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Kinetic Analysis of NemA and XenB by HPLC

https://static.igem.org/mediawiki/2014/9/9b/TNT-Standard-Curve-scaled.gif

(this will be figure X, a totally EPIC animation of a 2D absorbance graph revolving into a 3D HPLC absorbance graph to highlight the importance of having a 3rd axis for elution time)

Summary

NemA and Xen B are capable of catalysing the conversion of TNT to various products using NADH and FMN as cofactors. The binding affinity of each protein for this substrate (the Michealis Menten constant, Km) and the maximum reaction velocity (Vmax), were determined and are comparable to the published values; shown in figure 1. NemA and XenB are therefore suitable enzymes for use in our system and have been shown to function at the physiologically relevant pH of 7.

	NemA Vmax (TNT)	XenB Vmax (TNT)	NemA Vmax (Nitroglycerin)	XenB Vmax (Nitroglycerin)	NemA Km (TNT)	XenB Km (TNT)	NemA Km (Nitroglycerin)	XenB Km (Nitroglycerin)
Experimental Results
Published Values			8				15

Figure 1

Abstract

NemA and XenB are two proteins that the iGEM Exeter team propose will allow E.coli to degrade TNT, at concentrations above those normally toxic to the cell. Among many others proposed, NemA catalyses the reaction shown in figure 2.

Various hydroxylamino derivatives may be produced, as well as ammonium ions which could be used as a nitrogen source by the E.coli for growth. The following experimental account describes the protocol used to confirm the substrate target of NemA and XenB and analyse the respective kinetic capabilities of these enzymes.

Results

Choice of analytical procedure

NADH is a cofactor in the conversion of TNT to X. This opens up the possibility to simply measuring the catalytic rate of TNT degradation by following change in the absorbance at 340nm. However, very early in the process we realised that TNT also produces a significant absorbance at 340nm, a fact that would complicate our analytical procedure. We therefore chose to use High Performance Liquid Chromatography (HPLC) analysis. As can be seen (Fig X), HPLC separates TNT and NAD by elution time meaning that the overlap in absorbance values are not an issue.

TNT Standard Curve

A standard curve of TNT concentration was determined first. Integration of the area below the absorbance peak of TNT at the concentrations described, resulted in the standard curve shown in figure 3. For further details see materials and methods.

Determination of NemA activity

Purified NemA protein was assayed for its ability to degrade TNT at a range of concentrations: from 0 mM TNT up to 4.4 mM TNT. The reaction is a stopped enzyme assay. In this system the reaction is started by the addition of NemA to a reaction mix containing TNT, FMN and NADH. The reaction is stopped by the addition of hydrochloroacetic acid at predetermined intervals and the concentration of TNT is then assayed using the HPLC. These data are shown in figure 4. Full details can be seen in the materials and methods.

Figure 5 shows NemA reaction kinetics as a function of initial velocity, Vi, against initial TNT concentration (derived from taking a tangent at the steepest section of each series in figure 4). From this it can be concluded that the Vmax is...

The data can either be plotted as a Lineweaver-Burke plot (figure 6), or as a Hanes plot (figure 7) to more accurately determine the Km values. From these it can be concluded that the Km of NemA for TNT substrate is...

Materials and Methods

Detection of TNT, NemA and its cofactors

Pure samples were injected into an Agilent Technologies infinity reverse phase high performance liquid chromatograph C18 column and their absorbance peaks identified by comparison with blanks (1:1 water, acetonitrile). Preliminary experiments showed a TNT absorption maximum at 230 nm. The elution time for the TNT peak was reliably situated at 0.8 minutes in all runs using the mobile phase gradient shown in figure 8

This mobile phase profile with a flow rate of 0.4 ml min-1 as well as an injection volume of 20 μL (minimum available) was kept constant over the course of the experiment to maintain elution profile consistency. A disturbance in the force of liquid applied to the column was shown to affect elution profiles to such an extent that identification and quantification of peak areas was unreliable. Buffer A consisted of 95% water and 5% acetonitrile whilst buffer B consisted of 95% acetonitrile and 5% water; both HPLC grade. 100% acetonitrile was not used to prevent polymerisation and build up of acetonitrile in the pumps. Initial runs consisting of NADH, FMN and NemA (as individual samples) dissolved in a 1:1 solution of acetonitrile and water showed no retention. The presence of these compounds was confirmed by the appearance of new absorbance peaks in the solvent front; previously absent in blank samples of a 1:1 solution of acetonitrile and water; shown in figure X. The mobile phase composition, shown in figure 1, allowed sufficient separation of the absorbance peak of TNT from the broad peaks of the highly absorbent peptides and cofactors in the solvent front; necessary for accurate and precise integration of the TNT peak area.

Quantification of TNT concentration

50 μL of stock TNT (0.44 M) in acetonitrile was diluted up to a total volume of 50 mL, with HPLC grade acetonitrile and water (1:1) to produce a 4.4 mM TNT solution. Serial dilutions of these solutions were performed to achieve the concentrations shown in figure 9.

Tube No.	1	2	3	4	5	6	7	8	9	10
[TNT] /mM	4.4	2.2	1.1	0.55	0.275	0.138	0.069	0.034	0.017	0

Figure 9

The absorbances of samples of these concentrations were measured using the HPLC technique described in figure 8.

Purification of His-tagged NemA

Insert brief description of how NemA was expressed in cells i.e. IPTG concentration, OD at induction etc.

NemA Kinetic Analysis

His tagged NemA was separated from whole cell lysate by running through a nickel column and were subsequently stored at 5oC. The resulting aqueous protein was quantified by absorbance in a Thermo Scientific Nanodrop 2000c using its published extinction coefficient of … A master mix of FMN (0.14 mM), NADH (0.14 mM) and NemA (x mM) was created. To samples of this master mix, TNT was added to create final reaction mixture concentrations of: 4.4, 2.2, 1.1, 0.55, 0.28, 0.14, 0.07, 0.03 and 0.02 mM. After incubation at 20oC, each reaction was stopped after 1, 5, 10 and 30 minutes with the addition of 20 µl of trichloroacetic acid (240 mg/mL). The resulting mixture was quick-frozen and stored at −70°C before HPLC analysis.