Reducing Sugars Assay

Carbohydrate-derived radicals are generated by direct electron transfer, hydrogen abstraction or fission of weak bonds. Direct electron transfer from the enediolate of reducing sugars is the basis of most reducing sugar assays. 

1 Reducing Sugar Assays. Many traditional tests for reducing sugars involve the one-electron reduction of a transition metal complex by the sugar in 

Because the products of oxidation reactions are osones, they can tautomerise again to new ene-diolates, which can then in their turn be oxidised. This means that the exact titre depends on the sugar effective extinction coefficients for monosaccharides assayed by the 2,2 -bicinchoninate assay range over a factor of 3 for common sugars. Similar differences between sugars are found with PAHBAH. 

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Figure C1.2.5 Calibration curve for the 2,2 -bicinchoninic acid reducing sugar-assay with galac-turonic acid as the calibration standard and A560 as the analytical signal.

Calculated as the ratio of the calibration sensitivities (or slope m) of the different cellodextrin standard curves to the glucose standard curve. Thus, a ratio of 1 is expected, theoretically, for a true reducing sugar assay, which has the same molar color yield for a series of saccharides. 

The combined data provide a ready means by which to compare and select appropriate assays for application in cellulase-catalyzed cellulose saccharification experiments. Products in such experiments are expected to include glucose cellobiose and, potentially, some cellooligosaccharides. Optimum reducing sugar assays would have equivalent molar color yields for these soluble products. As shown in Table 3, this optimum situation only applies to the two copper-based assays (Nelson, BCA). Because of their importance with respect to the analysis of insoluble cellulose (discussed next), calibration curves reflecting the molar color yields for the DNS and BCA assays are presented in Fig. 1. 

Green, F., 3rd, Clausen, C. A., and Highley, T. L., Adaptation of the Nelson-Somogyi reducing-sugar assay to a microassay using microtiter plates. Anal Biochem 1989, 182 (2), 197-9. 

Assay Determine as directed under Reducing Sugars Assay, Appendix X. 

Figure 7.8 Chromophores from reducing sugar assays, (a) Tetrahedral Cu coordinated by 2,2 -bicinchoninate. (b) A PAHBAH complex of methylgloxal. (c) The Morgan-Elson test for hexosamines. The Morgan-Elson and PAHBAH reactions are of course heterolytic, but are included here for completeness.

For PASC hydrolysis assay, 10 pL sample was mixed with 190 pL solution containing 2.1 g/L PASC and 8 pM BSA in 50 mM Na-acetate of pH 5 in a 96-well plate. After 30 min at 50 °C, 50 pL 0.5 M NaOH was added to stop hydrolysis. After 5 min centrifiigation at 2000 rpm, 100 pL supernatant was subjected to PHBAH reducing sugar assay. 

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