Hello Every One,
I have taken UV spec readings (for 3 minutes) for the activity of Xylosereductase and xylose dehydrogenase by the method defined elsewhere by using NADPH/ NAD. from that particular OD values how one can calculate the enzyme activity in U/ml/min.
Thank you
Please refer the link below:
http://www.biotechnologyforbiofuels.com/content/2/1/9
Hi there,
The important point for enzyme activity determination is to run the assay in initial velocity condition (ie. linearity between substrate consumption/product appearance with time). For xylose reductase, the decrease of NAD(P)H (OD at 340nm) during the 3 minutes has to be linear. The other important point is to run the assay in substrate saturating condition. So both NAD(P)H and xylose concentration have to be large towards their respective Km. So from your data, you will be able to measure velocity expressed as variation of OD340 versus time. This can be converted into variation of NAD(P)H concentration versus time just by dividing the former expression of velocity by 6220. Knowing the volume of the assay you can then express velocity as mol of NAD(P)H consumed/min. And finally you know what volume of your enzyme solution has been used in the assay so you can calculate activity of your enzyme by divinding velocity by the volume of enzyme present in the assay. So activity will be expressed as mole of NAD(P)H oxidized per min per mL of enzyme solution. 1U is 1µmol/min so a simple conversion will give you the activity in U/mL. As there is 1 to 1 stoechiometry between NAD(P)H and xylose for xylose reductase, the activity expressed toward NAD(P)H will be the same as he one expressed toward xylose. About xylose deshydrogenase, the increase of NAD(P)H will be in accordance with xylose dissapearance.
Dominique gave the figure 6220. This is the molar extinction coefficient for the difference in 340 nm absorbance between NAD(P)H and NAD(P) for a pathlength of 1 cm. The change in absorbance is converted to concentration using Beer's Law:
Delta(Conc) = Delta(A340)/(path length x extinction coefficient).
Example: If the pathlength is 1 cm and the 340 nm absorbance changes (at a constant rate) by 0.622 in 10 minutes, that is 10 µM/min. Multiply by the volume of the reaction in liters to get µmoles/min, or units. Divide the the number of mg of enzyme in the reaction to get the specific ativity (µmoles/min/mg).
Note that this activity value is specific to the exact reaction conditions you used. If you change anything, such as the substrate concentrations, you could get a different activity. Ideally, you would determine Vmax so that you could calculate kcat. This would require a Michaelis-Menten analysis.
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