Difference between revisions of "Part:BBa K2346000"

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	<h2>Background</h2>		<h2>Background</h2>
		+
		+	Cytochrome P450 55B1 from Chlamydomonas reinhardtii<br>
		+	General Equation: $ \ce{2NO + NAD(P)H + H+ → N2O + NADP+ + H2O} $
		+
		+	[[Image:/wiki/images/6/6c/T--HFLS_H2Z_Hangzhou--img_enzyme_nor1.png]]
		+
		+	We found three assay methods to test the activity of nitric oxide reductase: using NO measurement Kit, titration and Gas-Phase Molecular Absorption Spectrometry. The first two methods were failed, and the details and reasons were recorded below. Due to limitations of lab equipment, we were not able to conduct the experiment using the third method.
		+
−	<img class="framed img" style="height: 30rem; width: auto;" src="/img/enzyme/nir1.png"></img>	+	[[Image:/wiki/images/6/64/T--HFLS_H2Z_Hangzhou--img_enzyme_nor2.png]]
		+	<div class="img-name">Nitric oxide reductase's tertiary structure</div>
		+
		+	<h2>Assay method 1: NO Measurement Kit</h2>
−	The first enzyme NiR is the product from expression of nirS gene amplified from Alcaligenes eutrophus DSM 530. The NiR catalyzes the reduction of nitrite (NO2-) to nitrogen monoxide(NO), as the first step of our substrate channeling system.	+	The NO kit we planned to use was developed by Nanjing Jiancheng Bioengineering Institute, and the concentration of NO in the solution can be calculated by the concentration of $\ce{NO3-}$, because NO will be reduced to $\ce{NO3-}$ in blood plasma of chicken or mice. We prepared the solution according to the method as below:
		+	<br><br>
		+	<table class="frameless ref-table">
		+	<tbody>
		+	<tr>
		+	<td><div class="no">1</div></td>
		+	<td>Remove the air in the flask by passing through nitrogen gas for 30 minutes.</td>
		+	</tr>
		+	<tr>
		+	<td><div class="no">2</div></td>
		+	<td>Pass through nitric oxide gas for 45 minutes in Flask C that contains ddH2O.</td>
		+	</tr>
		+
		+	<tr>
		+	<td><div class="no">3</div></td>
		+	<td>Flask A contains 30% H2O2 solution, and Flask B contains KMnO4 / NaOH solution, to oxidize and absorb the undissolved gas.[1]</td>
		+	</tr>
		+	</tbody>
		+	</table>
		+	<br>
		+	However, although the NO solution we prepared using this method was saturated, the concentration was too low for the verification to proceed.
−	<h2>Biology</h2>	+	[[Image:/wiki/images/5/59/T--HFLS_H2Z_Hangzhou--img_enzyme_nor3.png]]
		+	<div class="img-name">Fig 1. Setup of equipments for preparing NO solution</div>
−	<img class="framed img" style="height: 30rem; width: auto;" src="/img/enzyme/nir2.png"></img>	+	[[Image:/wiki/images/5/5a/T--HFLS_H2Z_Hangzhou--img_enzyme_nor4.jpg]]
−	<div class="img-name">Nitrite reductase's tertiary structure</div>	+	<div class="img-name">Fig 2. NO gas container</div>
−	We expressed the nitrite reductase with a His-tag in E.coli using a pET28a vector. The proteins are then extracted from the cells and purified using a HIS-trap column. After further purification, we ran electrophoresis to check the correct expression of NiR. The result of protein electrophoresis is shown below.	+	[[Image:/wiki/images/1/19/T--HFLS_H2Z_Hangzhou--img_enzyme_nor5.png]]
		+	<div class="img-name">Fig 3. Equipments setup</div>
−	<h2>Assay method</h2>	+	<h2>Assay Method 2: Titration</h2>
−	We used N-(1-naphthyl)ethylene diamine dihydrochloride spectrophotometric method, a common approach in waste water treatment, to measure the concentration of nitrite in the solution to track the reaction process. The nitrite concentration was measured using a color-showing reagent consisting of p-aminobenzenesulfonamide and 1g n-(1-naphthyl)-ethylenediamine dihydrochloride.	+
−	<br><br>	+
−	The reagent reacts with nitrite to form the redish pink diazo salt  in the solution, which has a maximum absorbance around 540nm. A standard curve was first made by measuring the absorbance at 540nm  of the reaction system under various nitrite concentration.	+
−	<br><br>	+
−	Stand curve between Absorbance-540nm and Sodium Nitrite concentration is shown below.	+
