1.You run an LDH assay on one of your samples, and obtain an activity of 1.5 A/m
ID: 712157 • Letter: 1
Question
1.You run an LDH assay on one of your samples, and obtain an activity of 1.5 A/min. You then dilute the sample 1:10, and obtain an activity of 1.2 A/min. Is this the activity that you would expect from a 1:10 dilution of your original solution? Why? What is happening in these assays?
2. You obtain a rate of 0.8 A/min from one of your samples (Sample A). You added 10 µl of a 1:10 dilution of Sample A to a total volume of 1.21 ml. What is the corresponding activity (in µmol/min/ml) in the original sample?
Explanation / Answer
1)
Lactate dehydrogenase (LDH) is an oxidoreductase enzyme that catalyzes the interconversion of pyruvate and lactate, in which we are worried with the activity of the enzyme, than it will not be changed under any dilution, provided all additional factors are constant during determination of the activity. Let us consider the change of activity of an enzyme preparation or sample, under circumstances of dilution of the source keeping all other parameters fixed, or identical, then the explanation is very simple. With dilution, there is less number of available enzyme molecules to meet and subordinate with a fixed number of substrate molecules to do the catalysis even if all other factors are present suitably above their individual half capacity constant under the setup.
The number of each enzyme protein will not change but the enzyme-substrate association will be reduced drastically leading to reduced enzyme- substrate complex formation essential for catalysis.
In defining enzyme activity, we count both the number of substrate molecules misplaced and the number of product molecules found during the catalysis. We don’t count the number of enzyme molecule involved. That means we don’t consider the molecular weight of the enzyme protein in determination of enzyme activity.
This condition generate the confusion over relationship between dilution and activity of the enzyme source and enzyme molecule
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