We are doing this assay, where we want to detect certain activity of an enzyme to degrade starch. One of the parameters also is to check for total sugar content of the enzyme reaction. What can you suggest to check for it? I want to be it as easy as possible and not using really harmful reagents, like acids, phenols etc. Thanks!
Enzymatic assay of glucose content using glucose oxidase/peroxidase (GOPOD) is very quick and easy. There are also commercially available kits by several suppliers (Megazyme, Sigma-Aldrich). All you have to do is add the GOPOD solution to a small fraction of your solution containing glucose, incubate at about 40 degrees Celsius for about 20 minutes then read the absorbance at 510nm. You can use a standard of known glucose concentration to calculate the glucose concentration of your sample.
Phenol-sulfuric acid method is a good method for total sugar quantification. It gives color with reducing and non-reducing sugars as well as with uronic acids.
See Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, D.A., Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356
It might be worth to try the methods from Drs. Alison Smith and Samuel Zeeman, if you do not like acids, phenols etc.
Nature Protocols 1, - 1342 - 1345 (2006)
http://www.readcube.com/articles/10.1038/nprot.2006.232
Good luck!
An easy way to analyse total sugar is as reducing sugar by using Lane & Eynon titration method. It is not that hazardous and what you have to do is to convert all sugars and starches into reducing sugars by complete acid hydrolysis and quantify the sugar content using the Lane & Eynon method.
You can use reducing sugar analysis method (Dinitrosalicylic acid method of Miller, G. L. (1958) : Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry. 31, 426-428).
Racgent ((5 g DNSA/200 ml NaOH 2M + 150 g double tartrate KNa / 500 ml water) / 1 liter water)
OD at 530 nm.
in horticultural products (like fruit juice) refractometric methods can be applied to estimate total soluble solid (TSS), a proxy for sugar, it is easy to use. But for research purpose it is good to use HPLC methods. Please read:-
Magwaza, Lembe Samukelo, and Umezuruike Linus Opara. "Analytical methods for determination of sugars and sweetness of horticultural products—A review." Scientia Horticulturae 184 (2015): 179-192.
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