Detecting Chloride, Phosphate, Nitrite and Other Anions—Section 21.2

Page Contents

• Fluorescent Chloride Indicators
• Premo Halide Sensor
• Cyanide Detection
• Phosphate and Pyrophosphate Detection
• Nitrite, Nitrate and Nitric Oxide Detection
• Data Table
• Ordering Information

This section describes fluorescent indicators for intracellular and extracellular chloride together with an assortment of analytical reagents and methods for direct or indirect quantitation of other inorganic anions, including bromide, iodide, hypochlorite, cyanide,nitrite, nitrate, phosphate, pyrophosphate and selenide.

Most of the fluorescent chloride indicators are 6-methoxyquinolinium derivatives, the prototype of which is 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ, ). Cl^– detection sensitivity has been improved by modifications of the quinolinium N substituent. Our current range of Cl^– indicators consists of:

• 6-Methoxy-N-(3-sulfopropyl)quinolinium (SPQ, M440)
• N-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE, E3101)
• 6-Methoxy-N-ethylquinolinium iodide (MEQ, M6886)
• Lucigenin (L6868)

All of these indicators detect Cl^– via diffusion-limited collisional quenching. This detection mechanism is different from that of fluorescent indicators for Ca²⁺, Mg²⁺, Zn²⁺, Na⁺ and K⁺. It involves a transient interaction between the excited state of the fluorophore and a halide ion—no ground-state complex is formed. Quenching is not accompanied by spectral shifts (Figure 21.2.1) and, consequently, ratio measurements are not directly feasible. Quenching by other halides, such as Br^– and I^–, and other anions, such as thiocyanate, is more efficient than Cl^– quenching. Fortunately, physiological concentrations of non-choloride ions do not significantly affect the fluorescence of SPQ and other methoxyquinolinium-based Cl^– indicators. With some exceptions, fluorescence of these indicators is not pH sensitive in the physiological range. Because Cl^–-dependent fluorescence quenching is a diffusional process, it is quite sensitive to solution viscosity and volume. Exploiting this property, SPQ has been used to measure intracellular volume changes.

The efficiency of collisional quenching is characterized by the Stern–Volmer constant (K_SV), defined as the reciprocal of the ion concentration that produces 50% of maximum quenching. For SPQ, K_SV is reported to be 118 M^-1 in aqueous solution and 12 M^-1 inside cells. For MQAE, in situ K_SV values of 25–28 M^-1 have been determined in various cell types, compared with the solution value of 200 M^-1. Intracellular Cl^– indicators are generally calibrated using high-K⁺ buffers and the K⁺/H⁺ ionophore nigericin (N1495) in conjunction with tributyltin chloride, an organometallic compound that acts as a Cl^–/OH^– antiporter. With the exception of diH-MEQ (see below), Cl^– indicators must be loaded into cells by long-term incubation (up to eight hours) in the presence of a large excess of dye or by brief hypotonic permeabilization. Because membranes are slightly permeable to the indicator, rapid leakage may occur. Experimentally determined estimates of leakage vary quite widely.

Measurement of intracellular Cl^– concentrations and the study of Cl^– channels have been stimulated by the discovery that cystic fibrosis is caused by mutations in a gene encoding a Cl^– transport channel, which is known as the cystic fibrosis transmembrane conductance regulator (CFTR). Cl^– permeability assays are used to detect activity of the CFTR and other anion transporters. In these assays, SPQ- or MQAE-loaded cells are successively perfused with chloride-containing extracellular medium followed by medium in which the Cl^– content is replaced by nitrate (NO₃^–). NO₃^– is used in this assay protocol because it produces no fluorescence quenching of the indicator, yet its channel permeability is essentially the same as that of Cl^– (Figure 21.2.2).

Figure 21.2.1 Fluorescence emission spectra of MQAE (E3101) in increasing concentrations of Cl–.

