BL 4820 Biochemical Techniques -- Lecture 2 - Characterizing an Acid Phosphatase
2. Finding a Valid Amount of Enzyme for Assay Conditions
To find the amounts of enzyme which will work well with the enzyme assay you are setting up for hexokinase, you must select a pH and [glucose] to start with. With these conditions set, you can then use different amounts of enzyme in the assay and find the conditions where you get a linear response in the assay:
The amount of glucose-6-P formed at different times in the reaction is measured using different concentrations of hexokinase. You can see in the above graphic that Enzyme concentrations of 1 and 2 are linear for the 30 min reaction time used, while at enzyme concentration 3, the amount of glucose-6-P falls off after 15 min. Clearly, only [Enz] = 1 and 2 give a valid assay; apparently at [Enz] = 3, the glucose is being used up or some other parameter is limiting production of the product.
When the amount of glcose-6-P produced in 30 min is used to calculate the rate of the reaction or the enzyme rate, a plot can be made to show which concentrations of enzyme are valid for the assay:
From the linear portion of this plot, you can then easily choose the valid assay conditions with respect to the amount of enzyme which can be used and still get results that can be compared to one another.