I am working with a flavin-dependent enzyme, when I am trying to measure kinetics for the enzyme over long time using UV-spectrophotometer. I can realize that the enzyme starts to precipitate all over the cuvette walls.
I used conc. ~ 20 uM for this of the protein in 100 mM sodium phosphate buffer plus ~ 10 % glycerol. I tried this with the enzyme mixed with either NADPH or NADPH+substrate (for reduction experiment). I got precipitate in both cases.
I tried to cntact the company to explain to me how much is the temperature can be when I am mausring ~ 0.5 second interval (light will be focused on the sample all the time), but They didn't give me specification about this.
I don't know if the light can do this and if this light from the spectrohpotometer is that much affecting.
this is the possibilities I can think of !!
Which kind of buffer do you utilize for enzyme or substrate solution? Do you have any positive ion in your protocol, such as Ca or Mg? Your precipitate, in this case could by a phosphate salt.
20 µM is an enormous concentration of enzyme, so if it is not very stable in the buffer you used it would not be surprising to see some precipitation. There may be a need to optimize the assay conditions to allow the enzyme concentration to be reduced and/or to make it more stable. You can optimize the conditions on the basis of the rate of the reaction.
Several things can be optimized, such as pH, ionic strength, type of buffer, and type of salt. Try organic buffers such as Tris-HCl and HEPES-NaOH instead of phosphate. Try including a little bit of non-ionic detergent such as Brij-35 or Tween-20 (0.005%) to help block hydrophobic interactions that could lead to protein aggregation.
Thank you for comments and Help
Dr. Maria Grazia Tozzi
It is just sodium phosphate buffer,pH 7.5. no metals. and the precipitation starts only after the recording of the spectra (exactly after 15 min or so] with no obvious precipitation of a control sample in the same room conditions.
Adam B Shapiro
Dr. I didn't mention that I have controls for this at room temperature [same mix of enzyme and substrates, and the enzyme alone as well].
the Enzyme stock (~100 uM) also is very stable when set at room temperature or 4 C for several weeks (at least in terms of precipitation, I have tested this too], it is nothing comparable to the test run on the spectrophotometer.
I have done the test on the UV spectrophotometer, because I use the same mixture for a stopped flow experiment (Temperature ~ 10 C) and sometimes I have some unexplained spectra by the end of the run ~ 16 min.
I am not sure now if the stability of the enzyme an issue if not affected at normal room temperature or 4C.
The precipitation only occurs at room temperature AND in the UV spectrophotometer? Have you measured the temperature inside the spectrophotometer sample chamber? It may be warmer than room temperature.
It is indeed very surprising that you see precipitation in your cuvette and the same reaction when set as control outside the UV-Vis spectrophotometer doesnt give any precipitation. I was wondering whether you have the controls setup with the respective ( reduced or oxidized) cofactors too depending on whether you moniter the forward or reverse reactions. It will also be benficial to have a control with all the components of the reaction mix kept outside the spectrophotometer. I have been working with an inhibitor that complexes with my cofactor after its oxidation and forms precipitate.
In spite of the fact that you mention controls, I agree with Adam that 20 uM enzyme is way too high to be used in an experiment; reducing it to nanomolar concentrations may be desirable to obey the conditions of initial velocity in interpreting your kinetics. I assume that your substrate and cofactor affinities for the enzyme are micromolar and that will lead to anamalous results at such high concentration of the catalyst.
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