Figure 21.2.2 Detection of cystic fibrosis transmembrane conductance regulator (CFTR) activity using 6-methoxy-N-(3-sulfopropyl)quinolinium, inner salt (SPQ, M440). Fluorescence of intracellular SPQ is quenched by collision with chloride ions, indicated by F0/F > 1 (F0 = fluorescence intensity in absence of chloride, F = fluorescence intensity at time points indicated on the x-axis). Upon addition of cyclic AMP to initiate channel opening, and exchange of extracellular Cl– (135 mM) for nitrate (NO3–), SPQ quenching decreases in CFTR-expressing cells (filled circles) as CFTR-mediated anion transport results in replacement of intracellular Cl– with nonquenching NO3–. Control cells with no CFTR expression (open circles) show no response.

SPQ

SPQ (M440, ) is currently in widespread use for detecting CFTR activity using the Cl^–/NO₃^– exchange technique described above. SPQ has also has been employed to investigate Cl^– fluxes through several other transporters such as the GABA_A receptor, erythrocyte Cl^–/HCO₃^– exchangers and the mitochondrial uncoupling protein. Although SPQ requires UV excitation (as do MQAE and MEQ), techniques for flow cytometric detection and calibration of the indicator using argon-ion laser excitation at 351 nm and 364 nm have been successfully demonstrated.

MQAE

MQAE (E3101, ) has greater sensitivity to Cl^– and a higher fluorescence quantum yield than SPQ; consequently, it is currently the more widely used of the two indicators. However, the ester group of MQAE may slowly hydrolyze inside cells, resulting in a change in its fluorescence response. MQAE has been used in a fluorescence-based microplate assay that has potential for screening compounds that modify Cl^– ion-channel activity. Other applications have included Cl^– measurements in cytomegalovirus-infected fibroblasts, smooth muscle cells and salivary glands, as well as in reconstituted membranes containing the GABA_A receptor or the mitochondrial-uncoupling protein (UCP-1).

MEQ and Cell-Permeant Dihydro-MEQ

The Cl^– indicator 6-methoxy-N-ethylquinolinium iodide (MEQ) can be rendered cell-permeant by masking its positively charged nitrogen to create a lipophilic, Cl^–-insensitive compound, 6-methoxy-N-ethyl-1,2-dihydroquinoline (dihydro-MEQ). This reduced quinoline derivative can then be loaded noninvasively into cells, where it is rapidly reoxidized in most cells to the cell-impermeant, Cl^–-sensitive MEQ (Figure 21.2.3). Using this technique, researchers have loaded live brain slices and hippocampal neurons with MEQ for confocal imaging of Cl^– responses to GABA_A receptor activation and glutamatergic excitotoxicity. Quenching of intracellular MEQ fluorescence by Cl^– has a K_SV of 19 M^-1, a value that is slightly higher than that reported for SPQ in fibroblasts. MEQ is available in solid form (M6886) and is supplied with a simple protocol (6-Methoxy-N-ethylquinolinium Iodide) for reducing it to dihydro-MEQ with sodium borohydride (not supplied) just prior to cell loading.

Figure 21.2.3 Intracellular delivery of the fluorescent chloride indicator 6-methoxy-N-ethylquinolinium iodide (MEQ, M6886), via oxidation of the membrane-permeant precursor dihydro-MEQ.

Lucigenin

The fluorescence of lucigenin (L6868, ) is quantitatively quenched by high levels of Cl^– with a reported K_SV = 390 M^-1. Lucigenin absorbs maximally at both 368 nm (ε = 36,000 cm^-1M^-1) and 455 nm (ε = 7400 cm^-1M^-1), with an emission maximum at 505 nm. Its fluorescence emission has a quantum yield of ~0.6 and is insensitive to nitrate, phosphate and sulfate. Lucigenin is a useful Cl^– indicator in liposomes and reconstituted membrane vesicles; however, because its fluorescence is reported to be unstable in the cytoplasm, it may not always be suitable for determining intracellular Cl^–. Lucigenin has been used to detect chloride uptake in tonoplast vesicles and to measure Cl^– influx across the pleural surface in perfused mouse lungs.

