Assaying Glycogen and Trehalose in Yeast

Background

Glycogen and trehalose are the two storage carbohydrates of yeast and many other organisms. In yeast, both these storage carbohydrates accumulate when the medium starts to be depleted and the rate of cell growth decreases. Methods for assaying storage carbohydrates in yeast date back at least to 1956 (Trevelyan and Harrison, 1956a and 1956b), and have been updated many times since (e.g., [Becker, 1978; Quain, 1981; Schulze et al., 1995; Parrou and Francois, 1997; Plata et al., 2013], among others). There are three basic steps in assaying these two storage carbohydrates: first, lysing or permeabilizing the cells; second, freeing glucose from the glycogen or trehalose; and third, assaying the resulting glucose.

Cells can be lysed mechanically (Schulze et al., 1995), but this is inevitably somewhat tedious and time-consuming, and tends to require larger numbers of cells. Cells can be permeabilized by alkali, but glycogen forms large, multi-branched granules, and can be difficult to extract, and so some protocols use both an alkali and an acid extraction (Trevelyan and Harrison, 1956a and 1956b; Quain, 1981). However, alkali treatment alone extracts the vast majority of the glycogen (and probably all of the trehalose) (Becker, 1978; Quain, 1981; Parrou and Francois, 1997); and it may allow enzymes such as amyloglucosidase access to the interior of the permeabilized cell, where it can liberate glucose from any residual glycogen, and alkali extraction alone is much easier than a dual alkali/acid extraction. Therefore, like Becker, and Parrou and Francois, we use only an alkali extraction. However, it is possible that this may fail to assay a relatively small amount of acid-extractable glycogen (Quain, 1981).

In older assays (e.g., [Trevelyan and Harrison, 1956a and 1956b]), glucose was released and/or assayed by purely chemical methods. However, these were relatively non-specific, and also assayed glucose present in other molecules, such as cell wall glucans. Therefore more modern methods use enzymes to liberate glucose from specific polysaccharides; e.g., amyloglucosidases are used to liberate glucose from glycogen (Becker, 1978), and trehalases are used to liberate glucose from trehalose (Parrou and Francois, 1997). A challenge to these methods is that some enzymes are contaminated with other activities. For instance, Parrou and Francois found that some amyloglucosidases were contaminated with trehalases. Therefore either purer enzymes need to be used, or less pure enzymes need to be used under conditions that inhibit the unwanted activities. Here, like Parrou and Francois, we use Aspergillus niger α-amyloglucosidase, which may also contain a trehalase activity (Parrou and Francois, 1997), depending on the specific preparation of enzyme, but we use it at high temperature (55 °C to 57 °C), approximately the optimum temperature for this enzyme, where the trehalase is inactive (Parrou and Francois, 1997).

Finally, the enzymatically-released glucose must be assayed. There are many well-developed assays for glucose. We use the glucose oxidase/peroxidase/o-dianisidine reagent of the Sigma-Aldrich glucose oxidase kit, which produces oxidized o-dianisidine, which has a pink/purple color, easily assayed by absorbance at 540 nm.

Our procedure is adapted from that of Parrou and Francois (1997). However, at most steps, we use smaller volumes of reagents, which make the assay easier in some respects. The small volumes allow us to adapt the procedure to 96-well microtitre dishes, which allows the assay to become moderately high-throughput. We give two procedures, one for 2 ml screw-capped tubes, and one for 96-well microtitre dishes.