strange enzyme assay - (Aug/14/2008 )
Hi all,
I've been working on an enzyme assay which utilize colorimetric method. The color formation correspond to the product formation. In preliminary experiment, I wanna to determine the appropriate amount of enzyme needed in the assay. So i've tried to perform the assay with different concentration of enzyme while holding other factors constant (substrates, etc.). However, the result seems to be quite strange. At lower concentration of enzyme (~50ng purified enzyme), a higher product formation was observed whereas at higher concentration of enzyme (~5ug purified enzyme), much lower product formation was observed! As far as I know, even if substrate isn't in excess, higher concentration of enzyme will only gives a similar product formation as with lower amount of enzyme. I reli can't think of the cause for this observation. Could anyone offer some help?
Thanks in advance~
we know this phenomenon for a kinase which tend to build aggregates at critical concentrations; trapped in this aggreates, kinase activity per mol is strongly lowered; work with the more diluted enzyme concentration which should be kept constant; rather, vary the substrate or co-substrate concentration
I dont think your observations are unusual, I have seen the same things when doing polymerase activity assays.
Thanks for replying~
But I'm not reli sure if this is the case for the enzyme i'm working on. From the assay, i've tried a range of enzyme concentration. While at a diluted conc., a maximum product production is observed; and with higher conc. the product production is much lower. Yet with a further increase in conc., there is a trend of gradual increase in product production, though it's far lower than the max (about 2-3 fold lower). If the enzyme build aggregates at critical concentration, i'd assumed it to have fewer product when the enzyme concentration further increased. Is my thought logical?
Please advise~
I'm not really good at enzyme kineticsSo what is the mechanism behind this observation?
Could you suggest the name of polymerase that gives the same observation?
It was the hepatitis c rna dependent polymerase, this protein was known to aggregate at higher concentrations and so the explanation would be the same as The Bearer. I assume you have repeated the experiment a few times and seen the same result. I might also be tempted to try a different preparation of the protein.Is your protein pure ? Could there be activity from a contaminent in your protein that could account for the shape of your graph ?
Yes, I've repeated a few times. Actually I doubt if that enzyme behave like the case that aggregate at higher concentrations as previous literature didn't report such phenomenon. I'm thinking if it could be due to reversible reaction as the enzyme can catalyze reversible reaction in vivo, but all reported experiments were performed in one direction in vitro as it is thermodynamically unfavored.
The proteins is his-tagged recombinant purified proteins. SDS-PAGE confirmed that it should be pure.