Sodium Carbonate is used as a stop solution in enzyme assays. How does it act on the enzyme substrate complex to stop the reaction?
Sodium carbonate is often added to stop an enzymatic reaction especially when using a chromophoric substrate (e.g. CNP-lactoside; PNP-glucoside). The reason is double (1) due to the shift in pH (above 10), most enzymes are inactive (due to an inappropriate charge on the AA acting as electrophile) and the enzyme reaction is stopped at each time interval you take a sample (2) the chromophore (e.g. PNP, ONP, CNP) that becomes released due to hydrolysis is almost completely present in its anionic form and becomes detectable in the VIS spectrum (e.g. yellow colour (A 405 nm) for ONP, PNP, CNP ... ). This increases the sensitivity of your assay. If you want to follow your enzyme reaction continuously, then you need to work at a pH above the pKa of the chromophore and that is not always feasible: the enzyme should be active at that pH.
Hope this helps
It depends on the enzyme. Probably shift in pH, it could be inhibition. What enzyme are we talking about?
what is the pH optimum? Do you know the pH of the solution or do you just dissolve the carbonate?
The pH optimum of the enzyme is 5.0 For Sodium Carbonate solution, I dont know the pH as I just dissolved it.
Then it's clear. Sodium carbonate is basic, not sure, what is the pH, but the pKa is about 10, so it will be quite high. Thus adding Na2CO3 will titrate your reaction buffer into high pH where the enzyme is not active anymore.
Boopathi,
You are only adding the salt. CO3(-2) is a base. So, if the normal concentration of Na2CO3 solution is much larger than the normality of your buffer, the final pH of the solution should be bigger than Na2CO3 pKa.
Na2 CO3 + H2O --> HCO3(-) + OH(-)+2Na(+)
A very high pH, probably denature your enzyme (e.g. changing Its tertiary and quaternary struture), stopping the reaction.
Sodium carbonate is often added to stop an enzymatic reaction especially when using a chromophoric substrate (e.g. CNP-lactoside; PNP-glucoside). The reason is double (1) due to the shift in pH (above 10), most enzymes are inactive (due to an inappropriate charge on the AA acting as electrophile) and the enzyme reaction is stopped at each time interval you take a sample (2) the chromophore (e.g. PNP, ONP, CNP) that becomes released due to hydrolysis is almost completely present in its anionic form and becomes detectable in the VIS spectrum (e.g. yellow colour (A 405 nm) for ONP, PNP, CNP ... ). This increases the sensitivity of your assay. If you want to follow your enzyme reaction continuously, then you need to work at a pH above the pKa of the chromophore and that is not always feasible: the enzyme should be active at that pH.
Hope this helps
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