First of all, I have narrow knowledge about antidiabetic assay as this is my first enzymatic test and I have no one to refer. I have a method but when I tried to google every each of the method detailed, the result showed that most of the method used a-glucosidase in solid form (mostly brand Sigma Aldrich) but mine was in liquid form from this brand (https://www.megazyme.com/alpha-glucosidase-yeast-maltase). The things is, I tried to study about unit of enzyme. But turn out to be quite complicated since it include activity, time, mass and so on.
Here is the method I referred:
Article In vitro evaluation of the α-glucosidase inhibitory potentia...
Can someone assist me on this? Any suggestion on method modification are welcomed.
Notes:
1. Sample: polyherbal extract
2. Phosphate buffer used: https://www.sigmaaldrich.com/MY/en/product/sigma/p5244
3. Acarbose will be used as a positive control at varying concentrations(12.5 to 400 μg/mL)
4. Will used Biotek multi-well plate reader
Thank you.
It is not an assay that I’ve done but I can give some general pointers on enzyme units.
I’m not surprised it is confusing, esp when the supplier itself doesn’t know what an enzyme unit is! Their definition is wrong as they have forgotten the time element. 1 unit is the amount of enzyme that will convert 1 micromol of substrate PER MIN under standard conditions (or more usually clearly defined conditions).
Whether you have solid or liquid (or in your case I suspect a fine suspension in Amm. sulfate) the aim is the same, to prepare a stock solution of a convenient ‘activity’, measured in units per ml. For example, if your solid contains 100 units, you might make that up in 1 ml to give 100 units per ml stock. Equally, if your enzyme is already a liquid, it will have a units per ml value on the label. All you need to consider then is whether to dilute further to create your stock.
With an ammonium sulfate suspension, you need to make sure the crystals are uniformly dispersed before taking out a known volume (and hence known number of units). Sometimes instead the crystals are centrifuged and resuspended in a buffer (esp. if ammonium sulfate needs to be substantially removed for the assay to work properly).
Make sure any methods you are using adopt the same definition of a unit. The main source of error comes from non standard definitions of the unit, sometimes 1 nmol substrate converted per min instead of 1 micro-mol per min. It is not a major issue if you check the definitions before starting the expt and adjust all unit/ml values to reflect one definition, either one is fine, I’d go with whatever gives numbers that are easier to handle.
Finally, don’t be too concerned with specific activity values. This is units per mg. It is more to do with enzyme purity. To get the right number of units in your assay only the unit/ml values of your stocks are relevant.
The product is supplied as a suspension at 1000 U/mL. You will have to dilute it a thousand-fold (final concentration in the assay) with the assay buffer since the paper you cited used it a 1 U/mL, assuming the specific activities and unit definitions are similar for the product you purchased and the one cited in the paper. Performing some preliminary experiments to test a range of enzyme concentrations would be a good idea.
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