Reactions are carried out in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl2 and 100 mM NaCl using 2 µM cysteine desulfurase (Nfs) in presence or not of 2 µM SufE1. BSA 2 µM can be used as control. In addition to enzymes, pyridoxal 5′-phosphate (10 μM) and DTT (1 mM) are used for all reactions. Reaction is initiated by adding 500 μM L-cysteine for 30 min at 25°C. Then the reaction is quenched by adding 50 μl of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride (prepared in 7.2 M HCl). The addition of 50 μl of 30 mM FeCl3 (prepared in 1.2 M HCl), followed by incubation for 20 min leads to formation of methylene blue, which is then measured at 670 nm. Na2S (1–100 μM) is used for calibration?
You can prepare solutions of sodium sulfide of known concentrations and put them through the same assay in place of your cysteine and desulfurase. You should get some methylene blue formation, and you can measure its absorbance at 670nm. If you make a graph of absorbance vs. concentration of sulfide, you should get a linear plot (note: if your sulfide concentration gets too high, the plot will deviate from linearity due to formation of a dimeric methylene blue species). You can then run your assay for a specified amount of time, quench it by forming methylene blue, measure its absorbance, and back calculate the corresponding sulfide concentration.
One additional issue that you may want to be aware of is that sodium sulfide is VERY hygroscopic. If you want a very accurate sulfide concentration, you may want to consider a less hygroscopic sulfide salt (I believe lithium sulfide fits this description).
A question. Is cysteine desulfurase very substrate specific? Can it desulfurate also DTT?
Some cysteine desulfurases can act on selenocystein, however they can’t act on DTT. The enzyme mechanism requires the presence of the amino group in order for the substrate to bind to the PLP cofactor and react properly.
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