (COX), were investigated by EPR spectroscopy at microwave frequencies 2.4-35 GHz. Our results support a Cu-Cu interaction in COX and N2OR. At least 10 lines in the 2.7-GHz, 12 lines in the 4.6-GHz and 14 lines in the 9.2 GHz spectra were resolved for N2OR. Eight copper lines at 2.7 GHz, about nine lines at 4.6 GHz and about six lines at 9.2 GHz were resolved for COX. Simulations of the EPR spectra were consistent with most of the resonances of the multiline spectra, including regions in the center of the spectra where overlap of the three seven-line patterns is proposed. These simulations indicated that Cu-Cu interaction, in a mixed-valence [Cu(1.5) ... Cu(1.5)], S = 1/2 site is consistent with, if not proof of, the unusual spectral features observed for N2OR and COX.

Animals↗
PubMed1992

Reaction of cyanide with cytochrome ba3 from Thermus thermophilus: spectroscopic characterization of the Fe(II)a3-CN.Cu(II)B-CN complex suggests four 14N atoms are coordinated to CuB.

Cytochrome ba3 from Thermus thermophilus reacts slowly with excess HCN at pH 7.4 to create a form of the enzyme in which CuA, cytochrome b, and CuB remain oxidized, while cytochrome a3 is reduced by one electron, presumably with the formation of cyanogen. We have examined this form of the enzyme by UV-visible, resonance Raman, EPR, and electron nuclear double resonance spectroscopies in conjunction with permutations of 13C- and 15N-labeled cyanide. The results support a model in which one CN- binds through the carbon atom to ferrous a3, supporting a low-spin (S = 0) configuration on the Fe; bridging by this cyanide to the CuB is weak or absent. Four 14N atoms, presumably donated by histidine residues of the protein, provide a strong equatorial ligand field about CuB; a second CN- is coordinated through the carbon atom to CuB in an axial position.

Binding Sites↗
PubMed1992

Properties of redox-inactivated bleomycins. In vitro DNA damage and inhibition of Ehrlich cell proliferation.

Blenoxane, bleomycin A2, bleomycin B2, and demethyl bleomycin A2 and products of their reactions with Fe2+ and oxygen were used to explore the relationship between their capacity to carry out in vitro DNA strand scission and their growth inhibitory activity against Ehrlich cells. Reaction of Fe2+, bleomycin and O2 in the absence of DNA decreased the subsequent effectiveness of various bleomycin congeners to degrade DNA in the presence of Fe2+ and oxygen. In comparison with controls, this loss of strand scission activity was not paralleled by equivalent decreases in growth inhibition. Demethyl bleomycin A2 retained full biological activity relative to bleomycin A2, despite being only 30% as effective as bleomycin A2 in its ability to cleave DNA in vitro. Prior reaction of bleomycins with Fe2+ did not alter their capacity to reduce oxygen or affect their ability to generate the activated intermediate which, for native bleomycin structures, is competent to cleave DNA in vitro.

Animals↗
PubMed1992

Reaction of Co(II)bleomycin with dioxygen.

The reaction of Co(II)bleomycin with dioxygen has been investigated. Dioxygen binds to the Co(II) complex within the time of mixing according to electron spin resonance and uv-visible spectroscopy and dioxygen analysis. Then, two dioxygenated cobalt centers react, releasing 1 mol of O2 and forming an intermediate characterized by a few highly shifted 1H NMR resonances and loss of the ESR spectrum. This is thought to be a dioxygen-bridged dimer of cobalt bleomycin molecules. Time-dependent absorbance and dioxygen measurements yield the same second order rate constant for this step of the reaction. According to uv-visible and NMR spectral analysis, the intermediate decays into diamagnetic products in a first order rate process. High performance liquid chromatography and 1H NMR studies demonstrate that the product contains two bleomycin species of equal concentration. One component is Co(III)bleomycin, designated Form II. The other is the peroxide adduct of Co(III)bleomycin, Form I, as determined by direct determination of hydrogen peroxide, which is slowly released from the product at low pH. In contrast, hydrogen peroxide is readily detected during the reaction of Co(II)Blm with O2. In isolation, Form I is unstable at pH 7 and is converted within 24 h into a mixture of Form I and Form II.

Bleomycin↗