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Biomedical subjects

P Saltman

Publications and source records attributed to P Saltman.

At least 19 recordsLinked to original sources
PubMed1999

Oxidation of deoxy myoglobin by [Fe(CN)6]3-.

The ability of myoglobin (Mb) to reversibly bind O2 and other ligands has been well characterized. Mb also participates with a variety of redox metals to form metmyoglobin (metMb). By using an anaerobic stopped-flow device we have measured outer-sphere oxidation by [Fe(CN)6]3 of native sperm whale myoglobin, recombinant wild-type Mb, and a series of mutant Mb proteins in which the distal His-64 was changed to Gly, Phe, Leu or Val. Second-order rate constants for oxidation of mutant proteins are 10-15 times greater than for recombinant or native (kox approximately 10(6) M-1 s-1). We attribute the reduced rate of oxidation of wild-type protein to a higher reorganization energy imposed by the presence of the unique water/His-64/heme interaction, which is absent in the mutant proteins.

Amino Acid Substitution↗
PubMed1999

Neuroelectric assessment of nutrient intake.

Electroencephalographic (EEG) activity and auditory event-related brain potentials (ERPs) were assessed in two groups (n = 12 each) of subjects. The 'food-nutrient' group had fasted from the night before and consumed a 500 cal nutrient drink; the 'control' group consumed breakfast but did not consume any nutrients during the recordings. All subjects were assessed every 15 min for six trial blocks at the same time of day, with the fast/nutrient group measured initially before and after consuming the nutrient drink. No effects of the nutrient drink were obtained on the post-stimulus EEG spectral power or mean frequency measures. However, the fast/nutrient group demonstrated less delta, theta, and alpha-1 power than the no-fast/control group. Increases in spectral power were generally observed across trial blocks especially for the alpha and beta bands, and are consistent with increases in arousal level. P300 amplitude was unaffected by the nutrient consumption, but target stimulus N100 amplitude was smaller for the food-nutrient compared to the control group. Taken together, the results suggest that nutrient consumption does not directly affect EEG or cognitive ERP measures.

Adolescent↗
PubMed1997

Copper-specific damage in human erythrocytes exposed to oxidative stress.

Ascorbate and complexes of Cu(II) and Fe(III) are capable of generating significant levels of oxygen free radicals. Exposure of erythrocytes to such oxidative stress leads to increased levels of methemoglobin and extensive changes in cell morphology. Cu(II) per mole is much more effective than Fe(III). However, isolated hemoglobin is oxidized more rapidly and completely by Fe(III)- than by Cu(II)-complexes. Both Fe(III) and Cu(II) are capable of inhibiting a number of the key enzymes of erythrocyte metabolism. The mechanism for the enhanced activity of Cu(II) has not been previously established. Using intact erythrocytes and hemolysates we demonstrate that Cu(II)-, but not Fe(III)-complexes in the presence of ascorbate block NADH-methemoglobin reductase. Complexes of Cu(II) alone are not inhibitory. The relative inability of Fe(III)-complexes and ascorbate to cause methemoglobin accumulation is not owing to Fe(III) association with the membrane, or its failure to enter the erythrocytes. The toxicity of Cu(II) and ascorbate appears to be a result of site-specific oxidative damage of erythrocyte NADH-methemoglobin reductase and the enzyme's subsequent inability to reduce the oxidized hemoglobin.

Copper↗
PubMed1996

Hemoglobin: a mechanism for the generation of hydroxyl radicals.

Oxyhemoglobin (HbO2) reduces Fe(III)NTA aerobically to become methemoglobin (metHb) and Fe(II)NTA. These conditions are favorable for the generation via Fenton chemistry of the hydroxyl radical that was measured by HPLC using salicylate as a probe. The levels of hydroxyl radicals generated are a function of both the percent metHb formed and the chemical nature of the buffer. The rates of formation of both metHb and hydroxyl radicals were dependent upon the concentration of Fe(III)NTA. Of the buffers tested, HEPES was the most effective scavenger of hydroxyl radicals while the other buffers scavenged in the order: HEPES > Tris > MPOS > > NaCL approximately unbuffered. The addition of catalase to remove H2O2 or bathophenanthroline to chelate Fe(II) inhibited virtually all hydroxyl radical formation. Carbonyl formation from free radical oxidation of amino acids was found to be 0.1 mol/mol of hemoglobin. These experiments demonstrate the ability of hemoglobin to participate directly in the generation of hydroxyl radicals mediated by redox metals, and provide insight into potential oxidative damage from metals released into the blood during some pathologic disorders including iron overload.

