We are conduction some experiments to test Glutathione-S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene (CDNB).
We used three control wells where the enzyme solution was replaced by buffer and saw a lot of conjugation (chances in absorbance at 340 nm)
In a second control we add the substrate solution alone and also recorded a lot of conjugation.
In the attached image you can see the slopes generated by spectrophotometer at 340nm)
From top to bottom (triplicate)
1) buffer + enzyme solution + substrate (final absorbance - initial absorbance = 0.9372)
2) buffer + substrate (final absorbance - initial absorbance = 0.4070)
3) substrate only (final absorbance - initial absorbance = 0.4439)
4) buffer (final absorbance - initial absorbance = 0.0011)
5) buffer (final absorbance - initial absorbance = - 0.1063)
In is obvious to think that the reaction number 2 and 3 should not present any changes in absorbance because there is no enzyme source in there! seems that CDNB is being conjugated.
we have replace all buffers and prepared all fresh reagents; the bad result still persists
any suggestion is helpful
thank you
moises
Hi Moises,
This is perfectly normal. GSH and CDNB do react spontaneously (i.e. in the absence of transferases). As you can see, the enzyme solution increased the rate in comparison to the assay without enzyme. Then, you just have to subtract it. To reduce the rate of the spontaneous reaction it is a common practice to user mildly acidic pH (6.5).
Take a look at the classical reference for the method:
Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139.
Other good reference:
Rice-Evans, Ziplock and Symons (1991) Techniques in free radical research. Elsevier.
An example:
Pannunzio TM, Storey KB (1998) Antioxidant defenses and lipid peroxidation during anoxia stress and aerobic recovery in the marine gastropod Littorina littorea. J Exp Mar Biol Ecol 221:277–292. doi: 10.1016/S0022-0981(97)00132-9
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