Alternative Detection Techniques for Halides

As mentioned above, the fluorescence of SPQ and related Cl^– indicators is quenched by collision with a variety of anions, including (in order of increasing quenching efficiency) Cl^–, Br^–, I^– and thiocyanate (SCN^–). For example, fluorescence of SPQ is partially quenched by the anionic pH buffer TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) but not by the protonated TES zwitterion, a property that has been exploited to measure proton efflux from proteoliposomes. Anion detectability using diffusional fluorescence quenching of these fluorophores is typically limited to the millimolar range. I^– quenches many other fluorophores and is commonly used to determine the accessibility of fluorophores to quenching in proteins and membranes.

In addition, halides can be oxidized to hypohalites (^–OCl, ^–OBr, ^–OI), which react with rhodamine 6G (R634, Probes for Mitochondria—Section 12.2) to yield chemiluminescent products. A cell produces ^–OCl by oxidizing Cl^– within the phagovacuole. ^–OCl also reacts with fluorescein (F1300, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) to yield fluorescent products, permitting analysis of ^–OCl levels in water.

Alternatively, 3'-(p-aminophenyl) fluorescein (APF) and 3'-(p-hydroxyphenyl) fluorescein (HPF) (A36003, H36004; Generating and Detecting Reactive Oxygen Species—Section 18.2) can be used for the selective detection of ^–OCl. Both of these fluorescein derivatives are essentially nonfluorescent until they react with the hydroxyl radical (HO·) or peroxynitrite anion (ONOO^–) (Figure 21.2.4). APF will also react with the hypochlorite anion (^–OCl), making it possible to use APF and HPF together to selectively detect hypochlorite anion. In the presence of these specific ROS, both APF and HPF yield a bright green-fluorescent product (excitation/emission maxima ~490/515 nm) and are compatible with all fluorescence instrumentation capable of visualizing fluorescein. Using APF, researchers have been able to detect the ^–OCl generated by activated neutrophils, a feat that has not been possible with traditional ROS indicators.

Figure 21.2.4 Detection of reactive oxygen species (ROS) with 3'-(p-hydroxyphenyl) fluorescein (HPF, H36004) and 3'-(p-aminophenyl) fluorescein (APF, A36003).

The fluorescent protein–based Premo Halide Sensor (P10229) is a pharmacologically relevant sensor for functional studies of ligand- and voltage-gated chloride channels and their modulators in cells. Chloride channels are involved in cellular processes as critical and diverse as transepithelial ion transport, electrical excitability, cell volume regulation and ion homeostasis. Given their physiological significance, it follows that defects in their activity can have severe implications, including such conditions as cystic fibrosis and neuronal degeneration. Thus, chloride channels represent important targets for drug discovery.

Premo Halide Sensor combines a yellow-fluorescent protein (YFP) variant sensitive to halide ions with the efficient and noncytopathic BacMam delivery and expression technology (BacMam Gene Delivery and Expression Technology—Note 11.1), yielding a highly sensitive, robust and easy-to-use tool for efficiently screening halide ion channels and transporter modulators in their cellular models of choice. Premo Halide Sensor is based on the Venus variant of Aequorea Victoria green-fluorescent protein (GFP), which displays enhanced fluorescence, increased folding, and reduced maturation time when compared with YFP. Additional mutations H148Q and I152L were made within the Venus sequence to increase the sensitivity of the Venus fluorescent protein to changes in local halide concentration, in particular iodide ions. Because chloride channels are also permeable to the iodide ion (I), iodide can be used as a surrogate of chloride. Upon stimulation, a chloride channel or transporter opens and iodide flows down the concentration gradient into the cells, where it quenches the fluorescence of the expressed Premo Halide Sensor protein (Figure 21.2.5, Figure 21.2.6). The decrease in Premo Halide Sensor fluorescence is directly proportional to the ion flux, and therefore the chloride channel or transporter activity. Premo Halide Sensor shows a similar excitation and emission profile to YFP (Figure 21.2.7) and can be detected using standard GFP/FITC or YFP filter sets. Halide-sensitive YFP-based constructs in conjunction with iodide quenching have been used in high-throughput screening (HTS) to identify modulators of calcium-activated chloride channels.

