I have purified enzymes cellulase and beta-glucosidase and performed SDS-PAGE. Therefore, I have characterized with metal ions at 2mM, 5mM and 10mM but at 5mM concentration lower activity than at 2 &10mM concentration? Can anyone help me to know the presumable reasons
thank you, sir, for your response Adam B Shapiro. The purified enzyme with metal ions was incubated for 1 h and then standard assay was performed to measure the activity. These standard experiments were performed in tripicates.
I have performed this experiment multiple times but still, 5mM concentration showed lower activity as compared to 2mM and 10mM.
One possibility is that the metal ions are mildly inhibitory, but the increase in the ionic strength of the solution containing the metal ions is mildly stimulatory. Try the experiment with ammonium salts.
Another thought is that the highest metal salt concentrations interfere in some way with the readout of the assays. This could be tested by replacing the enzyme with the product of the reaction.
Because this effect was seen for every single metal salt, including monovalent metals, I tend to favor the idea that there is a systematic error somewhere in the experiment. It could be something wrong with the equipment being used, such as pipettors or plate reader.
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