Amino Acids↗
PubMed1995

Low-level oxidative stress causes cell-cycle specific arrest in cultured cells.

Oxidative stress is involved in a multitude of pathological conditions. In the present study, cell cycle arrest was demonstrated in monolayer cultures of Chinese hamster ovary cells subjected to low-level oxidative stress induced by the addition of hydrogen peroxide. Fluorescence-activated cell sorting analysis characterized this arrest as occurring in both the G1 and G2/M phases of the cell cycle. Upon exposure to oxidative stress, cells in G1 arrest in G1, while S-phase cells complete DNA synthesis and subsequently arrest in G2/M. Nitroxide spin labels with differing permeabilities are able to protect the cells, which suggests that both the G1 and the G2/M arrest involve oxidation of targets located at the cell surface.

3T3 Cells↗
PubMed1995

Sites and mechanisms of low-level oxidative stress in cultured cells.

Oxidative stress is involved in a multitude of pathological conditions. In the present study, we investigated the cellular targets and the mechanisms of low-level oxidative stress in a Chinese Hamster Ovary cell culture. Oxidative stress was induced either by continuous enzymatic production of superoxide or by bolus addition of hydrogen peroxide (H2O2). Low-level oxidative stress irreversibly impaired the reproductive capacity of the cells in the absence of damage to membrane integrity or energy metabolism. Cells were protected by catalase but not by superoxide dismutase, indicating that H2O2, not superoxide, was the causative agent of cell damage. Nitroxide spin labels decreased hydroxyl radical (.OH) formation and protected cells from the oxidative stress. The differing membrane permeabilities of these spin labels suggest that the damage is localized on the cell surface. Oxidative stress to DNA and RNA was not significant, as shown by levels of guanine hydroxylation products. A mechanism is proposed whereby low-level oxidative stress acts at the cell surface to cause impairment of cell reproduction.

Animals↗
PubMed1994

Zinc(II) and copper(II) binding to serum albumin. A comparative study of dog, bovine, and human albumin.

Metal binding strategies employing low molecular weight chelators and equilibrium dialysis were used to investigate several unresolved aspects of zinc and copper binding to serum albumin. Direct measurement of histidine binding to bovine serum albumin when the histidine is presented either as a metal-chelate or alone provides no evidence for an albumin-metal-histidine ternary complex. Using previously determined intrinsic constants for Zn(II) and Cu(II), we have measured zinc binding to bovine serum albumin in the presence of saturating amounts of copper. The results of these experiments unambiguously show that zinc and copper bind at separate noninteracting sites on this protein. The intrinsic constants for zinc and copper binding to dog serum albumin have been determined. Contrary to previous reports, we find that dog serum albumin has a specific high affinity site for copper, log10K 10.17 for Cu(II) compared to 6.85 for Zn(II) at the separate site.

Animals↗
PubMed1994

Spinal bone loss in postmenopausal women supplemented with calcium and trace minerals.

The effects of calcium supplementation (as calcium citrate malate, 1000 mg elemental Ca/d) with and without the addition of zinc (15.0 mg/d), manganese (5.0 mg/d) and copper (2.5 mg/d) on spinal bone loss (L2-L4 vertebrae) was evaluated in healthy older postmenopausal women (n = 59, mean age 66 y) in a 2-y, double-blind, placebo-controlled trial. Changes (mean +/- SEM) in bone density were -3.53 +/- 1.24% (placebo), -1.89 +/- 1.40% (trace minerals only), -1.25 +/- 1.46% (calcium only) and 1.48 +/- 1.40% (calcium plus trace minerals). Bone loss relative to base-line value was significant (P = 0.0061) in the placebo group but not in the groups receiving trace minerals alone, calcium alone, or calcium plus trace minerals. The only significant group difference occurred between the placebo group and the group receiving calcium plus trace minerals (P = 0.0099). These data suggest that bone loss in calcium-supplemented, older postmenopausal women can be further arrested by concomitant increases in trace mineral intake.

Aged↗
PubMed1993

Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albumin.

