So the enzyme activity is calculated as either increase of product concentration [P] or decrease in substrate concentration [S].
In practise, it's determined often using e.g. spectrophotometer as a single value, either change of absorbance of substrate/product or consumption of cofactor etc.
But if you use e.g. HPLC for determination of the amount of product and substrate in the reaction mixture, you can either simply use the amount of product formed ( [P] ) or how much product is in total of substrate and product ( [P]/[S]+[P] ).
Like in the beginning, I was measuring cis-trans isomerisation. To avoid mistakes due to bad pipetting etc. I always calculated how much % the trans isomer is from the pool.
Now I performed some purification and got some inconsisent data, as in two samples the total amount of my substrate+product was decreased. Thus I don't know which way I should go?
If the data are available, [P]/([S]+[P]) is preferable because it increases the signal:noise versus increase in [P] or decease in [S]. The [P]/([S]+[P]) measurement typically requires a separation step such as chromatography or capillary electrophoresis so that [S] and [P] can be measured independently.
I will give you an example from my own experience of the sort of problem that can complicate a [P]/([S]+[P]) measurement, and how the problem was solved. An assay for the activity of DNA ligase used size exclusion HPLC to separate the ligated product (double-stranded DNA) from the unligated substrate (single-stranded DNA) under conditions that denatured the substrate but not the product. We used the fluorescence of a label attached to one of the DNA strands for quantitation. We observed that the quantum yield of the acceptor label was lower in the product than in the substrate. As a result, it appeared that [S]+[P] decreased as the reaction progressed. It was necessary to multiply the product fluorescence by a correction factor to account for the difference in quantum yield between S and P, so that [S]+[P] was constant.
So my advice is to look for a difference in detection or yield between substrate and product in your assay.
Hi there,
When using spectrophotometer, the reaction takes place in the measurement cuvette where you assume you always introduce the same amount of substrate from one sample to an other. Then activity is expressed as [P] or [S] variation in time. When you use HPLC, you analyze the content of samples in which reaction has taken place for a while and then has been stopped. Then one aliquot of the samples is injected in your HPLC system. In this case, you will separate and quantify both S and P and as you have to normalize the data, you will calculate the % of conversion of S into P. Then use this % to calculate the actual [P] formed in the samples(it will correspond to (% of P from HPLC) x [S]0). This process should erase the problem due to inaccurate injection (variation in S+P content of the sample). If S+P does vary a lot, you should be in trouble either with injection accuracy and/or sample preparation for injection (including reactants stability from stop of the reaction to actual injection).
PS : and always make sure you are measuring initial velocities if you are interested in the kinetics of the reaction.
Thanks for your answers. I just wanted to make sure that what I do is not some voodoo and something inappropriate.
Well, the initial velocities are always tricky, but I'll try to make the incubation as short as possible....
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