Journal
ELECTROPHORESIS
Volume 28, Issue 12, Pages 1921-1925Publisher
WILEY
DOI: 10.1002/elps.200700031
Keywords
CE; deoxyadenosylcobalamin; methylmalonic acidemia; methylmalonyl-coenzyme A mutase
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Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization Of L-methylmalonyl-coenzyme A to succinyl-coenzyme A. We described a method for methylmalonyl-CoA and succinyl-CoA separation by CE, suitable for the evaluation of MCM activity. The working conditions for optimal separation were obtained in order to achieve the best resolution in the shortest analysis time. The optimization of buffer composition together with other variables, such as injection time, separation voltage, migration time, and capillary temperature, resulted in a solution of 30 mM NaH(2)pO(4) containing 15 mM SDS, pH 3.2. Separations were carried out in an uncoated fused-silica capillary (55 cm, 50 mu m id) at -25 kV, reading at 254 nm. The method performance was evaluated by measuring total and holo-MCM activity in biological matrices such as rat liver and human peripheral blood lymphocytes (PBL). The mean MCM activity was expressed in nmol/h/mg protein of tissue/cell extract and was calculated from the amount of reaction product formed. The rapidity of analysis and utmost precision (repeatability and within-laboratory reproducibility) point out the potentialities of the proposed method for the differential diagnosis of methylmalonic acidemia, in relation to protein or coenzyme defects.
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