Intrinsic stoichiometric equilibrium constants were determined for zinc(II) and copper(II) binding to bovine and human serum albumin. Data were obtained from equilibrium dialysis experiments. Metals were presented to apoprotein as metal chelates in order to avoid metal hydrolysis and to minimize nonspecific metal-protein interactions. Scatchard analysis of the binding data indicated that the high affinity class for both zinc and copper was comprised of one site. Results of binding experiments done at several pH values suggested that while both histidyl and carboxyl groups appear to be involved in copper binding, histidyl residues alone were sufficient for zinc binding. These amino acid residues were used in combination to model several binding sites used in the formulation of equilibria expressions from which stoichiometric constants were calculated. The log10K for bovine serum albumin were calculated to be 7.28 for Zn(II) and 11.12 for Cu(II). Those for human serum albumin were determined to be 7.53 and 11.18 for Zn(II) and Cu(II), respectively. These constants were used in equilibria to simulate speciation of metal-albumin and metal-chelator and to illustrate relative binding affinities. This comparison of binding strengths was possible only through the calculation of an intrinsic stoichiometric binding constant.

Animals↗
PubMed1993

Thiols, gold-thiols, zinc-thiols and the redox state of hemoglobin.

The beta subunit of human hemoglobin can be oxidized site-specifically through beta-Cys-93 by Cu(II)(His)2. A series of thiol ligands, gold thiols and zinc(II) inhibit this oxidation. The thiol inhibitors formed a transient ternary intermediate involving Cu(I) with consequent inhibition of electron transfer from the Fe(II)-heme. The intermediate led to the formation of a disulfide at the beta-Cys-93 site. The most effective thiols achieved maximum inhibition at one equivalent per beta heme. Gold thiols and zinc complexes inhibited heme oxidation by competing with the Cu(II)(His)2 for the beta-Cys-93 site.

Binding Sites↗
PubMed1992

Site-directed mutagenesis of histidine residues involved in Cu(II) binding and reduction by sperm whale myoglobin.

Sperm whale myoglobin (Mb) reduces Cu(II) through a site-specific mechanism involving complexation by one or more surface histidine residues. Three mutants of Mb, derived from recombinant wild-type Mb, were designed in which surface histidine residues exhibiting strong Cu(II) binding were replaced with amino acids with comparatively poor metal binding characteristics. The kinetics of Cu(II)(Gly)2 reduction by native Mb, recombinant wild-type Mb, and the mutants were compared. Recombinant wild-type Mb reduced Cu(II) at a rate similar to that of native Mb. Two single mutations (His-48----Ala and His-116----Asp) decreased the rate by 31% and 7%, respectively, relative to wild-type Mb and decreased the rate by 38% and 16%, respectively, relative to native Mb. A double mutation (His-113----Ala, His-116----Asp) decreased the rate only slightly more than the single mutation at His-116. Previous NMR studies showed that His-113 exhibits the strongest Cu(II) binding of all surface histidines, but the present experiments suggest that it plays little or no role in the reduction of Cu(II) by Mb. His-48, located 12.7 A from the Fe(II)-heme, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role in the overall redox activity of Mb, but its involvement shows that Mb has the ability to reduce Cu(II) through a histidine residue located more than 20 A from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(II) can be mediated through multiple pathways in sperm whale Mb.

Animals↗
PubMed1992

Metal-ion-directed site-specificity of hydroxyl radical detection.

A wide variety of .OH detectors are in use for determination of biological .OH production. The chemical generation of .OH is site-specific with respect to the metal-binding site, and thus .OH detectors with metal-binding properties may affect the biological damage and bias .OH detection. The present study shows that both salicylate and phenylalanine, added as low molecular weight .OH indicators, decreased Cu(II) binding to erythrocyte ghosts. In a cell-free system, Cu(II) complexed to both salicylate and phenylalanine. Phenylalanine is a stronger Cu(II) chelator than salicylate, both when competing for Cu(II) bound to ghosts and when competing directly with each other. When OH radicals were generated by ascorbate and Cu(II), the amount of .OH detected as dihydroxybenzoates was proportional to the amount of .OH produced. However, when phenylalanine was added to this system, the efficiency of .OH detection by salicylate strongly decreased, concomitant with the transfer of Cu(II) binding from salicylate to the amino acid. This decrease was larger than that predicted by calculations for random competition of the two detectors for .OH. Deoxyribose and mannitol, which do not bind copper appreciably, competed poorly with salicylate for the .OH. Hydroxylation of phenylalanine, on the other hand, was only slightly affected by the presence of salicylate and unaffected by deoxyribose and mannitol. These results suggest that the detection of .OH by low molecular weight .OH indicators was related to the relative affinity of the detectors for the catalyzing metal, and thus partially site-specific. Furthermore, glutamate, which does not contain an aromatic ring but binds Cu(II) with considerable affinity, competed strongly with salicylate for the .OH, indicating that metal-binding properties rather than the presence of an aromatic ring were the cause of the deviation from random competition. The results indicate that .OH indicators with metal-binding properties affect the distribution of catalytic metal ions in a biological system, causing a shift of free radical damage and localizing a site-specific reaction of .OH on these detectors, with a resulting positive bias in the apparent .OH production.

