ascorbic-acid and methionine-sulfoxide

We examined the metal-catalyzed oxidation of brain-derived neurotrophic factor (BDNF) using the Cu(II)/ascorbate/O2 model oxidative system.. Electrospray ionization mass spectrometry, peptide mapping and amino acid analysis were utilized to determine the nature of the covalent modification induced by the metal-catalyzed oxidative system. Additionally, analytical ultracentrifugation, the Bradford assay, circular dichroism and ANSA dye-binding were used to determine the nature of any conformational changes induced by the oxidation.. Exposure of BDNF to the Cu(II)/ascorbate/O2 system led to the modification of ca. 35% of Met92 to its sulfoxide, and to subsequent conformational changes. The proteolytic digestion procedure was sensitive to this conformational change, and was unable to detect the modification. Chemical digestion with CNBr, however, was not sensitive to this change, and allowed for the identification of the site of modification.. The modification of Met92 to its sulfoxide rendered the oxidized BDNF inaccessible to proteolytic digestion, due to conformational changes associated with the oxidation.

Topics: Amino Acid Sequence; Anilino Naphthalenesulfonates; Ascorbic Acid; Brain-Derived Neurotrophic Factor; Catalytic Domain; Chromatography, High Pressure Liquid; Copper; Cyanogen Bromide; Endopeptidases; Fluorescent Dyes; Methionine; Molecular Sequence Data; Molecular Weight; Oxidation-Reduction; Oxygen; Protein Conformation; Protein Denaturation; Sequence Analysis, Protein; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Ultracentrifugation

The interaction between sodium ascorbate and dopamine was investigated by three different parameters: radical intensity, prooxidant action, and cytotoxicity induction. Sodium ascorbate and dopamine produced the doublet and quartet ESR signals under alkaline conditions (pH 8.0-9.5), respectively. Addition of increasing concentrations of sodium ascorbate completely scavenged the dopamine radical and replaced the latter with its own radical. Similarly, dopamine slightly, but significantly reduced the radical intensity of sodium ascorbate. These two compounds stimulated the methionine oxidation and hydrogen peroxide generation in culture medium, but in combination, their stimulation activities were weakened. Both of these two compounds dose-dependently reduced the viable cell number of human oral squamous carcinoma HSC-4 cells, and their cytotoxic activity was significantly reduced by catalase. When these two compounds were mixed together before adding to HSC-4 cells, both of their cytotoxic activities were diminished. The present study demonstrates the interaction between sodium ascorbate and dopamine, which might modify their biological activities and generation of nerve disorders such as Parkinson's disease.

Topics: Amino Acids; Ascorbic Acid; Catalase; Cell Survival; Dopamine; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Humans; Hydrogen Peroxide; Methionine; Oxidation-Reduction; Parkinson Disease; Tumor Cells, Cultured

Beta amyloid peptides (A beta), etiologically associated with Alzheimer's disease (AD), have been shown to inhibit both glutamine synthetase (GS) and creatine phosphokinase (CPK) in vitro. These two enzymes are affected in AD and are sensitive to oxidative stress. Residue 35 of the A beta 25-35, the most potent section of the 40-42 amino acid long fragment of amyloid precursor protein (APP), is a methionine, which has been reported to be oxidized to methionine sulfoxide presumably via a free radical oxidation process. We questioned whether methionine sulfoxide would inhibit GS and CPK directly and if this inhibition also involved free radical oxidative stress. In this report, we demonstrate that methionine sulfoxide inhibits GS by about 50% and CPK by about 25% at 20 mM concentration. Neither intact SOD, nor ascorbate inhibit the action of methionine sulfoxide completely, with regard to the inactivation of GS. These results indicate that the action of methionine sulfoxide may not be directly due to the oxidation of GS by free radicals. In fact, the presence of exogenous proteins, such as denatured SOD or catalase, inhibit the action of methionine sulfoxide as, or more effectively than, the addition of active free radical antioxidant enzymes.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Ascorbic Acid; Catalase; Cattle; Creatine Kinase; Enzyme Inhibitors; Free Radicals; Glutamate-Ammonia Ligase; Methionine; Nerve Tissue Proteins; Oxidation-Reduction; Peptide Fragments; Protein Denaturation; Serum Albumin, Bovine; Sheep; Superoxide Dismutase

The effect of primary structure and external conditions on the oxidation of methionine to methionine sulfoxide by the ascorbate/Fe3+ system was studied in small model peptides. Degradation kinetics and yield of sulfoxide formation were dependent on the concentration of ascorbate and H+, with a maximum rate observed at pH 6-7. Phosphate buffer significantly accelerated the peptide degradation compared to Tris, HEPES, and MOPS buffers; however, the formation of sulfoxide was low. The oxidation could not be inhibited by the addition of EDTA. Other side products besides sulfoxide were observed, indicating the existence of various other pathways. The influence of methionine location at the C terminus, at the N terminus, and in the middle of the sequence was investigated. The presence of histidine in the sequence markedly increased the degradation rate as well as the sulfoxide production. The histidine catalysis of methionine oxidation occurred intramolecularly with a maximum enhancement of the oxidation rate and sulfoxide production when one residue was placed between the histidine and the methionine residue.

Topics: Amino Acid Sequence; Ascorbic Acid; Buffers; Chlorides; Edetic Acid; Ferric Compounds; Histidine; Hydrogen-Ion Concentration; Methionine; Molecular Sequence Data; Oxidation-Reduction; Peptides; Temperature