Premo Halide Sensor (P10229) is pre-packaged and ready for immediate use. It contains all components required for cellular delivery and expression—including baculovirus carrying the genetically encoded biosensor, BacMam enhancer and stimulus buffer—in ten 96- or 384-well plates. Premo Halide Sensor has been demonstrated to transduce multiple cell lines including BHK, U2OS, HeLa, CHO, and primary human bronchial epithelial cells (HBEC), providing the flexibility to assay chloride permeable channels in a wide range of cellular models.

Figure 21.2.5 Schematic representation of Premo Halide Sensor expression and halide sensitivity. Baculoviral particles encoding Premo Halide Sensor Sensor (P10229) enter the cells via an endocytic pathway. Following cellular entry, the baculovirus moves to the nucleus where Premo Halide Sensor gene is expressed. Premo Halide Sensor protein is localized throughout the cytoplasm and is free to react with iodide ions upon chloride channel activation, resulting in a loss of fluorescence emission intensity.

Figure 21.2.6 Principle of Premo Halide Sensor Sensor (P10229): Iodide redistribution upon chloride channel activation. Basal fluorescence from Premo Halide Sensor is high when chloride channels are closed or blocked. Upon activation (opening) of chloride channels, the iodide ions enter the cell, down its concentration gradient, and quench the fluorescence from Premo Halide Sensor.

Figure 21.2.7 Quenching of Premo Halide Sensor fluorescence by increasing concentrations of iodide and chloride. U2OS cells were transduced with Premo Halide Sensor. After 24 hours, cells were trypsinized and lysed by resuspension in sterile distilled water. Fluorescence quenching of the lysate was examined using increasing concentrations of NaCl (A) and NaI (B). Iodide induces substantially greater quenching of Premo Halide Sensor fluorescence than chloride.

The homologous aromatic dialdehydes, o-phthaldialdehyde (OPA, P2331MP) and naphthalene-2,3-dicarboxaldehyde (NDA, N1138), are essentially nonfluorescent until reacted with a primary amine in the presence of excess cyanide or a thiol, such as 2-mercaptoethanol, 3-mercaptopropionic acid or the less obnoxious sulfite, to yield a fluorescent isoindole (Figure 21.2.8, Figure 21.2.9). Modified protocols that use an excess of an amine and limiting amounts of other nucleophiles permit the determination of cyanide in blood, urine and other samples.

We also offer the ATTO-TAG CBQCA (A6222) and ATTO-TAG FQ (A10192) reagents, which are similar to OPA and NDA in that they react with primary amines in the presence of cyanide or thiols to form highly fluorescent isoindoles (Figure 21.2.10). The ATTO-TAG CBQCA and ATTO-TAG FQ reagents should also be useful for detecting cyanide in a variety of biological samples.

We have found that our Thiol and Sulfide Quantitation Kit (T6060, Introduction to Thiol Modification and Detection—Section 2.1) also provides an ultrasensitive enzymatic assay for cyanide, with a detection limit of ~5 nanomoles. In this case, interference would be expected from thiols, sulfides, sulfites and other reducing agents.

Figure 21.2.8 Fluorogenic amine-derivatization reaction of o-phthaldialdehyde (OPA) (P2331MP).

Figure 21.2.9 Fluorogenic amine-derivatization reaction of naphthalene-2,3-dicarboxaldehyde (NDA) (N1138).

Figure 21.2.10 Fluorogenic amine-derivatization reaction of CBQCA (A6222, A2333).

