Sugars assay

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.

The HPLC sugar assay was developed and validated as an efficient screening technique. As a result, some limitations may be encountered. Samples high in salt content may create difficulties, particularly interfering with glucose or sucrose. In some cases, some of the salt can be removed with an HPLC clean-up column or the use of a column different from the one specified (e.g., anion-exchange or calcium-loaded column). The preferred solution is to analyze such samples using GC instead of HPLC. 

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. 

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. 

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

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. 

One of the major uses of colorimetric methods is in the monitoring of chromatographic columns. All the low pressure GPC columns can be connected to automated colorimetric assay systems based on any of the total sugar assay methods. There is a potential in the adoption of such systems (analogous to flow injection analysis) into HPLC systems for the specific and more sensitive (compared to RI and UV) detection of underivatized glucose oligomers. 

Subsequently, Schneider (94) suggested the addition of the hot trichloroacetic acid extraction step to the acid-soluble fraction remaining at the end of the ST procedure, thus permitting more direct isolation of the DNA fraction. This procedure (the Schmidt-Thannhauser-Schneider, or STS, procedure), which permits the use of either phosphorus or sugar assays as the measure of nucleic acid, has become a standard rapid tissue assay. Certain refinements necessary for conversion of a tissue assay method to an isotope assay method, involving more precise segregation of cellular constituents, can be regarded as- extensions or adaptations of the basic procedure. For these reasons, the procedure is described in detail. 

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