Hi everybody,
For LDH enzyme activity, I prepared to different series of buffers based on two protocols:
1-I prepared the solutions of 100 mM Tris, 3.6 mM Sodium pyruvate, 3.4 mM NADH separately. Then, I added these solutions separately to the cuvette
- 500 µL of Tris buffer
- 83 µL of Sodium Pyrovate
- 83 µL of NADH
- Right before the measurement: 333 µL Sample
2- I prepared a buffer containing 100 mM Tris, 4 mM sodium pyrovate and 3.6 mM NADH and pH=7.5. Then, I added 900 microliter from buffer and 100 microliter of the enzyme.
I examined the protein absorbance reduction over the 5 minutes at 340 nm in the kinetic mode.
The activity of enzyme based on protocol 1, was significantly higher than the protocol 2. Is there any interaction between NADH, Sodium Pyruvate and Tris buffer when I prepared it as a single buffer? the final concentration of sodium pyruvate in the protocol 2, in the final reaction mixture is higher than protocol 1, but still is not in the inhibitory range. the higher amount of pyrovate may lead to lower activity?
Also, I am measuring the activity of commercial enzyme. the results in protocol 2 is so close to the reported enzyme in comparison to protocol 1.
At the end, I want to say, by preparing serial dilution of enzyme, In higher dilution of enzyme solution, I achieved slightly higher enzyme activity. Is this trend acceptable?
Thank you in advance for your comments and recommendation
NADH is not very stable in solution over a period. If you are making the protocol 2 mixture and keeping it for long time (2 -3 hrs at RT) then your NADH is decomposing which would give lower rate of reaction. You can check this by monitoring the absorbance in cuvette at 340 nm without adding the enzyme.
It is always best to make separate NADH solution and keep it cold.
Beside potential problems with storage and the stability of NADH, as mentioned by Nitin, none of the components you describe in the assay mixtures is present at the same concentration in the two protocols, so it comes to no surprise that the results are different
Thank you @Nitin for your recommendation
@ Pierre,
The NADH content in two protocols are approximately the same, but the difference is in sodium pyruvate. whether the higher concentration of pyruvate lead to lower activity result?
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