P_iPer Phosphate Assay Kit

The P_iPer Phosphate Assay Kit (P22061) provides an ultrasensitive assay that detects free phosphate in solution through formation of the fluorescent product resorufin. Because resorufin also has strong absorption, the assay can be performed either fluorometrically or spectrophotometrically. This kit can be used to detect inorganic phosphate (P_i) in a variety of samples or to monitor the kinetics of phosphate release by a variety of enzymes, including ATPases, GTPases, 5'-nucleotidase, protein phosphatases, acid and alkaline phosphatases and phosphorylase kinase. Furthermore, the assay can be modified to detect virtually any naturally occurring organic phosphate molecule by including an enzyme that can specifically digest the organic phosphate to liberate inorganic phosphate.

In the P_iPer phosphate assay (Figure 21.2.11), maltose phosphorylase converts maltose (in the presence of P_i) to glucose 1-phosphate and glucose. Then glucose oxidase converts the glucose to gluconolactone and H₂O₂. Finally, with horseradish peroxidase as a catalyst, the H₂O₂ reacts with the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) to generate resorufin, which has absorption/emission maxima of ~571/585 nm. The resulting increase in fluorescence or absorption is proportional to the amount of P_i in the sample. This kit can be used to detect as little as 0.2 µM P_i by fluorescence (Figure 21.2.12) or 0.4 µM P_i by absorption.

The P_iPer Phosphate Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Concentrated reaction buffer
• Recombinant maltose phosphorylase from Escherichia coli
• Maltose
• Glucose oxidase from Aspergillus niger
• Horseradish peroxidase
• Phosphate standard
• Hydrogen peroxide
• Detailed protocols for detecting phosphatase activity (PiPer Phosphate Assay Kit) 

Each kit provides sufficient reagents for approximately 1000 assays using a reaction volume of 100 µL per assay and either a fluorescence or absorbance microplate reader.


Figure 21.2.11 Principle of the P_iPer Phosphate Assay Kit (P22061). In the presence of inorganic phosphate, maltose phosphorylase converts maltose to glucose 1-phosphate and glucose. Then, glucose oxidase converts the glucose to gluconolactone and H₂O₂. Finally, with horseradish peroxidase (HRP) as a catalyst, the H₂O₂ reacts with the Amplex Red reagent to generate the highly fluorescent resorufin. The resulting increase in fluorescence or absorption is proportional to the amount of P_i in the sample.

Figure 21.2.12 Detection of inorganic phosphate using the PiPer Phosphate Assay Kit (P22061). Each reaction contained 50 µM Amplex Red reagent, 2 U/mL maltose phosphorylase, 1 mM maltose, 1 U/mL glucose oxidase and 0.2 U/mL HRP in 1X reaction buffer. Reactions were incubated at 37°C. After 60 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Data points represent the average of duplicate reactions, and a background value of 43 (arbitrary units) was subtracted from each reading.

P_iPer Pyrophosphate Assay Kit

The P_iPer Pyrophosphate Assay Kit (P22062) provides a sensitive fluorometric or colorimetric method for measuring the inorganic pyrophosphate (PP_i) in experimental samples or for monitoring the kinetics of PP_i release by a variety of enzymes, including DNA and RNA polymerases, adenylate cyclase and S-acetyl coenzyme A synthetase. In the P_iPer pyrophosphate assay, inorganic pyrophosphatase hydrolyzes PP_i to two molecules of inorganic phosphate (P_i). The P_i then enters into the same cascade of reactions as it does in the P_iPer Phosphate Assay Kit (Figure 21.2.11). In this case, the resulting increase in fluorescence or absorption is proportional to the amount of PP_i in the sample. This kit can be used to detect as little as 0.1 µM PP_i by fluorescence or 0.2 µM PP_i by absorption (Figure 21.2.13).

The P_iPer Pyrophosphate Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Concentrated reaction buffer
• Recombinant maltose phosphorylase from Escherichia coli
• Maltose
• Glucose oxidase from Aspergillus niger
• Horseradish peroxidase
• Inorganic pyrophosphatase from baker's yeast
• Pyrophosphate standard
• Detailed protocols for detecting pyrophosphatase activity (PiPer Pyrophosphate Assay Kit)

Each kit provides sufficient reagents for approximately 1000 assays using a reaction volume of 100 µL per assay and either a fluorescence or absorbance microplate reader.


Figure 21.2.13 Detection of pyrophosphate using the P_iPer Pyrophosphate Assay Kit (P22062). Each reaction contained 50 µM Amplex Red reagent, 0.01 U/mL inorganic pyrophosphatase, 2 U/mL maltose phosphorylase, 0.2 mM maltose, 1 U/mL glucose oxidase and 0.2 U/mL HRP in 1X reaction buffer. Reactions were incubated at 37°C. After 60 minutes, A) fluorescence was measured in a fluorescence-based microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm or B) absorbance was measured in an absorption-based microplate reader at 576 ± 5 nm. Data points represent the average of duplicate reactions. In panel A, a background value of 78 (arbitrary units) was subtracted from each reading; in panel B, a background absorbance of 0.011 was subtracted from each reading.

EnzChek Phosphate Assay Kit

The EnzChek Phosphate Assay Kit (E6646), which is based on a method originally described by Webb, provides an easy enzymatic assay for detecting P_i from multiple sources through formation of a chromophoric product (Figure 21.2.14). Although this kit is usually used to determine the P_i produced by a wide variety of enzymes such as ATPases, kinases and phosphatases (Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3), it can also be used to specifically quantitate P_i with a sensitivity of ~2 µM P_i (~0.2 µg/mL) (Figure 21.2.15). Moreover, this colorimetric assay has proven useful for determining the level of P_i contamination in the presence of high concentrations of acid-labile phosphates using a microplate reader. Because the sulfate anion competes with P_i for binding to purine nucleoside phosphorylase (PNP), this kit can be adapted for measurement of sulfate concentrations between 0.1 and 10 mM in the presence of a low (<100 µM) fixed P_i concentration.

The EnzChek Phosphate Assay Kit contains:

• 2-Amino-6-mercapto-7-methylpurine riboside (MESG)
• Purine nucleoside phosphorylase (PNP)
• Concentrated reaction buffer
• KH₂PO₄ standard
• Detailed protocols for detecting and quantitating P_i (EnzChek Phosphate Assay Kit)

Each kit provides sufficient reagents for about 100 phosphate assays using 1 mL assay volumes and standard cuvettes.


Figure 21.2.14 Enzymatic conversion of 2-amino-6-mercapto-7-methylpurine riboside (MESG) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine by purine nucleoside phosphorylase (PNP), reagents supplied in the EnzChek Phosphate Assay Kit (E6646). The accompanying change in the absorption maximum (Abs) allows quantitation of inorganic phosphate (P_i) consumed in the reaction.

Figure 21.2.15 Quantitative analysis of inorganic phosphate using the EnzChek Phosphate Assay Kit (E6646). KH2PO4 was used as the source for the inorganic phosphate, and the absorbance at 360 nm was corrected for background absorbance. The inset shows an enlargement of the standard curve, demonstrating the lower range of the assay; the units are the same.

EnzChek Pyrophosphate Assay Kit

In the EnzChek Pyrophosphate Assay Kit (E6645), we have adapted the method provided in the EnzChek Phosphate Assay Kit to permit the sensitive spectrophotometric detection of PP_i, which is converted by the enzyme pyrophosphatase to P_i. Because two moles of P_i are released per mole of PP_i consumed, the sensitivity limit of the EnzChek Pyrophosphate Assay Kit is 1 µM PP_i (~0.2 µg/mL). This assay has been modified to continuously detect several enzymes that liberate PP_i such as aminoacyl-tRNA synthetase, luciferase, cytidylyl transferase and S-acetyl coenzyme A synthetase and potentially DNA and RNA polymerases, adenylate cyclase and guanylyl cyclase.

