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.

The authors have no competing financial interests or other conflicts of interest.

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 &amp;beta;-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&#039;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 &amp;mu;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 ...

anaerobic-protein-purification-kinetic-analysis-via-oxygen-electrode

Research

JoVE Journal

Biochemistry

8.4K Views.  Wesleyan University. 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.

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

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

Bioengineering

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Cancer Research

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Changing Mitochondrial Function for Therapeutic and Bioinsecticide Applications

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

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

Summary

Explore More Videos

Chapters in this video

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays