Nitrate Reductase Assay - (Jun/03/2009 )

I am attempting to measure nitrate reductase activity from frozen corn leaf samples. I have leaves that were rapidly frozen, I have chemical reagents to allow measurement of Nitrite (the product of nitrate reductase) and a I have a nitrite standard. The color agent and sample are mixed together 1:1 and when nitrite is present the sample turns pink and has a peak absorbance at 540nm.

My problem is figuring out a way to standardize the measurements using real plants because the green color interferes with the measurement. The sample never turns pink, or even close, it looks as though I just diluted it with water. I have tried rapidly boiling plants and mixing the green extract with known standards, but haven't had great luck with it. At this point my samples and my standards are not comparable at all.

Anyone have any great suggestions??

-Tyler

you could try the protocol given by worthington:

nitrate reductase assay

in their protocol, instead of using a killed enzyme preparation as a blank they use an assay without nitrate as a blank.