Falvin - 15-1-2023 at 23:20
I prepared a solution of n-benzoyl L-tyrosine p-nitroanilide (BTNA) in dimethyl sulfoxide for use in an enzymatic assay. However, mixing it with a
solution of phosphate buffer produced a color change for unknown reasons. Here is what I did:
A 1.4mM solution of BTNA in DMSO was prepared.
5mM phosphate buffer was prepared. This was done by dissolving dipotassium phosphate in distilled water and adjusting the pH to 7.59 by addition of
hydrochloric acid.
Into a cuvette was added 0.4mL of the BTNA solution, followed by addition of 0.1mL of the phosphate buffer solution.
When the BTNA solution and phosphate buffer were mixed, the mixture took on a yellow color. I tried mixing the BTNA solution with distilled water
without any phosphate added to see if, perhaps, the act of mixing water with the BTNA solution is responsible for the color change. In this case, the
mixture did not change color.
So, what is going on here? What is the reaction between phosphate and BTNA?
Tsjerk - 16-1-2023 at 01:01
Maybe use Tris.HCl as buffer? But also, you measure absorption at around 380 nm right? I don't know what the yellow is, but maybe it doesn't
interfere.
Falvin - 25-1-2023 at 00:23
Absorbance is measured at 410.
Falvin - 27-1-2023 at 11:51
Update:
I ended up using HEPES as the buffer for the enzyme assay, but I still want to know what is going on here.
I tried adding concentrated NaOH to the BTNA to see what would happen. Solution turned yellow and a white precipitate formed. There also seems to be
some phase separation occuring. The yellow color was more intense than with phosphate. I then tried adding slightly less concentrated NaOH. Same
result. I then took some HEPES solution, added NaOH to raise its pH to 11.3 , and then added it to the BTNA solution. No precipitate formed, but
solution still turned yellow.
I then tried adding HCl to the BTNA solution. Nothing happened. I then took some phosphate solution and added HCl to lower to pH to about 2.6, then
added it to the BTNA. Nothing happened.
So, it seems that this is not due to phosphate itself, but rather the fact that the solution is akaline. So, is it simply that the OH group gets
deprotonated and the resulting ion happens to have a yellow color?
[Edited on 27-1-2023 by Falvin]
[Edited on 27-1-2023 by Falvin]