The EnzChek Pyrophosphate Assay Kit contains:

• Inorganic pyrophosphatase
• 2-Amino-6-mercapto-7-methylpurine riboside (MESG)
• Purine nucleoside phosphorylase (PNP)
• Concentrated reaction buffer
• Na₂P₂O₇ standard
• Detailed protocols for detecting and quantitating PP_i (EnzChek Pyrophosphate Assay Kit)

Each kit provides sufficient reagents for about 100 PP_i assays using standard 1 mL assay volumes and standard cuvettes.

With the discovery of the role of nitric oxide in signal transduction (Probes for Nitric Oxide Research—Section 18.3), assays for nitrite (NO₂^–) have assumed new importance. Because inorganic nitrite is spontaneously produced by air oxidation of nitric oxide, the same reagents that have been utilized to detect nitric oxide production in cells should be useful for detecting nitrite in aqueous samples. Furthermore, inorganic nitrate (NO₃^–) can be reduced to NO₂^– by both chemical and enzymatic means, permitting the quantitative analysis of NO₃^– in samples.

Measure-iT High-Sensitivity Nitrite Assay Kit

The Measure-iT High-Sensitivity Nitrite Assay Kit (M36051) provides an easy and accurate method for quantitating nitrite. This kit has an optimal range of 20–500 picomoles nitrite (Figure 21.2.16) , making it up to 50 times more sensitive than colorimetric methods utilizing the Griess reagent. Nitrates may be analyzed after quantitative conversion to nitrites through enzymatic reduction.

Each Measure-iT High-Sensitivity Nitrite Assay Kit contains:

• Measure-iT nitrite quantitation reagent (100X concentrate in 0.62 M HCl)
• Measure-iT nitrite quantitation developer (2.8 M NaOH)
• Measure-iT nitrite quantitation standard (11 mM sodium nitrite)
• Detailed protocols (Measure-iT High-Sensitivity Nitrite Assay Kit)

Simply dilute the reagent 1:100, load 100 µL into the wells of a microplate, add 1–10 µL sample volumes and mix. After a 10-minute incubation at room temperature, add 5 µL of developer and read the fluorescence. The assay signal is stable for at least 3 hours, and common contaminants are well tolerated in the assay. The Measure-iT High-Sensitivity Nitrite Assay Kit provides sufficient material for 2000 assays, based on a 100 µL assay volume in a 96-well microplate format; this nitrite assay can also be adapted for use in cuvettes or 384-well microplates.

Figure 21.2.16 Linearity and sensitivity of the Measure-iT high-sensitivity nitrite assay. Triplicate 10 µL samples of nitrite were assayed using the Measure-iT High-Sensitivity Nitrite Assay Kit (M36051). Fluorescence was measured using excitation/emission of 365/450 nm and plotted versus picomoles of nitrite. Background fluorescence was not subtracted. The variation (CV) of replicate samples was <2%.

Griess Reagent Kit

Under physiological conditions, NO is readily oxidized to NO₂^– and NO₃^– or it is trapped by thiols as an S-nitroso adduct. The Griess reagent provides a simple and well-characterized colorimetric assay for nitrites—and nitrates that have been reduced to nitrites—with a detection limit of about 100 nM. The Griess assay is suitable for measuring the activity of nitrate reductase in a microplate. Nitrite reacts with the Griess reagent to form a purple azo derivative that can be monitored by absorbance at 548 nm (Figure 21.2.17).

The Griess Reagent Kit (G7921) contains all of the reagents required for NO₂^– quantitation, including:

• N-(1-Naphthyl)ethylenediamine dihydrochloride
• Sulfanilic acid in 5% H₃PO₄
• A concentrated nitrite quantitation standard for generating calibration curves
• Detailed protocols for spectrophotometer- and microplate reader–based assays (Griess Reagent Kit for Nitrite Determination) 

Both the N-(1-naphthyl)ethylenediamine dihydrochloride and the sulfanilic acid in 5% H₃PO₄ are provided in convenient dropper bottles for easy preparation of the Griess reagent. Sample pretreatment with nitrate reductase and glucose 6-phosphate dehydrogenase is reported to reduce NO₃^– without producing excess NADPH, which can interfere with the Griess reaction. NO that has been trapped as an S-nitroso derivative can also be analyzed with the Griess Reagent Kit after first releasing the NO from its complex using mercuric chloride or copper (II) acetate.

