We would like to thank Dr. Camille Keller of Wesleyan University for technical support. Special thanks to Professor Lindsay D. Eltis and Jenna K. Capyk from the University of British Columbia, as well as Christian Whitman from the University of Texas at Austin, for their advice regarding anaerobic protein purification methods and the use of an O2-sensitive electrode.

1. General Materials and Methods
<ol>
	<li>Prepare all the required media as described in Table 1. Autoclave at 120 &#730;C for 15 min. Sterile filter the SOC solution, after the addition of MgCl2 and glucose, by passing it through a 0.2 &#181;m filter. Adjust the pH of the Miller&#39;s Lysogeny Broth (LB media) solution prior to autoclaving. Supplement the LB-Amp media solution after autoclaving with sterile solutions of 0.2 mM L-cysteine, then 0.1 mM ferrous ammonium sulfate to enhance pro...

The critical steps in obtaining active, purified DesB protein involve the forming and maintaining of the reduced Fe(II) active site in the enzyme. As such, correct performance of the induction, purification, concentration, and desalting steps are essential to successfully obtaining active enzyme. Inducing protein expression in the presence of 1 mM ferrous ammonium sulfate ensures that Fe(II) is correctly incorporated into the active site of DesB. This method is inspired by studies like those with amidohydrolase metalloen...

Enzymes that utilize iron or other metals to activate oxygen are often susceptible to inactivation during the purification process because of their removal from the reducing environment of a cell. Therefore, these proteins must be used as cell lysates, be subjected to external reducing agents, or be purified anaerobically to ensure that they have optimal enzymatic activity1,2,3,4. For those enzymes that are oxygen-sensitive (specifically iron-containing enzymes), performing all the purification and characterization steps while maintaining anaerobic conditions is necessary to fully characterize them. This has led researchers to develop entire laboratory set-ups within the confines of anaerobic chambers for studies ranging from protein expression through crystallography5,6,7,8.
Herein, we report methods for the anaerobic purification and kinetic characterization of the enzyme DesB using an oxygen electrode system. DesB is a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6 that is related to LigAB, a protocatecuate dioxygenase from the same organism. Both enzymes belong to the type II protocatechuate dioxygenase (PCAD) superfamily which has not been extensively studied to date9, likely in part due to enzymes of this superfamily being susceptible to inactivation when purified using standard aerobic protein purification methods. Since some of the PCAD enzymes display substrate promiscuity while others are substrate-specific2,10, further characterization of this superfamily is necessary to identify specificity determinants. As has been observed in several enzyme superfamilies11,12,13,14,15, small molecules can alter activity via direct competitive inhibition or the binding of molecules to separate allosteric pockets which causes an increase or decrease in enzymatic activity16. While kinetics alone cannot differentiate the binding location of a modulator, determining the magnitude of an activity change is important for understanding the effects. As such, methods for kinetic characterization of native DesB activity and its activity in the presence of 4-nitrocatechol (4NC), a compound commonly used to characterize and inhibit dioxygenase enzymes2,17,18, are shown.
DesB is able to break down gallate, a lignin-derived aromatic compound, via an extradiol dioxygenase (EDO) reaction in which ring opening is catalyzed using oxygen as one of the substrates10,19. This enzymatic reaction occurs within the context of the breakdown of lignin, an aromatic heteropolymer found in the cell wall of plants. Lignin can be depolymerized, yielding a variety of aromatic compounds that can be further broken down into central metabolites3,20,21,22,23,24,25,26,27,28,29,30,31,32,33. Extradiol dioxygenases (EDO) catalyze a ring opening reaction on dihydroxylated aromatic compounds, where cleavage occurs adjacent to a metal-coordinated diol; in contrast, intradiol dioxygenases cleave analogous aromatic compounds between the two hydroxyl groups (Figure 1). EDOs, like many other metalloenzymes, have a divalent metal center for coordinating Fe(II) composed of a two-histidine, one-carboxylate triad9,34,35. These metalloenzymes become oxidized, through either autoxidation or mechanism-based inactivation, whereas the enzyme is rendered inactive2,36,37,38.
In the experimental procedures described in this manuscript, we utilize DesB, a member of the PCAD superfamily from the bacterium Sphingobium sp. SYK-6, to catalyze the addition of oxygen across the C4-C5 bond of gallate (Figure 2A). The regiochemistry of this cleavage is analogous to LigAB, which is a protocatechuate-4,5-dioxygenase (Figure 2B). Thus far, investigations of this gallate dioxygenase include no reports of compounds that inhibit DesB10,19,39. With the use of aerobic purification methods, DesB exhibited variable activity, while with the use of anaerobic methods we were able to consistently obtain protein with reproducible activity. The kinetic studies described here show the methods for anaerobic purification of DesB, kinetic characterization of the reaction of DesB with gallate, and the inhibition of DesB by 4-nitrocatechol (4NC).

