we extracted crude alpha-Glucosidase from alfalfa seeds, how to calculate its enzyme units??
alpha glucosidase acts on starch to release glucose. So first prepare glucose standard graph and compare OD of glucose released by enzyme with standard graph. With this you will get concentration of product released by enzyme. Then convert this to molar concentration by dividing product concentration with molecular weight of glucose. Then calculate molar concentration of product/glucose released per ml per minute. Now you can write this as ___ Units of enzyme activity. You can further divide this protein concentration of enzyme extract in mg to get specific activity i.e unit /mg protein
alpha glucosidase acts on starch to release glucose. So first prepare glucose standard graph and compare OD of glucose released by enzyme with standard graph. With this you will get concentration of product released by enzyme. Then convert this to molar concentration by dividing product concentration with molecular weight of glucose. Then calculate molar concentration of product/glucose released per ml per minute. Now you can write this as ___ Units of enzyme activity. You can further divide this protein concentration of enzyme extract in mg to get specific activity i.e unit /mg protein
If you want to use a coupled enzyme assay and the extract is not excessively crude then you can measure glucose release at the same time the glucosidase is acting on the starch. This is done by adding an excess of another enzyme that acts on the released glucose and also uses a cofactor such as NADP or NAD that absorbs in the UV as it is goes through redox. A kinase could also be used with excess ATP and spectrophotometric monitoring of the redox conversion of glucose phosphate.
You can express your result (Activity Units) as micro moles of glucose released per min per ml of the sample.
So first convert micro grams (from standard graph) of glucose released to micro moles of glucose released and then multiply with dilution factor (If the sample is diluted while performing assay) and divide it by incubation time.
All the best
3/24/16
Dear Sheeraz,
Other ways to measure a-glucosidase (AG) utilize maltose as a substrate (easier to prepare than starch)...requires no boiling to get it into solution, and can be stored frozen indefinitely. Can measure the increase in reducing power, but must correct for the very high reducing power of maltose, which will be in much higher concentrations than the glucose released. Better to measure the release of glucose from maltose with glucose oxidase.
Another way is to use para-nitrophenyl-alpha-D-glucoside as a substrate (available from Sigma, etc.). AG breaks this down to release p-nitro-phenol which immediately produces a yellow color in the assay mix (provided the pH is > pH ~5.5) and is easily measured spectrophotometrically @ 420 nm. This is easier & quicker to measure vs. glucose. Use p-nitro-phenol (PNP) as a standard. Moles PNP released/min = moles substrate hydrolyzed/min.
I hope this information helps you.
Bill Colonna, Center for Crops Utilization Research, Dept. Food Science &Human Nutrition, Iowa State University, Ames, IA USA [email protected]
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes