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COUPLED ENZYME SYSTEM [1 record]

Record 1 2010-12-23

English

Subject field(s)
  • Biological Sciences
CONT

Enzymes are commonly used in clinical and research laboratories in coupled enzyme systems to determine the activity of another enzyme. Ex: the enzyme to be assayed, asparate aminotransferase, produces oxaloacetate which is a substrate for the second enzyme, malate dehydrogenase. This oxidizes its other substrate, reduced NAD, so that the reaction is readily followed by the decrease in extinction at 340 nm. If the assay is to be accurate the oxaloacetate must not accumulate; only if it is used as soon as it is formed will the rate of formation of NAD be a true measure of the activity of asparate aminotransferase. Since the second enzyme has to function with a minute substrate concentration, a great excess has to be present in the assay mixture.

OBS

It has been calculated that even with a 100-fold excess of the second enzyme, compared to the first, there was likely to be a 4% error in the initial rate measurements. Thus these coupled enzyme systems are likely to be very expensive, and it is essential that the second enzyme be free from any contaminating activities that might interfere.

French

Domaine(s)
  • Sciences biologiques
CONT

Les réactions enzymatiques couplées peuvent servir au dosage du substrat S ou du produit P d'une enzyme ou encore à la mesure de l'activité d'une enzyme. Par exemple lorsque l'enzyme E, dont on mesure l'activité, n'est pas une déshydrogénase à NAD [exposant+] ou NADP [exposant+], et que ni le substrat S, ni le produit P ne possèdent de propriétés spectrales particulières, on couple la première réaction à une deuxième, catalysée par une déshydrogénase dont P est le substrat. Dans certaines conditions, mesurer l'activité de la deuxième équivaut à mesurer l'activité de la première. La deuxième réaction est dite «réaction indicatrice» de la première, appelée «réaction principale».

OBS

Dans ces conditions la réaction principale est limitante pour la seconde, les vitesses des deux réactions sont alors égales. Pour cela, le NADH, H (exposant+] doit être en large excès et l'activité potentielle de E2 doit être grandement supérieure à celle de E1 (environ cent fois plus).

Spanish

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