Enzymatic analysis

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The term enzymatic analysis refers to two different methods in analytical chemistry :

principle

Enzymatic reactions follow the general scheme:

Here the enzyme has the function of a catalyst , which accelerates the conversion of the substrate to be determined with a coenzyme . The enzymes here have different specificities that are only effective for one substance and are group-specific (e.g. only for oligosaccharides ) or stereospecific (conversion of only one stereoisomeric form).

Absorption curves of NADH and NAD +

The coenzyme, however, is often identical. Frequently occurring coenzyme pairs are NADH / NAD + or NADPH / NADP + . The reduced form (NADH or NADPH) shows an additional absorption maximum at 340 nm compared to the oxidized form (NAD + / NADP + ), which can be evaluated photometrically . Because the amount of coenzyme converted is equivalent to the substrate, an indirect determination can be made.

If no suitable dye-forming substrate is available, the enzymatic detection can also be coupled to the reactions of other enzymes that generate a color change (for example via NAD + ). This principle is called by Otto Warburg as composite enzymatic detection .

execution

As a rule, the blank value and the analysis sample are measured after the enzyme has acted for 5 to 10 minutes and the extinction difference calculated, which is a measure of the consumption of coenzyme. Some bioanalysis service providers offer UV test kits with ready-made reagents.

application areas

Since the enzymatic substrate determinations are very specific and sensitive (measurement in the ppm range is possible), enzymatics are increasingly used in food analysis. It combines the requirement of reference methods, rapid methods and routine methods. The analysis of carbohydrates (especially glucose , fructose , mannose , sucrose ), organic acids (especially malic acid , citric acid ), alcohols and nitrogen compounds is of particular importance when determining the substrate .

Determining the enzyme activity to check for the presence of enzymes can indicate the beginning of spoilage and thus be an indicator of the freshness status.

Individual evidence

  1. a b c d e f Reinhard Mattisek, Gabriele Steiner, Markus Fischer: Food analysis . 4th edition. Springer, Berlin 2010, ISBN 978-3-540-92205-6 , pp. 365-370 .