When you use NADH to measure enzyme activity, you need to watch the variation of absorbency at 340nm. But sometimes the absorbency doesn't decrease as you expected. For example, my absorbency usually decrease from 1.0XX to 0.5XX. But, sometimes it decrease from 1.0XX to 1.0XX while using the same kind of method and content.
Hello Anqi,
I think we will need more details to better answer your question and provide help... with only this information I'd say you have no (or very low) activity analyzing the samples in which you don't see the absorbance change. Please keep in mind that the activity can be quite low due to a very small content of the particular enzyme in the preparation. If that's the case, increasing the amount of sample would help to see a higher decrease in absorbance. On the other hand, you can increase the concentration of the other substrates or check other additives necessary to measure the activity correctly. On top of this, it might be that you're not reaching saturating concentrations; i.e. not reaching the Vmax. I would not consider to increase the NADH concentration, since you already have an initial absorbance of more than 1, meaning you have already more than 100 uM in your assay (if the spectrophotometer is calibrated correctly and the NADH is also at the correct concentration).
Besides, to have a better idea about your results, please provide the specific activity values (mU/mg protein or U/mg protein). If your samples are cell extracts or membrane preparations, just divide the rates to the protein amounts you are using in your assay. You can also measure the activity of another control enzyme that do not change by the treatments you're doing and use them to normalize all the NADH-related enzyme activities you are interested in.
Best, Alfredo
I agree with answer Mr. Alfredo . You must measure the protein concentration (mg/ml) .
I agree with answer Mr. Alfredo . You must measure the protein concentration (mg/ml) .
Alfredo Cabrera-Orefice Thank you so much, Alfredo. I am a bachelor without a lot of experiment experience. But every time when I measure the activity of my enzyme I experience an unsteady enzyme activity. I did measure the concentration of each enzyme using Coomassie Brilliant Blue every time. Usually, the concentration doesn't change so much each time after putificaion, but the activity change a lot each time. And I used the same enzyme to measure the activity for three times. Within those threee times, the activity varies a lot.
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And sometimes even after a few days putting in the fridge(-40℃), my enzyme activity decrease to zero. My labmates used the same enzyme without this bad stability.
Maybe I will never measure activity again in the future, but i am still curious. Why those problems happen? Have you ever met up with those problems before??
Anqi
Dear Anqi,
I have had issues with gradual loss of activity of purified enzymes after several freeze-thaw cycles or long storage. To avoid these issues some supplements should be added to the enzyme, of course depending on its nature (soluble or membrane) and catalytic features; e.g. DTT, detergents, glycerol. The idea is to prevent, for instance, protein oxidation and/or unfolding. Depending on your enzyme, dilution and/or loss of quaternary structure or ligands would also result in low activities. This happens quite often in purified soluble oligomeric enzymes.
My suggestion for your case would be to double-check the procedures you're doing while handling your enzyme with those from your colleagues. Sometimes a simple but critical step is what makes the difference, particularly since they don't have the problems you're sharing with us. Besides, check again if the composition, pH and chemical concentrations in the solution you normally use to dissolve your enzyme and/or do the assays are correct. Try to get input from the experienced labmates and discuss step-by-step what you do to figure out where the problem comes from.
Good luck!
Alfredo