I have a beta barrel protein with a single interior disulfide. I would like to use Ellman's reagent (DTNB) to determine at what temperature the disulfide breaks via the absorbance of TNB at 412 nm. Since I know the protein doesn't start unfolding until close to 50° C, I would expect relatively little change in absorbance at 412 nm until that temperature, then a significant increase when the disulfide breaks followed by little change in absorbance following complete denaturation of the disulfide, resulting in a sigmoidal curve of absorbance at 412 nm versus temperature. However, I get something that looks more like an exponential curve. What could be causing this?
Firstly, I don't think the disulfide bond will break as a result of protein unfolding in the absence of a reductant. Secondly, since Ellman's reagent is not a very sensitive method, you must have a pretty high protein concentration in your experiment, probably several micromolar at least. If this much protein is aggregating/precipitating as it is heated, this will cause the absorbance to increase due to light scattering. To test for this, just leave out the Ellman's reagent and see if you get the same effect.
Firstly, I don't think the disulfide bond will break as a result of protein unfolding in the absence of a reductant. Secondly, since Ellman's reagent is not a very sensitive method, you must have a pretty high protein concentration in your experiment, probably several micromolar at least. If this much protein is aggregating/precipitating as it is heated, this will cause the absorbance to increase due to light scattering. To test for this, just leave out the Ellman's reagent and see if you get the same effect.
Dear Brian,
I agree with Adam, increase in temperature itself should not lead to breakage of the disulfide bond. If the disulfide bond is intact then ideally you should not see any change in absorbance as you change the temperature.
Reason for exponential curve: If you carry out a labeling reaction on a free cysteine, then you will get an exponential curve. So cysteine in your case might be free, and that is why you get the exponential curve. So please check the oxidation status of your protein first.
If your aim is to determine Tm of the protein, why don't you use circular dichroism (CD) measurements. CD measurements will give you good measure of Tm. If you have a buried tryptophan in the folded protein, you can also try tryptophan fluorescence as a probe to calculate the Tm for your protein. Differential scanning calorimetry could be another option.
Best,
Jogender
Hi Brian, I agree with Adam and Jogender about the stability of protein disulfide bonds in regards to increasing T.
However, since you are probably using many-fold molar excess of DTNB, you might be observing breakdown of the DTNB.
Have you tried running the DTNB alone with increasing T?
Steingrimur, that was also my thought as well. The increasing temperature may be promoting cleavage of the S-S bond in the DTNB. I have not tested this yet though.
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