Nessler's reagent is an alkaline solution of K2HgI4. I use a commercial preparation that, when it is fresh, has no precipitate, but after a period of time it displays a precipitate which has no effect on the reagent's properties. So, the soluble form is normal !

Dear karamanos, thank you for your response.

Can I use nessler`s reagent before appearing precipitate for quantitative assay of asparaginase?

I compared nessler`s reagent without precipitate with nessler`s reagent including precipitate by adding the reagents to equal volume of 3M ammonium nitrate solution (contain ammonia) separately which resulted in formation of precipitation in both of them, but nessler`s reagent without precipitate formed it less. So, Is Existence of precipitate effective on quantitative assay?

The Nessler's reagent is very good for qualitative detection and it should be used cautiously for quantitative determinations. A clear precipitate is found in commercial preparations, but I think this is due to KOH (crystallization?). I have always used the clear, slightly yellow supernatant without any visible problem. If you are able to obtain a good quality standard curve, you will be able to obtain good assessment of your activity. Now, when high amounts of ammonium are present in the assay, a brown precipitate will occur rapidly after the addition of the reagent in your assay, and this will influence your result (decrease of the response). I have followed the color development with a plate reader (at A = 520 nm), and when we are in the presence of a precipitate the A520 decreases. You can have a look to the attached document to see this phenomenon.

Yannis