−	<img class="framed img" src="/img/enzyme/nir3.png"></img>	+	By measuring the concentration of nitrous oxide—the final product of the reduction of nitric oxide, the amount of nitric oxide reacted can be calculated, and therefore the activity of nitric reductase can be verified. When the reaction is complete, acidic potassium permanganate is added to the solution, and the highly oxidative potassium permanganate reacts with nitrous oxide. We then use oxalic acid to titrate the remaining potassium permanganate. Therefore, the amount of potassium permanganate used in the reaction with nitrous oxide can be calculated, and the amount of nitrous oxide produce can be found.
−	<div class="img-name">Standard curve between Absorbance-540nm and Sodium Nitrite concentration</div>	+
−	<h2>Reactivity assay in vitro</h2>
−	To assay the reactivity of the protein. We put the enzyme, sodium nitrite and pbs buffer together  to establish a reaction system. We recorded the nitrite concentration over 24 hours, under temperature of 37 degree Celcius. A standard curve was first made(as seen above) by measuring the absorbance at 540nm of the reaction system under various nitrite concentration.
	<br><br>		<br><br>
−	According to our results, the absorbance dropped from 0.633 to 0.008 over 24 hours, which indicates a drop of nitrite concentration from 0.196 mg/L to roughly zero. Currently we are still testing samples with shorter and various reaction time-length to plot the closest curve of the reactivity.
−	<img class="framed img" src="/img/enzyme/nir4.png"></img>	+	However, even for the most stable solution of nitrous oxide—the standard solution of 1.00mg/ml, nitrous oxide will escape in gas form, and the escaping speed is not constant. As a result, the method of directly measuring the amount of nitrous oxide is not valid.
−	<div class="img-name">NiR reactivity assay in vitro</div>	+
−	Since the application of these enzymes will be in vivo, we are satisfied with NiR’s reactivity was verified. We are much more interested in NiR’s ability to react in vivo with much greater concentration of nitrite.	+	<img class="framed img" src="/img/enzyme/nor6.png"></img>
−		+	<div class="img-name">Fig 4. Standard N2O solution</div>
−	<h2>Reactivity assay in vivo</h2>	+
−	<h3>Protocols</h3>	+	<h2>Assay Method 3: Gas-Phase Molecular Absorption Spectrometry</h2>
		+
		+	The turnover of the overall reaction of nitric oxide reductase was determined by monitoring the NADH consumption rate under various concentrations of NO, where the NO concentration was controlled by bubbling the NO/N2 mixed gas produced with the gas divider (ESTEC SGD-SC-0.5L). Unfortunately, since we could not find access to the required gas divider, we were not able to verify the results. The results from the original paper was shown in Fig 4.
		+	<br>
		+
		+	<h3>Protocol</h3>
	<table class="frameless ref-table">		<table class="frameless ref-table">
	<tbody>		<tbody>
	<tr>		<tr>
−	<td><div class="dot"></div></td>	+	<td><div class="no">1</div></td>
−	<td>Add IPTG into prepared cultures of E.coli (IPTG concentration: 0.1mg/L)</td>	+	<td>Prepare the buffer: 0.1 M sodium phosphate at pH 7.2.</td>
	</tr>		</tr>
	<tr>		<tr>
−	<td><div class="dot"></div></td>	+	<td><div class="no">2</div></td>
−	<td>Induce at 16 °C, 150 rpm overnight</td>	+	<td>Add reduced form of NADH.</td>
	</tr>		</tr>
	<tr>		<tr>
−	<td><div class="dot"></div></td>	+	<td><div class="no">3</div></td>
−	<td>Add sodium nitrite solution at various concentration (Target concentration: ~65mg/L)</td>	+	<td>Purge the air in the buffer solution by nitrogen gas to prevent formation of NO2 before adding NO.</td>
	</tr>		</tr>
	<tr>		<tr>
−	<td><div class="dot"></div></td>	+	<td><div class="no">4</div></td>
−	<td>Culture the bacteria at 37 °C,150 rpm</td>	+	<td>Introduce NO gas into the sample solution after passing 1 M KOH and the buffer solutions. </td>
	</tr>		</tr>
	<tr>		<tr>
−	<td><div class="dot"></div></td>	+	<td><div class="no">5</div></td>
−	<td>Take samples (500uL) of the cultures at various time points</td>	+	<td>Incubate the buffer solution containing NO (2.5, 2.0, 1.6, 1.5, 1.2, or 1.0 mM) and NADH (0.16 mM) in the optical cell at 10°C for 5 min.</td>
	</tr>		</tr>
	<tr>		<tr>
−	<td><div class="dot"></div></td>	+	<td><div class="no">6</div></td>
−	<td>Measure optical density(600nm) of the samples</td>	+	<td>Measure the absorbance (340nm) of the solutions.</td>
−	</tr>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>Centrifugate at 6,000 rpm for 60s </td>	+
