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.