Several articles highlight that red blood cells express a functional endothelial nitric oxide synthase. How can I assay eNOS activity in RBCs? What are the different techniques used?
This is challenging research area!
There are several NOS activity assays.
The most classic utilizes the conversion of radiolabled Arg to Cit. This may be your best bet.
Alternately, one can look for NO related species, using various tools, but this can be hard in the presence of Hb. Finally, one can use the conversion of OxyHb to metHb by NO.
I recommend starting with the literature, such as the Blood article from the Kelm group that extensively assessed RBC NOS function:
http://www.ncbi.nlm.nih.gov/pubmed/23007404
Thank you Peter for mentioning our paper:) - For red cell eNOS activity I would recommend the citrullin assay - but please do not use cell lysates - but just whole cells (in the presence of an arginase inhibitor like NorNOHA) or membrane preparations (please refer to Wood, Cortese-Krott et al for the latest protocol). For pitfalls please refer to Cortese-Krott redox biology 2014. I am happy to help further if you need.
Best,
Miriam
Indeed, the works of Klienbongard, Rassaf and Kelm are the best. They compare multiple techniques and find that the chemiluminiscent technique is the most sensitive of all. We did that in mouse RBCs http://www.ncbi.nlm.nih.gov/pubmed/19515903
If you want to have a relative comparison of the NOS activity in two different conditions, it is relatively easy: one has to look into the nitrite and nitrate levels in RBCs and extracellular medium (plasma) the presence and absence of L-NMMA. Not to forget the L-arginine supplementation to the medium! In mice (in contrast to humans) one may even check the hypothesis by comparing the data in wild type and eNOS-deficient animals. In humans, I am afraid, one has to take care of substate supplementation (L-argrinine and O2) and control the intracellular Ca2+ levels (using fluorescent dyes) and redox state of cells (NAD(P)H is also a substrate to take care of). Some of NO produced this way will be bound to hemoglobin. So, if you want to get a "true" eNOS capacity, one has to be careful getting this one released. Hemolysis will interfere with the measurements. In order to reduce the artifacts related to it a special buffer is used to achieve rapid sedimentation of it (see our paper for its composition).
Have you used the Sievers Nitric Oxide Analyzer (NOA 280i) for NO analysis in liquids? We first used its earlier version (Ahmed et al, Lab Invest 1997) and more recently the NOA 280i; it can detect very low levels of NO in condition media.
http://www.geinstruments.com/products-and-services/nitric-oxide-analyzer
i have another question if you don't mind
are there a way to determine the NOS activity by measuring the NADPH consumption (1.5 µM coresponding to 1 U)
Hi Amine Allaoui,
NADPH is not a substrate for NOS, so it couldn't used. If there is an indirect method (quite sure there is none) the detection will be non-specific
thank you Ayman for your reply
for NADPH, it is used in the reaction
1 mole to convert 1 mole arginine --> 1 mole hydroxyargine
0.5 mole to go from hydroxyarginine --> citruline
please, see the joined document
now, about the non specific detection, can you be more clear please
thank you again
me too i have a question about Nos activity assay. i want to measure it but using a colorimetric method (no kit).
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