−	</tr>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>Take 100uL samples from supernatant and dilute with 900uL ddH2O</td>	+
−	</tr>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>Take 100uL samples from the diluted solution and dilute again with 900 uL ddH2O</td>	+
−	</tr>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>Add 20uL of chromogenic agent to the solution</td>	+
−	</tr>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>Stand the solution for 20 mins and wait for the color to develop. </td>	+
−	</tr>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>Measure the absorbance(540nm) of the solutions </td>	+
−	</tr>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>Record data in Excel forms</td>	+
	</tr>		</tr>
	</tbody>		</tbody>
	</table>		</table>
		+
		+	<h2>Results</h2>
−	<br>	+	Due to limited access to equipments, we could not directly measure the reactivity of NOR. However, experiments had been carried out for the same enzyme expressed also expressed in E.coli, with method listed above. For the continuation of our project, we had to draw conclusion from other’s experiment results.
−	*Prepare the chromogenic agent as follows:	+
−	<table class="frameless ref-table">	+	[[Image:/wiki/images/7/74/T--HFLS_H2Z_Hangzhou--img_enzyme_nor7.png]]
−	<tbody>	+	<div class="img-name">Fig 5. Time course of the absorption change of the reduced form of NADH at 340 nm in the NO reduction reaction catalyzed by P450nor. [2]</div>
−	<tr>	+
−	<td><div class="no">1</div></td>	+
−	<td>In 500mL beaker	+
−	<table class="frameless ref-table">	+
−	<tbody>	+
−	<tr>	+
−	<td><div class="dot"></div></td>	+
−	<td>50mL Phosphoric Acid (d=1.70g/m)</td>	+
−	</tr>	+
−	<tr>	+	<h2>Conclusion</h2>
−	<td><div class="dot"></div></td>	+
−	<td>20.0g p-aminobenzenesulfonamide (C6H8N2O2S)</td>	+
−	</tr>	+
−	<tr>	+	<b>We delightedly found out that the curve became smooth and parallel to the x-axis just within the course of five minutes. This represented nearly all the substrates had been converted to products in an extremely rapid rate: much more rapid than nitrite reductase. This means that whenever NO is released from nitrite reductase as product, NOR can utilize it as substrate quickly.</b>
−	<td><div class="dot"></div></td>	+
−	<td>1g n-(1-naphthyl)-ethylenediamine dihydrochloride</td>	+
−	</tr>	+
−	<tr>	+	<h2>Reference</h2>
−	<td><div class="dot"></div></td>	+	<table class="frameless ref-table">
−	<td>250mL ddH2O</td>	+	<tbody>
−	</tr>	+
−	</tbody>	+
−	</table>	+
−	</td>	+
−	</tr>	+
	<tr>		<tr>
−	<td><div class="no">2</div></td>	+	<td><div class="no noind">1</div></td>
−	<td>Transfer the solution to a  500mL volumetric flask and dilute with ddH2O to standard volume.</td>	+	<td>程得胜, 黄曜, 黄郁芳. 一氧化氮标准储备水溶液浓度的测定及稳定性分析[J]. 分析测试学报, 2007, 26(4):566-569.</td>
	</tr>		</tr>
−	</tbody>
−	</table>
−	<b>NOTICE: We diluted the solution x100, so you have to time 100 the corresponding nitrite concentration using standard curve.</b>
−
−	<h3>Results</h3>
−	<img class="framed img" src="/img/enzyme/nir5.png"></img>
−	<div class="img-name">Nitrite conc. against reaction time</div>
−
−	<table class="frameless ref-table">
−	<tbody>
	<tr>		<tr>
−	<td><div class="dot noind"></div></td>	+	<td><div class="no noind">2</div></td>
−	<td>According to our results, the absorbance at 540nm dropped from 0.410 to 90.105 over 45.5 hours, which indicates a drop of NaNO2 concentration from 62.0(mg/L) to 14.9(mg/L).</td>	+	<td>Shiro Y., Fujii M., Iizuka T., Adachi S., Tsukamoto K., Nakahara K., Shoun H. 1995. Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism. J. Biol. Chem. 270, 1617–162310.1074/jbc.270.4.1617.</td>
	</tr>		</tr>
−		+
	<tr>		<tr>
−	<td><div class="dot noind"></div></td>	+	<td><div class="no noind">3</div></td>
−	<td>According to the graph, the reactivity of NiR is the highest during 20-30hours. About 76% of total nitrite is degraded at the end of assay. Also, there is no obvious reduction in nitrite concentration in control group.</td>	+	<td>Shoun H, Fushinobu S, Jiang L, et al. Fungal denitrification and nitric oxide reductase cytochrome P450nor[J]. Philosophical Transactions of the Royal Society of London, 2012, 367(1593):1186.</td>
	</tr>		</tr>
	</tbody>		</tbody>
	</table>		</table>

