You can look at the published literature on the enzyme to see what values have been reported by others. If you use the same substrate, the same enzyme (from the same species) and run the reaction under the same conditions (temperature, pH, salts, buffer), you should be able to get a similar value.
Although I have not determined PP2A activity yet, Km values should be determined by sensitive HPLC method (file attached). Essential points of Km determinations are as follows; i.e., please determine the initial velocity with protein concentrations (containing enzyme) less than 0.1 mg/mL (final concentration), substrate concentrations should be around the Km value. Since the Km values depends upon the glycochain structures of the enzyme (our unpublished observation), cell homogenization and storage should be done carefully.
Dear Sir Adam B Shapiro
YEs I was trying to found some published literature, but the problem is in my case, I obtained different Km value. I am using electrochemical rotating disk electrode technique for determining Km values. Is there any difference can observe from different methods?
Thank you so much for your answer.
Dear Kou Hayakawa Sensei
Thank you so much for your answer.
In my case the method is electrochemical rotating disk electrode and I want to stabilize the impacts of rotation rate on the detection by using Km values. Hence my objective is to calculate the Km values at different rotation rates with various concentrations of substrates.
I have no experience with the technique you are using, but I'll try to make some suggestions. Excuse me if they are naive.
I have already mentioned what I consider to be the key factors affecting Km measurement. In your case, I would add that you should make sure that the substrate is well-stirred so that there is no unstirred boundary layer that would cause an error in the substrate concentration. Be careful not to cause denaturation of the enzyme by excessive turbulence from the rotation, and make sure the temperature of the sample is correctly measured and is the desired temperature.
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