Ascorbic Acid↗
PubMed1991

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha.

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.

Binding Sites↗
PubMed1991

Cardiac reperfusion damage prevented by a nitroxide free radical.

Experimental evidence is presented that directly links ischemia/reperfusion injury to the formation of oxygen-derived free radicals. 2,2,6,6-Tetramethylpiperidine-N-oxyl (TEMPO)--a stable nitroxide radical that disproportionates superoxide radicals and oxidizes reduced metal ions required for OH. formation--was tested for its ability to prevent reperfusion damage in the isolated rat heart subjected to regional ischemia. Severe reperfusion arrhythmia--ventricular fibrillation and ventricular tachycardia--were prominent in control hearts, and their duration was significantly reduced by the presence of 0.4 or 1 mM TEMPO. TEMPO also repressed both postischemic release of lactate dehydrogenase and OH. formation. TEMPO slowed the heart rate, but compensatory pacing did not alter the dramatic effect of the nitroxide on reperfusion arrhythmia. TEMPO was partially protective when introduced at the end of ischemia but had no effect when added 1 min into reperfusion. It was concluded that both reperfusion arrhythmia and cell damage were directly related to oxidative damage incurred during the critical first minute of reperfusion. TEMPO strongly protected against reperfusion injury by preventing the formation of OH. and not by decreasing heart rate or by direct suppression of arrhythmia.

Animals↗
PubMed1991

Spinal bone density and calcium intake in healthy postmenopausal women.

Dietary calcium intake and bone mineral density (BMD) of the lumbar spine (L2-L4) were determined in 131 healthy free-living postmenopausal women (aged 64.7 +/- 7.6 y, means +/- SD). The calcium consumption for the total population was 606 +/- 302 mg/d. Subjects consuming less than the population mean of dietary calcium had significantly lower BMDs than did subjects with intakes above the mean (P less than 0.009); these two groups did not differ in basic demographic characteristics. Additional analyses using a stepwise univariate regression model demonstrated that BMD was significantly associated with body weight (P less than 0.001) and dietary calcium intake (P less than 0.02). These data support the hypothesis that dietary calcium intake is a determinant of skeletal health in postmenopausal women.

Aged↗
PubMed1990

Different cellular targets for Cu- and Fe-catalyzed oxidation observed using a Cu-compatible thiobarbituric acid assay.

The widely used thiobarbituric acid (TBA) assay for oxidative damage to biomolecules fails in Cu2(+)-containing solutions due to the formation of a cloudy precipitate. The chelation of Cu2+ ions with EDTA or Chelex was investigated. Both prevented precipitate formation, but only Chelex allowed proper color development in the TBA assay. The Chelex modified assay could be adapted to a variety of systems, and was applied to the detection of Cu2+/ascorbate dependent deoxyribose breakdown and oxidative damage in erythrocyte ghosts, lysates and whole cells. Using this method, it was shown that Cu2+/ascorbate caused membrane damage in ghosts but not in whole red blood cells (RBC). Fe3+/ascorbate, on the other hand, caused formation of TBA-reactive products even in whole RBC. When Cu2+ and Fe3+ were presented to isolated hemoglobin as their 1:1 nitrilotriacetate complexes, the protein bound 10-12 cupric ions per molecule, but no ferric ions. It is suggested that oxidative damage catalyzed by copper or iron ions has different cellular targets, determined by the different binding properties of the two metals to various cellular components.

Chelating Agents↗