Determination of beta-hydroxyacyl CoA-dehydrogenase activity in meat by electrophoretically mediated microanalysis.
The combined use of an in-tube enzyme assay and capillary electrophoresis for determining beta-hydroxyacyl CoA-dehydrogenase (beta-HADH) activity in meat was investigated. Beta-HADH is a significant mitochondrial enzyme in food muscle; thus, the determination of its activity is important in food analysis. The enzymatic assay and the separation of the reaction products were carried out by electrophoretically mediated microanalysis (EMMA) using a plug-plug reaction mode at variable potential. For the quantification of beta-HADH activity, the rate of conversion of reduced beta-nicotinamide adenine dinucleotide (NADH) to beta-nicotinamide adenine dinucleotide (NAD+) was calculated by determining NAD+ at 260 nm. A calibration curve for NAD+ concentration versus normalized areas showed a highly significant (p < 0.001) linear relationship (R2 = 0.993). Accurate quantification of beta-HADH activity was achieved since on-line monitoring allowed us to account for the NAD+ produced from NADH degradation by applying a correction factor. An average reaction time of 0.66 +/- 0.06 sec was determined for a protein concentration in the range of 0.1-0.5 mg protein/mL. The assay was reproducible since coefficients of variation of less than 6.2% were calculated for triplicate analyses.
Centro de Investigación en Alimentación y Desarrollo, A.C. (CLAD), Dirección de Tecnologia de Alimentos de Origen Animal, Hermosillo, Sonora, México. vallejo@cascabel.ciad.mx
Belinda
B
Vallejo-Cordoba
Miguel A
MA
Mazorra-Manzano
Aarón F
AF
González-Córdova
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000032:analysis* / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D008460:Meat / Q000032:analysis*; D008832:Microchemistry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015094:3-Hydroxyacyl CoA Dehydrogenases