Two different methods are used for determining the total sugar concentration (gl

Question

Two different methods are used for determining the total sugar concentration (glucose/fructose) in samples. The first method is a DNS Reducing Sugar Assay, which tests for the presence of a free carbonyl group (seen in both glucose and fructose) and utilizes a standard curve for known sugar concentrations to determine (by taking the absorbances and using the standard curve equation) the final sugar content in unknown samples. The second method is a sugar-specific enzyme assay which uses enzymes from the Glycolytic and Pentose Phosphate pathways (Hexokinase, G6P Dehydrogenase, and PGI) and measures (by taking the absorbance differences after certain enzymes are added and using Beer's Law) the amount of NADPH produced, which is stoichiometric to the amount of glucose or fructose produced.

What might account for a difference in the results of each of these assays?

Explanation / Answer

The DNS Reducing Sugar Assay is a direct method for estimation of total amount of glucose/fructose present in the given sample. The sugar-specific enzyme assay is an indirect method for estimation of monosaccharides. By using DNA, we can only estimate one parameter, but by using enzymatic methods, we can assess the activity of enzyme and how much reducing equivalents are releasing in a metabolic pathway, etc. In DNA method the conversion of moles of DNA equals to the moles of glucose. It assumes that unknown will respond in assays the same as the known; however it varies with enzymatic assays.

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