---

Latest revision as of 01:47, 2 November 2017

Nitric oxide reductase (NOR)

Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). We are still working on that. Since nitric oxide is nearly insoluable in water, it’s hard for us to create a aquatic reaction system for assaying the enzyme reactivity. We are still working on that. But currently we plan to create a reaction environment in a sealed container filled with solution, enzyme, and nitric oxide gas, By constant shake over, let’s say, two hours, we’ll then measure the change in the concentration of NO using the Gas-Phase Molecular Absorption Spectrometry to see if the presence of the enzyme made any difference on the concentration.

Sequence and Features

Background

Cytochrome P450 55B1 from Chlamydomonas reinhardtii
General Equation: $ \ce{2NO + NAD(P)H + H+ → N2O + NADP+ + H2O} $

File:/wiki/images/6/6c/T--HFLS H2Z Hangzhou--img enzyme nor1.png

We found three assay methods to test the activity of nitric oxide reductase: using NO measurement Kit, titration and Gas-Phase Molecular Absorption Spectrometry. The first two methods were failed, and the details and reasons were recorded below. Due to limitations of lab equipment, we were not able to conduct the experiment using the third method.

File:/wiki/images/6/64/T--HFLS H2Z Hangzhou--img enzyme nor2.png

Assay method 1: NO Measurement Kit

The NO kit we planned to use was developed by Nanjing Jiancheng Bioengineering Institute, and the concentration of NO in the solution can be calculated by the concentration of $\ce{NO3-}$, because NO will be reduced to $\ce{NO3-}$ in blood plasma of chicken or mice. We prepared the solution according to the method as below:

<tbody> </tbody>

1	Remove the air in the flask by passing through nitrogen gas for 30 minutes.
2	Pass through nitric oxide gas for 45 minutes in Flask C that contains ddH2O.
3	Flask A contains 30% H2O2 solution, and Flask B contains KMnO4 / NaOH solution, to oxidize and absorb the undissolved gas.[1]

However, although the NO solution we prepared using this method was saturated, the concentration was too low for the verification to proceed.

