Evelyn L. McGown

Reduction of extracellular methemoglobin by erythrocytes.

Erythrocytes, suspended in a glucose-containing buffer, catalyzed the partial reduction of extracellular methemoglobin. Physiological concentrations of ascorbic acid or dehydroascorbic acid greatly enhanced the rate of reaction and the ultimate extent of reduction. The relationship between erythrocyte concentration and initial reaction rate was nonlinear, which suggested that the rate limiting factor was not an erythrocyte membrane enzyme. Also, significant dehydroascorbate-stimulated reduction occurred even when the erythrocytes and methemoglobin were separated by a dialysis membrane. The above observations indicate that the transfer of reducing equivalents across the erythrocyte membrane and reduction of extracellular methemoglobin can be accomplished by release and recycling of ascorbic acid.

In vitro amino acid incorporation by the post-mitochondrial supernatant from rat liver

Abstract Interest in the use of the post-mitochondrial supernatant (PM-supernatant) for the study of in vitro amino acid incorporation arose from experiments indicating that the ribosomal aggregation of the PM-supernatant system more nearly represented that of the intact cell than did other ribosomal preparations. The PM-supernatant system was found to be 25 or 50 % more active than the microsomal and ribosomal amino acid incorporation systems. For PM-supernatant in vitro incorporation of l -[ 14 C]valine into acid insoluble material, ATP, GTP, an energy generating system, Mg 2+ , K + , and a mixture of l -amino acids were necessary. Sephadex G-25 column chromatography of the PM-supernatant, pH 7.4, and presence of glutathione during homogenization of the tissue were necessary for optimal PM-supernatant in vitro incorporation. Under these conditions the rate of l -valine incorporation by the PM-supernatant from 0.17 to 0.20 nmoles/min per mg RNA for a liver from a fed rat. Fasting (20 h) prior to sacrifice, resulted in a 30–40 % decrease in amino acid incorporation by the PM-supernatant as well as a decrease in ribosomal aggregation. The decrease in PM-supernatant incorporation by fasting was found to be associated with both polysomal and cell sap factors.

One-dimensional and two-dimensional nuclear magnetic resonance studies of the reaction of phenyldichloroarsine with glutathione

14C-labeled phenyldichloroarsine (PDA) enters the red blood cell and forms a 1:2 adduct with intracellular glutathione. Upon gel filtration of the hemolysate, [14C]PDA was recovered with the glutathione-containing fractions. One-dimensional and two-dimensional nuclear magnetic resonance spectroscopy were used to confirm the structure of the adduct and elucidate its stereochemistry, stability, and reactivity.

Anomalies in polysome profiles caused by contamination of the gradients with Cu2+ or Zn2+

The presence of 30 μM Cu2+ or Zn2+ in sucrose gradients used to determine the extent of ribosome aggregation in rat liver preparations resulted in profiles which were abnormally aggregated. This problem was eliminated by inclusion of EDTA in the gradient or by the use of detergents other than deoxycholate.

Regeneration of functional hemoglobin from iron(III) hemoglobin by reduction with hydrogen and a heterogeneous catalyst

Functional hemoglobin was regenerated from partially autoxidized hemoglobin by reduction with molecular hydrogen in the presence of a heterogeneous catalyst consisting of elemental platinum embedded in an electroactive polymer. The visible spectrum of the regenerated hemoglobin was identical to that of native iron(II) hemoglobin. The regenerated hemoglobin displayed highly cooperative oxygen-binding characteristics. P50 values for oxidized-regenerated hemoglobin samples were not different from native hemoglobin. The Hill coefficients for regenerated hemoglobin were slightly lower than the controls, possibly because of small amounts of irreversibly oxidized hemoglobin arising during the initial autoxidation. The advantages of the reduction system include: (1) the heterogeneous catalyst avoids the problem of protein adsorption onto bare platinum, (2) catalyst and reducing agent are easily removed from the protein, and (3) the by-product H+ is buffered easily.

Reaction of cysteine(s) with phenyldichloroarsine

Abstract : In order to further our studies dealing with the interaction of organic arsenicals with biological sulfhydryl-containing molecules, we investigated the reaction of phenyldichloroarsine with L-cysteine, and two derivatives of L-cysteine in methanol-d4. We found that cysteine (in contrast to glutathione in H2O) formed a mono-cysteine adduct when the L-Cys/PDA ratio was < or = 1:1 (2). When cysteine was present in two molar equivalents, the expected 2:1 adduct was formed via chelation of the two sulfur atoms of cysteine to the arsenic atom (3). Both L-cysteine methyl ester and N-acetyl-L-cysteine reacted like L-cysteine and formed a 1:1 adduct when the ratio was < or =. (jg)

Regeneration of functional hemoglobin from partially oxidized hemoglobin in the presence of molecular hydrogen and a multicomponent redox catalyst

Publisher Summary This chapter discusses the regeneration of functional hemoglobin from partially oxidized hemoglobin in the presence of molecular hydrogen and a multicomponent redox catalyst. Molecular hydrogen offers a significant advantage over the reducing agents because it is mild and its oxidation product, H + , can be buffered easily. Polymeric redox catalysts offer a promising approach to the use of molecular hydrogen for methemoglobin reduction. Multicomponent redox catalysts are synthesized in which atoms of metallic platinum are imbedded in an electroactive polymer synthesized from a monomeric bipyridyl derivative. For convenience, the polymeric catalyst is coated onto an inert material with high surface area. With such a catalyst, molecular hydrogen regenerates functional hemoglobin from partially oxidized hemoglobin. Reversible O 2 binding is restored for regenerated ferrous hemoglobin, with only slight reductions in cooperativity and hemoglobin stability. The advantages of the reduction system include the following considerations: (1) the heterogeneous catalyst avoids the problem of protein adsorption onto bare platinum, (2) catalyst and reducing agent are easily removed from the protein by simple filtration, and (3) the by-product, H + is buffered easily.

Preparation, structure, and solution dynamics of phenyldichloroarsine-thio sugar adducts

In order to study the interactions betwen trivalent organic arsenicals and biologically relevant molecules, we investigated the structure and solution dynamics of the 1:2 adducts formed between phenyldichloroarsine (PDA) and the two thio sugars, 1-thio-β-D-glucopyranose and 1-thio-β-D-galactopyranose

Kinetic Study of the Reduction of Methemoglobin with Ascorbate Using a COBAS-FARA Centrifugal Analyser

The reduction-kinetics of methemoglobin with ascorbic acid were monitored using a COBAS-FARA centrifugal analyzer. The data from the initial rates for the reduction of hemoglobin were monitored as a function of the ascorbate concentration. The results show a first order dependence of the reaction rates on the ascorbate concentration and the rate constant was found to be 1.34 × 10 −2 s −1 M −1 . Ascorbic acid is a one electron donor in this process and consequently forms a stable radical. The presence of oxyhemoglobin interferes with the reduction of methemoglobin by ascorbate

Reaction of trans-2-Chlorovinylarsine Oxide with Polydeoxynucleotides

Trans-2-chlorovinylarsine oxide (in DCl/acetone-d6) was added to various polydeoxynucleotides. The arsenical did react with poly[dG].poly[dC], releasing guanine, and resulting in a partial apurinic duplex.