Nitric oxide synthase (NOS) has been found to play many diverse physiological roles ranging from neurotransmitter to vasodilator to cytotoxic agent. It has been implicated in many normal and pathophysiological processes. Three isoforms of NOS exist, though all three isoforms are present in many different tissues and cell types. The enzyme synthesizes nitric oxide (NO) during the conversion of L-arginine to L-citrulline in the presence of oxygen and the cofactors NADPH, FAD, FMN and BH
4.
Determining the activity of NOS and thus, the amount of NO produced in cells or tissues is rapidly becoming a commonly used technique in many areas of research. Several companies have developed colorometric and fluormetric NOS activity assays which measure the concentration of nitrate and nitrite. These assays are based on the fact that NO is converted to nitrate and nitrite; therefore, measuring the concentrations of these molecules is a measure of the total NO in the cell or tissue. These types of assays work well; however, they are an indirect measure of NOS activity and have detection limits of about 2.5 uM. NOS activity can be directly measured by quantifying the production of citrulline from arginine.
The Calbiochem NOS Activity Assay Kit is based on the conversion of arginine to citrulline, using arginine labeled with 3H or 14C. Calbiochem claims the kit can detect down to the picomole level. The kit can be used for cell or tissue extracts and comes with enough spin columns for 50 reactions. The kit contains all reagents except for the radiolabeled arginine. The sample is mixed with equilibrated resin which binds to un-reacted arginine and is then applied to spin cups, allowing citrulline to elute through the column completely. The NOS activity is then determined by quantifying the radioactivity in the eluate with a liquid scintillation counter. The advantage of using this kit is that the spin cups replace column chromatography, which is traditionally used to separate arginine from citrulline. These columns are time consuming to prepare and use. Thus, a simple spin column to which the resin and sample is applied and centrifuged for 30 seconds is a major advantage.
We were measuring NOS activity in fibroblasts and HUVEC (human umbilical vein endothelial cells). We purchased several of these kits and have processed over 200 samples, each consisting of one 10 cm plate of 100% confluent cells. The kit protocol says to use 100 ul equilibrated resin per sample. We found that this was not enough to bind all the unreacted arginine and started using 300 ul equilibrated resin per sample. Though the kit came with enough reagents for 50 reactions, using more resin per sample allowed us to process only 16 reactions per kit. As the kit is expensive to start with, this necessary modification made the kit too expensive to use. Rat cerebellum extract is provided in the kit as a positive control. No activity was detected in any of the rat cerebellum extract samples from any of the kits, even when thawed within 3 minutes of analysis. Purified NOS worked well as a positive control and verified the reaction was working. Overall, we were not able to detect NOS in our samples using this kit, even though Calbiochem claims it detects to the picomole level. This may be due to the fact that we have medium to low levels of NOS in our samples; however, we were surprised that even after combining six 10 cm plates of cells into one sample, we still did not observe activity. We were, however, able to detect NOS activity in our samples by fluorescence microscopy using DAF-2/DA (Calbiochem) in which the intensity of the fluorescence indicated a high level of activity, although it was not quantified using this method. We also measured expression of NOS in these cells using Applied Biosystems TaqMan® Gene Expression Assays specific for the three isoforms of NOS. We found NOS II was expressed at approximately 103 copies/ul and NOS III was expressed at approximately 105 copies/ul. Although expression cannot be directly related to activity, the positive results from these other experiments indicate possible problems with this kit.
Naomi Rowland
Research Assistant
Western Kentucky University
Department of Biology