glycogen branching assay to measure the degree of glycogen branching

Glycogen Branching Assay to Measure the Degree of Glycogen Branching

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.

glycogen-branching-assay-to-measure-the-degree-of-glycogen-branching

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

EoE Sub Articles

eoe sub articles

1. Qualitative assessment of glycogen branching
<ol>
	<li>Prepare saturated calcium chloride solution by adding 74.5 g of anhydrous calcium chloride to ~40 mL of water and stirring. Add a little more water and continue to stir. Make the volume up to 100 mL with water and continue stirring until the CaCl2 is fully dissolved.</li>
	<li>Prepare a working stock of iodine/CaCl2 color reagent by mixing 50 &#181;L of KI/iodine stock solution with 13 mL of saturated CaCl2 solution in a 15 mL tube. Mix well and store at 4 &#176;C, protected from the light. The solution is stable for at least 1 week under these conditions.</li>
	<li>Determination of branching
	<ol>
		<li>In a 1.5 mL tube, combine 650 &#181;L of iodine/CaCl2 color reagent stock with 100 &#181;L of water and mix thoroughly. Transfer the solution to a disposable methacrylate cuvette. 
		NOTE: The solution in the cuvette should be clear and pale yellow in color.</li>
		<li>Place in the spectrophotometer and, running in a wavelength scanning mode, collect a background spectrum from 330 nm to 800 nm.</li>
		<li>In a 1.5 mL tube, combine 650 &#181;L of working iodine/CaCl2 color reagent with 50 &#181;g of oyster glycogen. Bring the final volume to 750 &#181;L with water and mix thoroughly. Transfer the solution to a disposable methacrylate cuvette. 
		NOTE: The solution in the cuvette should be clear and a deep orange/brown color.</li>
	</ol>
	</li>
	<li>Place in the spectrophotometer and collect an absorption spectrum from 330 nm to 800 nm.</li>
	<li>Repeat steps 1.3.3 through 1.4 with 50 &#181;g of amylopectin and 30 &#181;g of amylose. 
	NOTE: The amylopectin sample should be yellow/green and the amylose sample should be green/blue. Both samples should be clear. The colored complexes formed are stable, with no change in the absorption spectrum, for at least 1 h at room temperature.</li>
	<li>To obtain an indication of the branched structure of an uncharacterized glycogen sample, combine 25 &#181;g to 50 &#181;g of glycogen with 650 &#181;L of working iodine/CaCl2 color reagent. Proceed as above, bringing the volume to 750 &#181;L with water, mixing thoroughly, and transferring to a methacrylate cuvette. 
	NOTE: The glycogen sample should yield a yellow/orange to orange/brown color depending upon the degree of branching (length of outer chains) of the glycogen present. Again, the sample should be clear.</li>
	<li>Collect the absorption spectrum.</li>
</ol>

<table><tbody><tr><td>Amylopectin (amylose free) from waxy corn</td><td>Fisher Scientific</td><td>A0456</td><td></td></tr><tr><td>Amylose</td><td>Biosynth Carbosynth</td><td>YA10257</td><td></td></tr><tr><td>Glycogen, Type II from oyster</td><td>MilliporeSigma</td><td>G8751</td><td></td></tr><tr><td>Methacrylate cuvettes, 1.5 mL</td><td>Fisher Scientific</td><td>14-955-128</td><td>Methacrylate is required since some procedures are conducted at 340 nm or below</td></tr></tbody></table>

Source: Wilson, W. A., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63046">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J. Vis. Exp. (2021).
In this video, we describe a qualitative spectrophotometric assay to determine the extent of branching in a glycogen sample with an uncharacterized structure. Comparing the absorbance maxima of the uncharacterized sample with that of polysaccharides with known branched structures indicates the presence of branching in the uncharacterized sample.

Source: Wilson, W. A., Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism. J. Vis. Exp. (2021).
In this video, we describe a ...

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.

38 vues. Test de ramification du glycogène pour mesurer le degré de ramification du glycogène

Glycogen Branching Assay to Measure the Degree of Glycogen Branching

Transcription

Glycogen Branching Assay to Measure the Degree of Glycogen Branching