File:/wiki/images/5/59/T--HFLS H2Z Hangzhou--img enzyme nor3.png

File:/wiki/images/5/5a/T--HFLS H2Z Hangzhou--img enzyme nor4.jpg

File:/wiki/images/1/19/T--HFLS H2Z Hangzhou--img enzyme nor5.png

Assay Method 2: Titration

By measuring the concentration of nitrous oxide—the final product of the reduction of nitric oxide, the amount of nitric oxide reacted can be calculated, and therefore the activity of nitric reductase can be verified. When the reaction is complete, acidic potassium permanganate is added to the solution, and the highly oxidative potassium permanganate reacts with nitrous oxide. We then use oxalic acid to titrate the remaining potassium permanganate. Therefore, the amount of potassium permanganate used in the reaction with nitrous oxide can be calculated, and the amount of nitrous oxide produce can be found.

However, even for the most stable solution of nitrous oxide—the standard solution of 1.00mg/ml, nitrous oxide will escape in gas form, and the escaping speed is not constant. As a result, the method of directly measuring the amount of nitrous oxide is not valid.

<img class="framed img" src="/img/enzyme/nor6.png"></img>

Assay Method 3: Gas-Phase Molecular Absorption Spectrometry

The turnover of the overall reaction of nitric oxide reductase was determined by monitoring the NADH consumption rate under various concentrations of NO, where the NO concentration was controlled by bubbling the NO/N2 mixed gas produced with the gas divider (ESTEC SGD-SC-0.5L). Unfortunately, since we could not find access to the required gas divider, we were not able to verify the results. The results from the original paper was shown in Fig 4.

Protocol

<tbody> </tbody>

1	Prepare the buffer: 0.1 M sodium phosphate at pH 7.2.
2	Add reduced form of NADH.
3	Purge the air in the buffer solution by nitrogen gas to prevent formation of NO2 before adding NO.
4	Introduce NO gas into the sample solution after passing 1 M KOH and the buffer solutions.
5	Incubate the buffer solution containing NO (2.5, 2.0, 1.6, 1.5, 1.2, or 1.0 mM) and NADH (0.16 mM) in the optical cell at 10°C for 5 min.
6	Measure the absorbance (340nm) of the solutions.

Results

Due to limited access to equipments, we could not directly measure the reactivity of NOR. However, experiments had been carried out for the same enzyme expressed also expressed in E.coli, with method listed above. For the continuation of our project, we had to draw conclusion from other’s experiment results.

File:/wiki/images/7/74/T--HFLS H2Z Hangzhou--img enzyme nor7.png

Conclusion

We delightedly found out that the curve became smooth and parallel to the x-axis just within the course of five minutes. This represented nearly all the substrates had been converted to products in an extremely rapid rate: much more rapid than nitrite reductase. This means that whenever NO is released from nitrite reductase as product, NOR can utilize it as substrate quickly.

Reference

<tbody> </tbody>

1	程得胜, 黄曜, 黄郁芳. 一氧化氮标准储备水溶液浓度的测定及稳定性分析[J]. 分析测试学报, 2007, 26(4):566-569.
2	Shiro Y., Fujii M., Iizuka T., Adachi S., Tsukamoto K., Nakahara K., Shoun H. 1995. Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism. J. Biol. Chem. 270, 1617–162310.1074/jbc.270.4.1617.
3	Shoun H, Fushinobu S, Jiang L, et al. Fungal denitrification and nitric oxide reductase cytochrome P450nor[J]. Philosophical Transactions of the Royal Society of London, 2012, 367(1593):1186.