I did an indirect coupled enzymatic assay for Benzoylformat decarboxylase enzyme. I had sample before desalting and a sample after desalting. the enzyme activity in the desalted sample is about 5 folds larger than the other. does it have an effect or I just have an error somewhere ?
Thanks in advance
Enzymes often have requirements for specific ions. They can also be inhibited by certain ions. Furthermore, they can have an optimum ionic strength. So it is not surprising that the enzyme had higher activity after desalting. To study these effects, you can start with the desalted, enzyme, and test the effect on its activity of performing the reaction in a range of concentrations of many different salts, one at a time. For example, you could test ammonium, potassium, and sodium salts of acetate, chloride, and sulfate from 0 to 300 mM. To see whether there is an effect of specific ions or just ionic strength, overlay the activity versus ionic strength plots of all 9 salts. The calculation of ionic strength is described here: https://calculator.tutorvista.com/ionic-strength-calculator.html
I think there are some Q you should be thought, if you use two buffer, the activity must be different. You should think about pH, salting strength, the ions. If all conditions is same, i think the activity will be similarity. Oh maybe the temperature, if you enzyme is unstable.
Do you know if this enzyme has specific requirements for pH and or salt concentration?
It is very likely that desalted enzyme has much higher activity. How much salt did you have in the "salted" condition?
Calculating enzyme concentration/activity make sure you also correct for the different volumes you may have before and after the desalting.
Dong Li the conditions were the same for both samples, same buffer, temperature and pH.
Anna M Senczuk the enzyme works best at pH 8. the buffer used for all sample has a concentration of 50mM (phosphate buffer).
so desalting does enhance the activity of the enzyme ?
how can i correct for the difference in volume? there are differences in volume, may be thats why the enzyme activity and specific activity look so different.
For our enzyme, desalination increases activity by 2 times. Some properties of the enzyme change during desalting, but it is necessary for the enzyme to remain frozen for a day.
Enzymes often have requirements for specific ions. They can also be inhibited by certain ions. Furthermore, they can have an optimum ionic strength. So it is not surprising that the enzyme had higher activity after desalting. To study these effects, you can start with the desalted, enzyme, and test the effect on its activity of performing the reaction in a range of concentrations of many different salts, one at a time. For example, you could test ammonium, potassium, and sodium salts of acetate, chloride, and sulfate from 0 to 300 mM. To see whether there is an effect of specific ions or just ionic strength, overlay the activity versus ionic strength plots of all 9 salts. The calculation of ionic strength is described here: https://calculator.tutorvista.com/ionic-strength-calculator.html
All osmolytes (salts, sugars, amino acids, glycerol...) reduce the activity of water, and hence protein internal mobility. Thus the addition of osmolytes will reduce enzymatic activity, but increases stability and crystalisability of proteins. In addition, you may have specific interactions between salt and enzyme, if those have been described you can look them up in BRENDA (https://www.brenda-enzymes.info/).
It does not seem an error. As Adam B Shapiro mentioned enzymes respond to certain ions, and also are affected by Ionic strength and solvent polarity ( as mentioned by Engelbert Buxbaum ). Some enzymes are also sensitive to dilution, because the tend to aggregate when concentrated and/or dissociate when diluted. Depending on the enzyme, desalting may cause and increase, a loss of activity, or no change.
In some cases, if the sample comes from a crude extract, desalting will remove metabolites and ions present in the original biological fluid and extracted with the enzyme. When this compounds affect enzyme activity, desalting will result in a change.
If you are interested in determining the cause of your change in activity. Try desalting parallel samples in several different conditions.
Best wishes,
Rogelio
As previously mentioned by Adam B Shapiro and Rogelio Rodríguez-Sotres is not an error. Enzymes are pH, temperature, and ionic strength sensitive. Halophilic enzymes show high activity when buffers contain high salt concentration but the activity decreases if salt concentration is decreased. Your case is just the opposite. As suggested by Adam, you can carried out a nice/easy experiment in which the activity can be measured using the same buffer with different slat concentration or even changing the salt (some ions have specific effects on the enzymatic activities). You can also combine temperature and salt concentration in a single assay in which you can monitor enzymatic activity (usually the higher the salt concentration is, the higher activity is observed at high temperatures).
All the best,
Rosa María
My recollection of working with this enzyme was that it contained some bound TPP which was lost on dialysis.
We found that including 100microM TPP in the dialysis buffer helped. It might be worth a try.
I realise I mis-read your question. I thought you were losing activity!
Activation I have not seen. It could be due to loss of some inhibiting species, change of buffer pH, ion removl etc.
Might also be worth looking at your coupling enzyme which I assume is an aldehyde dehydrogenase. It may have been activated by a reducing agent for example. You could use a direct assay.
Desalting we do to remove sulphate ions that normally interferes in the measurement of enzyme activity . Try desalting using column of Sephadex G25 or G10 instead of dialysis it will save time and you get good recovery. Sometimes co-factors needed for enzyme activity are lost during desalting and it reduces activity. You need to check it.
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