Dissertation > Mathematical sciences and chemical > Chemistry > Analytical Chemistry > Instrument analysis ( physics and physical chemistry ) > Capillary analysis,analysis of electrocapillary

Quantitative Analysis of L-Methionine Sulfoxide in Proteins by Micellar Capillary Electrophoresis

Author TongGuoFu
Tutor HuangKaiXun;PengHong
School Huazhong University of Science and Technology
Course Organic Chemistry
Keywords Capillary electrophoresis Methionine Sulfoxide Amino Acids Protein Hydrolysis Oxidation Desalination
CLC O657.8
Type Master's thesis
Year 2011
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Oxidation of free or protein-bound methionine residues to a diastereomeric mixture of the R- and S-isomers of methionine sulfoxide by ROS genernated in organism is a kind of ubiquitous phenomenon in protein modification.However,there are still no rapid and effective methods to quantitative analyze the R- and S-isomers of methionine sulfoxide.High performance capillary electrophoresis (HPCE or CE) is a powerful separation technique, which is combination of traditional electrophoresis with chromatography. Owing to its flexible separation modes, versatile separation buffers and low consumption of buffers, CE was a powerful enantioseparation technique, especially successfully used for separating amino acid enantiomers, peptides, pharmaceutical drugs and other chiral substances.This article is aimed at separating two diastereomers of L-Met-O and determining its content in proteins by CE-UV after derivatizing with dansyl chloride. The main results are shown as follow:(1) CE-UV was used to separate and determinate L-Met-O in the 20 kinds of standard amino acid mixture. Analytical conditions such as temperature, voltage and the composition of running buffer were optimized. Baseline separation was obtained between two diastereomers of L-Met-O( L-Met-R-O,L-Met-S-O) with other amino acids, where analytic temperature was chosen at 15°C, voltage at 20 kV and running buffer composed of borax buffer(20 mM, 9.5), sodium dodecyl sulfate (SDS) (120 mM),β-cyclodextrin (β-CD) (16 mM).(2) The sample recovery experiments of L-Met-O in the 20 amino acids mixture was employed by CE at the above optium ananlytic condition and its recoveries were obtained by adding different concentration of standard L-Met-O. According to standard curve (R2=0.9996),the recoveries were 113.7±2.9%, 109.8±3.8% and 102.4±1.5%, when the concentration of standard Met-O added in amino acids mixture were 30μM, 60μM, and 90μM, respectively.(3) In order to investigate the amount of methionine sulfoxide in bovine serum albumin (BSA) by CE, hydrolysis of proteins and desalination of protein hydrolyzate were [1] Nielsen H K, Loliger J, Hurrell R F, Reactions of proteins with oxidizing lipids: 1. Analytical measurements of lipidoxidation and of amino acid losses in a whey protein-methyl linolenate model system. British Journal of Nutrition, 1985, 53, 61-73[2] Davies M J, The oxidative environment and protein damage. Biochimica et Biophysica Acta, 2005, 1703, 93-109[3] Kim H Y, Gladyshev V N, Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals. Biochemical Journal, 2007, 407, 321-329[4] Stadtman E R, Moskovitz J, Berlett B S, et al., Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism. Molecular and Cellular Biochemistry, 2002, 234, 3-9[5] Moskovitz J, Singh V K, Requena J, et al., Purification and Characterization of Methionine Sulfoxide Reductases from Mouse and Staphylococcus aureus and Their Substrate Stereospecificity. Biochemical and Biophysical Research Communications, 2002, 290, 62-65[6] Schallreuter K U, Rubsam K, Gibbons N C, et al., Methionine Sulfoxide Re-ductases A and B are deactivated by Hydrogen Peroxide (H2O2) in the Epidermis of Patients with Vitiligo. Journal of Investigative Dermatology, 2008, 128, 808-815[7] Stadtman E R, Remmen H V, Richardson A, et al., Methionine oxidation and aging. Biochimica et Biophysica Acta, 2005, 1703, 135-140[8] Wells-Knecht M C, Lyons T J, Mccance D R, et al., Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes-Evidence against a generalized increase in oxidative stress in diabetes. Journal of Clinical Investigation, 1997, 100, 839-846[9] Ruan H, Tang X D, Chen M L, et al., High-quality life extension by the enzyme peptide methionine sulfoxide reductase. Proceedings of the National Academy of Sciences, 2002, 99, 2748-2753

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