Figure 21.2.17 Principle of nitrite quantitation using the Griess Reagent Kit (G7921). Formation of the azo dye is detected via its absorbance at 548 nm.

DAF-FM

DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein, D23841; ) and its diacetate derivative (DAF-FM diacetate, D23842, D23844; Probes for Nitric Oxide Research—Section 18.3) have significant utility for measuring nitric oxide and nitrite production in live cells and solutions. The fluorescence quantum yield of DAF-FM is reported to be 0.005 but increases about 160 fold to 0.81 after reacting with nitrite (Figure 21.2.18). DAF-FM has some important advantages over the similar nitric oxide sensor, DAF-2, and other aromatic diamines:

• Spectra of the NO (NO₂^–) adduct of DAF-FM are independent of pH above pH 5.5.
• NO₂^– adduct of DAF-FM is significantly more photostable than that of DAF-2.
• DAF-FM is a more sensitive reagent for NO₂^– than is DAF-2; the NO and NO₂^– detection limit for DAF-FM is ~3 nM versus ~5 nM for DAF-2.)
• The higher absorptivity and greater water solubility of the NO₂^– adduct of DAF-FM should make this assay much more sensitive than detection with 2,3-diaminonaphthalene (see below) or other aromatic diamines.

Because the reaction of DAF-FM with NO requires a preliminary nonspecific oxidation step, it is important to also perform control experiments with nitric oxide synthase inhibitors to confirm the source of the fluorescent species.

Figure 21.2.18 Fluorescence emission spectra of DAF-FM (D23841, D23842, D23844) in solutions containing zero to 1.2 µM nitric oxide (NO).

2,3-Diaminonaphthalene

We also offer 2,3-diaminonaphthalene (D7918, ), which reacts with NO₂^– to form the fluorescent product 1H-naphthotriazole. A rapid, quantitative fluorometric assay that employs 2,3-diaminonaphthalene can reportedly detect from 10 nM to 10 µM NO₂^–, and is compatible with a 96-well microplate format. Nitrate (NO₃^–) does not interfere with this assay; however, NO₃^– can be reduced to NO₂^– by bacterial nitrate reductase and then detected using the same reagent. A detailed protocol for measuring the stable products of the nitric oxide pathway (NO₂^– and NO₃^–) using 2,3-diaminonaphthalene has been published and is shown to be approximately 50 times more sensitive than the Griess assay.

NBD Methylhydrazine

NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M20490) has been used to measure NO₂^– in water. Reaction of NBD methylhydrazine with NO₂^– in the presence of mineral acids leads to formation of fluorescent products with excitation/emission maxima of ~468/537 nm. This reaction serves as the principle behind a selective fluorogenic method for the determination of NO₂^– (Figure 21.2.19). The assay is suitable for measurements by absorption or fluorescence spectroscopy or by fluorescence-detected HPLC.

Figure 21.2.19 Reaction scheme illustrating the principle of nitrite detection by NBD methylhydrazine (M20490).

Other Nitrate Detection Reagents

Rhodamine 110 (R6479) has proven useful in a fluorescence quenching method for determining trace nitrite. This sensitive assay takes advantage of the reaction of the green-fluorescent rhodamine 110 with nitrite at acidic pH to form a nitroso product that exhibits much weaker fluorescence. With a linear range of 1 × 10^-8 to 3 × 10^-7 moles/L and a detection limit of 7 × 10^-10 moles/L, this assay has been used to measure nitrite in tap water and lake water without any prior extraction procedures.

Efficient quenching of SPQ or MQAE fluorescence (M440, E3101; see above) by nitrite (but not nitrate) has been used for direct measurement of NO₂^– transport across erythrocyte membranes and for functional assays of bacterial nitrite extrusion transporters.