Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis.
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-methionine R-sulfoxide is used as a substrate. The method provides baseline resolution of the substrates and, therefore, can be used to easily determine the purity of the substrates. The method is rapid ( approximately 20min sample to sample), requires no column regeneration, and uses very small amounts of buffers. Separation was performed by using a 75-mum internal diameter polyimide-coated fused silica capillary (no inside coating) with 60cm total length (50cm to the detector window). Samples were separated at 22.5kV, and the separation buffer was 25mM KH(2)PO(4) (pH 8.0) containing 0.9ml of N-lauroylsarcosine (sodium salt, 30% [w/v] solution) per 100ml of buffer. Prior to use, the capillary was conditioned with the same buffer that also contained 25mM sodium dodecyl sulfate. The CE method is compared with high-performance liquid chromatography (HPLC) as determined by comparing results from measurements of hepatic enzyme activities in mice fed either deficient or adequate selenium.
Uthus
Eric O
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Grand Forks Human Nutrition Research Center, U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), Grand Forks, ND 58202, USA. eric.uthus@ars.usda.gov
D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods*; D008099:Liver / Q000201:enzymology; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D012643:Selenium / Q000494:pharmacology
D008715:Methionine; D051150:Methionine Sulfoxide Reductases; D012643:Selenium; C013111:methionine sulfoxide