Im doing enzyme assay for the proteases of sars cov 2 . However with different concentrations of the protein, the solution did not change colour to yellow. Im wondering if these substrates have no reaction colour? can someone verify this?
Hi there,
The most obvious reason for such results is that your proteins are not active. Either they are inactive from the start or the enzyme assay condition is not appropriate for the enzyme stability.
Thank you Dr. Dominique Liger . I forgot to mention that I tested the proteases with other substrates ;
1. L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide(PFLNA) - with PLPRO
2. N-CBZ-glycine p-nitrophenyl ester -with both proteases
these substrates reacted well and give out yellow colour. What do you think?
The chromogenic chemistry of the substrates is the same as that of L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide(PFLNA), which did work. Therefore, the problem is either that the enzyme does not recognize these peptides as substrates, or the reagent you have is not what it is supposed to be.
Thank you Dr Adam B Shapiro for your comments. I even increased the substrates concentration in different protein concentrations also no colour change but the absorbance is increasing and after 24 hours the solution turn cloudy (phosphate buffer). I diluted both of the substrates in DMSO. Do u think it might be the cause? thank you for your feedback.
It sounds like the substrate is insoluble in the assay buffer. Over time, it is precipitating, leading to the absorbance increase without color change, which is due to cloudiness.
It is OK to dissolve the substrate in DMSO, as long as the final DMSO concentration in the reaction is not so high that it inhibits the enzyme. However, the substrate is probably much more soluble in DMSO than it is in phosphate buffer, so it comes out of solution when the DMSO is diluted with buffer.
It's surprising that FRLKGG-pNa has poor solubility. It should have positive charge at neutral pH. What concentration of this peptide are you trying to use in the reaction? Have you tried lower concentrations? Have you checked that the pH in the reaction is what it is intended to be? What is the final DMSO concentration?
Hi,
Another possibility is the potential mutations in the proteases, which makes it unable to be recognized its substrate.
Thank you,
Amal.
Dr. Adam B Shapiro : For FRLKGG-pNa assay : 50mM phosphate buffer (pH 7.4) , 0.5mM substrate concentration, Temp: 30 C. final DMSO concentration would be 25% (100 ul in 400 ul total reaction). Reference : Article Structural basis for catalysis and ubiquitin recognition by ...
hi Amal Senevirathne , I also tested the proteases with other substrates and reacted well. Only the 2 substrates did not.
My thinking is that 0.5 mM peptide may be too high. Many peptides have low solubility. Also, 25% DMSO is probably killing the enzyme. I suggest trying a much lower peptide concentration and a much lower DMSO concentration. I consider 2% DMSO to be acceptable, although some enzymes can tolerate more. You can find out the maximal tolerable DMSO concentration by testing the enzyme with a substrate that is giving a signal. You can probably get by with 0.1 mM peptide and still get a useful absorbance change, especially if you measure absorbance continuously.