NITRATE REDUCTASE ASSAY ON RADISH SPROUTS Nitrate is one of many ions in the soil yet it has profound affects on plant metabolism and growth and serves both as a nutrient and signal Nitrate is an important source of nitrogen for plants and most devote a significant portion of their carbon and energy reserves for its uptake and assimilation On a global scale it has been estimated that plants assimilate 20 billion tons of nitrate per year Plants have evolved intricate mechanisms to detect nitrate and integrate its assimilation with nitrogen metabolism photosynthesis and carbon metabolism Without these mechanisms plants would be at the mercy of environmental fluctuations and would be unable to control carbon nitrogen ratios reductant levels ionic and pH balances and cell growth Nitrate itself is not incorporated into carbon but instead is first reduced to ammonia in two steps Nitrate is reduced to nitrite by nitrate reductase NR and then nitrite is reduced to ammonia by nitrite reductase NiR Eight reducing electrons and several protons are required for these steps in addition to the carbon skeletons needed for the incorporation of ammonia Nitrate and nitrite reduction occur in both roots and shoots of plants Because the flux of nitrogen through this pathway can be very significant large amounts of energy carbon and protons must be provided by the plant s metabolism Regulatory mechanisms control these transactions so that the plant can accommodate the needs of the nitrate assimilation pathway The first committed step in the nitrate assimilation pathway is the reduction of nitrate to nitrite The reaction is shown below NO3 NAD P H H NO2 NAD P H2O NADH or NADPH serve as the reductant and a proton is consumed in the reaction This reaction is catalyzed by NR which is a complex metalloenzyme that forms homodimers and homotretramers with each subunit being almost 1000 amino acids long NR has two active sites one to bind NAD P H and one to bind nitrate Three cofactors FAD heme Fe and MoCo provide the redox centers that facilitate the transfer of electrons as shown below NAD P H FAD heme Fe MoCo NO3Each subunit of NR has one each of the cofactors Most forms of plant NR use NADH but some are bispecific using either NADPH or NADH The MoCo is a molybdenum complexed with a small organic molecule called a molybdopterin 1 In these experiments we will study the physiological regulation of the nitrate reductase enzyme by examining how the level of enzyme responds to different environmental conditions We will determine enzyme levels by measuring enzyme activity using a simple enzymatic assay for nitrate reductase Day 1 Measure nitrate reductase activity in protein extract from radish sprouts to become acquainted with nitrate reductase assay Day 2 Study the effect of different environmental conditions on the level of nitrate reductase by measuring NR activity in the cotyledons of radish seedlings grown under a variety of different conditions Each team will come up with conditions to test and examine the effect on NR levels The NR assay measures the production of nitrite per minute in a reaction that contains the NR enzyme usually in the form of a protein extract nitrate and NADH in a buffered solution Nitrate is the substrate and is reduced to nitrite NADH provides the electrons for the reduction and is oxidized during the reaction Both NADH and nitrate are provided at a concentration that is saturating ie much higher than the Km so the the enzyme will be working at Vmax NR is activated two fold by phosphate and has a pH optimum around 7 5 therefore a phosphate buffer is used at pH 7 5 The nitrite that is produced is then quantified by a two step reaction First sulfanilamide in a 3N HCl solution is mixed with the nitrite The nitrite modifies the amino group to produce a very reactive diazonium ion Next N 1naphthylethylenediamine dihydrochloride NED in water is added which reacts with the diazonium group coupling the two aromatic compounds The resulting azo compound has enough conjugation that it absorbs visible light and is thus colored pink The amount of azo compound and the amount of color is directly related to amount of nitrite present and is measured in a spectrophotometer By using a standard of known concentration of nitrite one can determine the amount of nitrite produced in the reaction One normalizes the activity expressed as umoles nitrite produced per minute to the amount of protein in the protein extract to obtain specific activity or level of NR 2 Day 1 Nitrate reductase assay on protein extracts from radish sprouts Extract Cut off 4 radish sprout tips and place into the large glass homogenizer containing 0 75 ml of homogenization buffer Homogenizer and buffer should be on ice Grind on ice until no more chunks of tissue can be seen Pipette 1 ml with a glass pipette to a chilled eppendorf tube then spin at room temperature for 5 min Then transfer the supernatant to another tube on ice Assay In a large eppendorf tube on ice combine and mix 175 ls water with 25 ls of 10X reaction mix and 50 ls of protein extract Incubate for 15 min at 30 C Then add 250 ls sulfanilamide in 3 M HCl mix well Add 250 ls NED in H2O mix well then incubate at room temp for 15 mins Spin the solution for 5 minutes put supernatant into cuvette and read absorbance at 540nm along with the absorbance standards and background level sample Absorbance standards Use 5 eppendorf tubes containing 50 ls 10X reaction mix and 0 0nmoles 2 5nmoles 5nmoles 10nmoles and 20nmoles nitrite NaNO2 brought up to a final volume of 250 ls with H2O Your 0 nmoles tube will serve as a blank for standard and assay and background assay Then add 250 ls sulfanilamide and 250 ls NED and incubate at room temp for 15min Background Assay In a large eppendorf tube on ice combine and mix 175 ls water with 25 ls of 10X reaction mix and 50 ls of protein extract DO NOT Incubate for 15 min at 30 C just add 250 ls sulfanilamide in 3 M HCl mix well Add 250 ls NED in H2O mix well then incubate at room temp for 15 mins Spin the solution for 5 minutes put supernatant into cuvette and read absorbance at 540nm along with the absorbance standards and background level sample To determine protein concentration of protein extract add 5 ls of protein extract to 1ml of protein stain mix then measure absorbance at 595 nm Use 0 2 5 g 5 g 10 g 20 g and 40 g BSA as a standard to make a standard curve Notes Before you leave tell your TA what treatment you would like to