<table>
	<tbody>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranodise</td>
			<td>Gold Bio Technologies</td>
			<td>I2481C50</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad</td>
			<td>161-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide</td>
			<td>Bio-Rad</td>
			<td>161-0158</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;tetramethyl-ethylenediamine</td>
			<td>Bio-Rad</td>
			<td>161-0801</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose Resin High Flow</td>
			<td>New England Biolabs</td>
			<td>E8022S</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) competent Escherichia coli cells</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>gallic acid</td>
			<td>Sigma Aldrich</td>
			<td>G7384</td>
			<td></td>
		</tr>
		<tr>
			<td>4-nitrocatechol</td>
			<td>Sigma Aldrich</td>
			<td>N15553</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrous ammonium sulfate</td>
			<td>Mallinckrodt</td>
			<td>5064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dithionite</td>
			<td>Alfa Aesar</td>
			<td>33381-22</td>
			<td></td>
		</tr>
		<tr>
			<td>wheaton serum bottles</td>
			<td>Fisher Scientific</td>
			<td>06-406G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm Acrodisc PF Syringe Filter with Supor Membrane</td>
			<td>Pall Corportation</td>
			<td>4187</td>
			<td></td>
		</tr>
		<tr>
			<td>400 mL Amicon Stirred Cell Concentrator</td>
			<td>EMD Millipore</td>
			<td>UFSC40001</td>
			<td></td>
		</tr>
		<tr>
			<td>76 mm Millipore Ultracel 10 kDa cutoff reconsituted cellulose membrane filter</td>
			<td>EMD Millipore</td>
			<td>PLGC07610</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-dithiothreitol</td>
			<td>Gold Bio Technologies</td>
			<td>DTT50</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G-25 coarse desalting gal column</td>
			<td>GE Healthcare</td>
			<td>17-0033-01</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Crimp-Top Vials</td>
			<td>Fisher Scientific</td>
			<td>03-391-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygraph Plus Electrode Control Unit</td>
			<td>Hansatech Instruments</td>
			<td>OXYG1 Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Eletrode Chamber</td>
			<td>Hansatech Instruments</td>
			<td>DW1</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Disc</td>
			<td>Hansatech Instruments</td>
			<td>S1</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE (0.0125 mmX25mm) 30m reel</td>
			<td>Hansatech Instruments</td>
			<td>S4</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode cleaning Kit</td>
			<td>Hansatech Instruments</td>
			<td>S16</td>
			<td></td>
		</tr>
		<tr>
			<td>Spacer paper</td>
			<td>Zig Zag</td>
			<td>available at any gas station</td>
			<td></td>
		</tr>
		<tr>
			<td>He-series Dri-Lab glove box</td>
			<td>Vacuum/Atmospheres Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HE-493 Dri-Train</td>
			<td>Vacuum/Atmospheres Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Ended Micro-Tapered Stainless Steel Spatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-10</td>
			<td></td>
		</tr>
		<tr>
			<td>DWK Life Sciences Kimble Kontes Flex Column Economy Column</td>
			<td>Fisher Scientific</td>
			<td>k420400-1530</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L, Model 701 N SYR, Cemented NDL 26s ga, 2 in, point stlye 2 syringe</td>
			<td>Hamilton</td>
			<td>80300</td>
			<td></td>
		</tr>
		<tr>
			<td>DWK Life Sciences Kimble Kontes Flex Column Economy Column</td>
			<td>Fisher Scientific</td>
			<td>K420401-1505</td>
			<td></td>
		</tr>
		<tr>
			<td>Emulsiflex-C5 high-pressure homogenizer</td>
			<td>Avestin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B-PER Complete Bacterial Protein Extraction Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>89821</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme from chicken egg white</td>
			<td>Sigma Aldrich</td>
			<td>12650-88-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>7177-48-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher Scientific</td>
			<td>BP-1421-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Fisher Scientific</td>
			<td>BP684-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>G46-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

anaerobic protein purification and kinetic analysis via oxygen electrode for studying desb dioxygenase activity and inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Shown is the SDS-PAGE gel analysis of individual fractions from purification of the DesB-maltose binding protein (MBP) fusion construct (Figure 3). The gel reveals that the protein is pure (MW = 91.22 kDa), except for the presence of DesB (MW = 49.22 kDa) and MBP protein domain (42 kDa) cleaved from each other. Fractions E2 and E3 were selected for concentration (step 4.2).
Reproducible results from...

Watch this Scientific Journal Video about Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition at JoVE.com

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...