I want to find level of arginase produced in human macrophage. I am referring following protocol.
1) Lysis of human macrophages in lysis buffer (0.1% triton-x-100+ protease inhibitor cocktail) 15 min at RT.
2) Incubate lysate with arginase activation solution (10mM MnCl2, 50mM Tris-Cl, pH7.5). Heat at 55 degree Celsius 10 min. Remove debris by pelleting.
3) Add equal volume of arginase substrate solution (0.5M L-arginine with pH 9.7). Incubate at 37 degree Celsius for 1 hr.
since I don't have urea detection kit. Please anyone suggest me protocol for quantifying urea in above cell lysate.
You might also consider detecting the other product of the arginase reaction, which is ornithine. It's more stable than urea, which will probably break down at the high pH of 9.7. How you do it depends upon what equipment you have available to you. An enzyme-coupled spectrophotometric assay, HPLC with pre-column derivatization, and mass spectrometry are some options. Can you use radioactive arginine? If so, HPLC with a radiometric detector might be convenient.
Hi Adam,
I have tried ornithin detection by using Ninhydrin. Since it will also react with amine group of protein. Hence, to avoid that false reading I had stop reaction at different time point. Initial OD should be of my total protein along with ornithin but later reading is only of my ornithin (form due to arginine to ornithin conversion by human arginase) which will be continuously increasing.
am I on right track?
I encountered one problem in this assay. I am getting brown insoluble precipitate along with my ornithin red soluble precipitate in both cell lysate as well as water (used as a negative control). Negative control was not showing reddish tinge in it. Still this precipitate reduces sensitivity of my assay. How do I get ride with this problem?
A potential problem with using the ninhydrin reaction to detect the production of ornithine is that there will be a very high background due to the high arginine concentration. I don't know what causes the precipitate, but I think a more selective approach would be preferable.
You can try using a-ISPF which is a colourimetric indicator for urea.
This paper by Corraliza is where I based my protocol off of.
http://www.ncbi.nlm.nih.gov/pubmed/8083527
Dear Leonard,
Thanks for your reply. Will you please send me copy of this article. I dont have subscription for this paper.
Dear Durgesh,
here is the paper by Corraliza for arginase activity. By the way, should you stop the reaction of converting arginnine bby an acid mixture, even when you use an urea detection kit instead of ISPF?