Author
 |
Uthus, Eric |
|
Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 2/11/2010 Publication Date: 6/8/2010 Citation: Uthus, E.O. 2010. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis. Analytical Biochemistry. 401:68-73. Interpretive Summary: Methionine, one of the amino acids that makes up proteins, contains sulfur. This sulfur can be oxidized making a compound called methionine sulfoxide (Met-O). If Met-O is not repaired, the protein containing this oxidized methionine may stop functioning. To repair the Met-O, animals and humans have an enzyme system called methionine sulfoxide reductase (Msr). This enzyme system is comprised of two enzymes, MsrA and MsrB. However, MsrB enzyme is a selenoprotein meaning that it contains selenium and is affected by dietary intake of selenium. MsrA is not affected by dietary selenium. Because selenium is anti-carcinogenic and the Msr enzyme system is important in oxidative defense, it is important to be able to distinguish between MsrA and MsrB. Most methods presently used to determine the activities of these enzymes use a substrate that can be impure. The research presented in this paper describes a method to obtain higher purity substrates. The method to determine substrate purity and enzyme activity is based on capillary electrophoresis, which uses electrical current to separate out and detect the compounds of interest. Results obtained by using the capillary electrophoresis method compare favorably with other methods that are currently used. However, the capillary electrophoresis method is faster (requires approximately 20 min from one sample to the next), necessitates easier sample preparation, and requires much lower use of buffers (keeping costs low and solvent disposal at a minimum). Because the capillary electrophoresis method also monitors substrate purity, it easily and completely distinguishes MsrA activity from MsrB activity. Technical Abstract: A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the product of the enzymatic reactions when either dabsyl L-methionine S-sulfoxide or dabsyl L-methioinine R-sulfoxide are used as substrates. The method also provides baseline resolution of the substrates and it therefore can be used to easily determine the purity of the substrates used. The method is rapid (approximately 20 min sample to sample), requires no column regeneration as does HPLC, and uses very small amounts of buffers. Separation was performed by using a 75 µm ID polyimide coated-fused silica capillary (no inside coating) with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV and the separation buffer was 25 mM KH2PO4, pH 8.0 containing 0.9 mL N-lauroylsarcosine [sodium salt, 30% (w/v) solution] per 100 mL buffer. Prior to use, the capillary was conditioned with the same buffer that also contained 25 mM sodium dodecyl sulfate. The CE method compares with HPLC as determined by comparing results from measurements of hepatic enzyme activities in mice fed either deficient or adequate selenium. |
