CDNB in GST activity determination

7 posts / 0 new
Last post
Liu Ping
Liu Ping's picture
CDNB in GST activity determination

Hello,
I'm trying to detect the GST activity in liver of goldfish. I chose CDNB as my substrate. But when I measured OD of the blank solution which including 2.8ml of 0.1M PB (pH 6.5), 0.1ml of 30mM GSH and 0.1ml of 30mM CDNB at 340nm, I found the absorbance was beyond the highest detected limitation of the spectrophotometer. That means the substrate has intensive background absorption. Normally the absorption of the blank solution is nearly to zero. When I decreased the concentration of the substrate, the OD decreaed too. However I diluted the CDNB solution 100 dilutions, the OD was still higher than 1.0.

I bought CDNB and GSH from Sigma, and dissolved CDNB in ethanol while GSH in 0.1M PB (pH 6.5). I measured OD of the blank solution on two different spectrophotometers, so it was not the problem of the instruments. I don't know why, how to deal with the problem. Anyone can help me?  

Looking forward to the replies. Thank you! 

R Bishop
R Bishop's picture
Liu Ping,

Liu Ping,

That sounds like quite a dilemma. You are right the absorbance of the "blank" should be zero. So its time for a little troubleshooting. Its odd that your "blank" has an absorbance beyond the detection limit of the spectrophotometer.  It seems that when you blank the spec that number should automatically go to zero.  Since that doesnt seem to be happening.....

1. The most likely thing is the concentrations of your stock solution are not correct.  On occasion, I have made stocks that were off by an order of magnitude, just through my own personal errror. This might lead to high absorbance in a spec. This would explain why your serial dilutions are decreasing in OD as well.

2. So that bring us to "I diluted the CDNB solution 100 dilutions, the OD was still higher than 1.0. " Do you mean that you diluted to 1:100 or 100 serial dilutions? Either way this seems to point to your GSH or CDNB concentrations being too high.

So I would recommend checking your math on your solutions and remaking them. 

Let us know what you figure out.

Bishop

GST ASSAY PROTOCOL:

  1. Prepare samples in a total 100 µl volume with GST Sample Buffer, including a negative control with 100 µl of GST Sample buffer only and a positive control with 2 µl of GST and 98 µl of Sample Buffer. Note: We recommend preparing several dilutions of your sample and performing duplicate wells for each measurement.
  2. Add 10 µl of Glutathione to each well containing the sample or control above.
  3. Prepare Substrate Mix by adding 10 µl CDNB Solution + 90 µl of GST Assay Buffer for each sample including the standard.
  4. Mix well and transfer 100 µl of the Mix into each sample (including the standard) well.
  5. Carefully shake the plate to start the reaction.
  6. Read the absorbance once every minute at 340 nm using a plate reader to obtain at least 5 time points.
Sami Tuomivaara
Sami Tuomivaara's picture
Liu Ping,

Liu Ping,

By definition, the absorbance of your blank is zero. Thus you have to zero the spec using your blank solution, after that, the absorbance reading is zero, as defined...

The absorbance reading of the blank solution before zeroing is irrelevant. It might be very high if the previous zeroing was done using air or solution that doesn't absorb at all at that wavelength.

Cheers,

R Bishop
R Bishop's picture
Suola's point is a good one.

Suola's point is a good one.

Bishop

Liu Ping
Liu Ping's picture
Bishop,

Bishop,
 
Thank you for your reply.
 
I have thought about the concentrations of CDNB and GSH before. In most of the literatures about GST activity determination the concentrations of CDNB and GSH are the same with mine and the final concentrations of CDNB and GSH are both 1mM. To "I diluted the CDNB solution 100 dilutions" I mean I diluted to 1:100, so the final concentration of CDNB in the reaction system is about 10μM. I haven't seen such low concentration of the substrate in GST activity determination in any literatures I found. Even at  the low concentration of CDNB, the absorbance was still high than 1.0. However I can try it.

There's no plate reader in our lab so I can't use the GST assay protocol you provided.  Thank you very much!

Liu Ping
Liu Ping's picture
Suola,

Suola,

Thanks a lot for your answer.

I used a UV-Vis double-beam spectrophotometer. I chose 0.1M PB as the reference solution and the blank solution I mentioned as control. I put one quartz  cuvette with 0.1M PB into reference cell and the other with the blank solution into sample cell as control. Then the OD of blank solution was 5.0 ( the highest detected limitation of the spec). I tried to zero the spec but it didn't work.

Sami Tuomivaara
Sami Tuomivaara's picture
Liu Ping,

Liu Ping,

I'm not sure if I understand the problem correctly... you said that you couldn't zero the spec? In that case, the problem is with the spec, not your samples!

Try the experiment as follows: (1) reference (=  blank, = zero) cell contains substrate and buffer. The absorbance of this cell is _set_ to zero. (2) Sample cell contains substrate, buffer and enzyme. (3) As soon as you add the enzyme, start the stopwatch. (4) Proceed normally with the absorbance measurement in various time points.

Make sure that the substrate concentration is the same in both cells, whatever volume of enzyme solution you put in the sample cell, you add the same volume of buffer in the reference cell, so that the substrate concentration is identical in both cells. Now, the initial absorbance (immediately after you add the enzyme, before significant amount of product has formed) in both cells is the same (Proteins don't absorb at 340 nm, so you don't have to worry about its contribution to the absorbance).

Now, the reference cell absorbance stays constant (without the enzyme) and the sample cell absorbance increases as the amount of product increases. The spectrophotomer is good at measuring _differences_ in absorbances of the two cells, even if both cells have high _absolute_ absorbance values.

Hope this helps.

Cheers,