Glycogen Branching Assay to Measure the Degree of Glycogen Branching

Branched polysaccharides, such as glycogen, comprise glucose residues linked linearly via alpha-1,4-glycosidic bonds and in branches via alpha-1,6-glycosidic bonds.

To determine the degree of branching in polysaccharides, begin with an aqueous color reagent containing potassium iodide, iodine, and calcium chloride. Under aqueous conditions, potassium iodide and iodine yield iodide ions.

In separate microcentrifuge tubes, mix the reagent with polysaccharides of known branch structures and a glycogen sample with an uncharacterized structure.

Polysaccharides adopt helical structures via their alpha-1,4-glycosidic linkages. The iodide ions bind to this helical core, forming linear polyiodide chains. This results in charge-transfer complex formation between the polysaccharides and the polyiodide chains, resulting in colored polysaccharide-iodide complexes.

Unbranched polysaccharides with longer polyiodide chains develop a blue color, whereas branched polysaccharides with shorter polyiodide chains develop yellowish to orange-brown complexes. The calcium chloride in the reagent intensifies complex color.

Transfer the mixture into disposable cuvettes. Using a spectrophotometer, measure the absorbance spectrum from 330 to 800 nanometers.

Unbranched complexes with a blue color absorb light at longer wavelengths, demonstrating a rightward shift in the absorbance maxima. In contrast, branched complexes absorb light at shorter wavelengths, with the leftward shift in the absorbance maxima indicating higher branching in the structure.

The absorbance maxima of the uncharacterized glycogen sample at approximately 450 nanometers confirms its branched structure.

To determine the glycogen branching, combine 650 microliters of iodine/calcium chloride color reagent stock with 100 microliters of water in a 1.5-milliliter tube, and mix the solution thoroughly before transferring to a disposable methacrylate cuvette. Place the cuvette in the spectrophotometer to conduct a run in a wavelength-scanning mode for collecting the background spectrum from 330 to 800 nanometers.

In a separate 1.5-milliliter tube, combine 650 microliters of the working iodine/calcium chloride color reagent with 50 micrograms of the oyster glycogen, and make up the final volume to 750 microliters with water. After blending the mixture thoroughly, transfer the solution to a disposable methacrylate cuvette to collect an absorption spectrum from 330 to 800 nanometers.

Similarly, obtain an absorption spectrum with 50 micrograms of amylopectin and 30 micrograms of amylose as described before.

To obtain an indication of the branched structure of an uncharacterized glycogen sample, combine 25 to 50 micrograms of the glycogen with 650 microliters of the working iodine/calcium chloride color reagent, and proceed as explained earlier to acquire the absorption spectrum.