Hello everyone. I am doing an experiment relates to enzyme alpha glucosidase. I extracted this enzyme from 7 strains of thermophilic bacteria from a hot spring. Now I test the enzyme activity of these strains and would like to compare the activity amongst them. This is my mixture reaction: 0.9ml phosphate buffer, 0.1 ml enzyme solution. After 10 min of reaction, I add 1ml Na2CO3 1M to stop the reaction and measure the absorbance of p-nitrophenol released by employing spectrophotometer (Abs 400nm). But the concentration of crude enzyme extracts varies among these bacterial strains, so I think that I need a certain enzyme concentration (e.g. 1mg/ml) in order to be able to compare the activity amongst them. But I cannot find any articles or papers about the concentration of enzyme alpha glucosidase used in activity assay.
Please give me some help or direction to find the suitable enzyme concentration in enzyme assay.
Thank you for reading my question and I really appreciate if you can help me!!!
You can calculate the specific activity of each preparation, which is the enzyme activity (µmoles/min under a specified set of conditions, preferably at saturating substrate concentration) divided by the total amount of protein (mg) in the reaction. It has units of µmoles/min/mg. However, this does not tell you the amount of alpha-glucosidase protein in each preparation, only the amount of alpha-glucosidase activity under a specific set of conditions.
It is more challenging to figure out how much of the protein is in each preparation. If there is a very potent inhibitor of the enzyme available, you can use active site titration. The idea is that the inhibitor is so potent that every molecule of inhibitor inhibits one molecule of the protein (actually, one active site--inactive protein is not detected). By measuring the amount of inhibition as a function of the inhibitor concentration, you can calculate the concentration of enzyme active sites present.
If you purify the protein from each strain, you can measure the activity of the purified protein under the standard enzyme assay conditions and compare the activity in the extract to the purified enzyme. Then you could calculate how much enzyme was in the extract.
Another way to measure the amount of a specific protein in an extract is by a quantitative immunological method such as an ELISA. This requires a lot of effort to get the antibody and the purified protein standard. You might need a different antibody for each strain of bacteria. The advantage of the immunological method is that it does not require that the protein be active in the enzyme assay.
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