Anaerobic Heme Recycling by Gut Microbes: Important Methods for Monitoring Porphyrin Production

Abstract

Heme is the most abundant species of iron inside the human body and an essential cofactor for numerous electron/chemical group transfer reactions and catalyses, especially those involving O₂. Whole anaerobic biomes exist that also depend on heme but lack widespread, O₂-dependent pathways for heme synthesis and breakdown. The gastrointestinal tract is an anaerobic ecosystem where many microbes are auxotrophic for heme, and where the abundant members of the Bacteroidetes phylum convert heme into iron and porphyrins. Working with mixtures of these hydrophobic compounds presents challenges for analyses, especially when their source is biological. In this brief chapter, we detail a handful of important methods and point out caveats necessary for their concurrent detection, separation, and quantification.

1. Introduction

Sometimes, biochemistry astounds. Every second, the human body generates 2.5 billion new red blood cells, each one packed with O₂-transporting hemoglobin (Muckenthaler et al. 2017). The new cells replace old ones which have been damaged, destroyed, and then scavenged by macrophages. The heme thus recovered is scrapped so that its iron can be recovered, limiting our dietary iron requirements to manageable amounts. Heme oxygenases catalyze the critical reaction in the iron recovery process, using O₂ to convert the heme into biliverdin, CO, and Fe²⁺.

Yet, heme also serves essential roles and heme homeostasis must be maintained in anaerobic niches where heme oxygenases are largely unavailable. The human gastrointestinal (GI) tract is anaerobic, and the major species of the GI microbiome are anaerobes from the Bacteroidetes and Firmicutes phyla. Most lack a complete biosynthetic pathway for heme, though many require heme as a nutrient for growth.

We recently showed that the hmu operon, encoding a pathway for heme uptake and metabolism, is largely confined to and ubiquitous within the Bacteroidetes phylum. We observed that a representative of this phylum, Bacteroides thetaiotaomicron, can use heme as a sole source of iron, provided the 6-gene hmuYRSTUV pathway is intact (Meslé et al., 2023). The products of heme catabolism by these species include Fe(II), protoporphyrin IX (PpIX), and possibly other, modified porphyrins. In addition, some pathogenic species of the GI tract, such as Helicobacter pylori, secrete porphyrins (Hamblin et al. 2005), while others like Salmonella enterica thrive on the iron derived from heme (Sebastiàn et al., 2022). The GI tract is consequently an environment populated by heme and porphyrins, compounds with significant influence over both microbial and human health.

Understanding anaerobic heme and porphyrin metabolism requires methods for concurrently quantifying heme, PpIX, and other porphyrins, over time and in the context of other biological molecules. Importantly, heme and porphyrins have different solubilities and absorbance/fluorescence properties that complicate their co-analysis. The following chapter describes methods for the extraction, detection, and quantification of heme, PpIX, and related porphyrins, from microbial cultures and other biochemical extracts. These methods may be useful for understanding heme and porphyrin metabolism in anaerobic microbes and microbiomes.

2. Methods

A. Preparing heme and porphyrin-containing extracts from biological materials

Heme and PpIX are not soluble in water, making it necessary to use other solvents to solubilize them, such as DMSO and alkaline water (1 M NaOH) (Meslé et al. 2023). This section presents solvents that can be used to extract heme and PpIX from cellular lysates and to solubilize concentrated heme and porphyrins for use in enzymatic assays. In the case of more dilute enzymatic assays that consume or produce heme or porphyrins, addition of methanol (followed by centrifugation) is typically sufficient to precipitate the proteins and solubilize the tetrapyrrole substrates/products for analyses.

1. Equipment

FastPrep Lysis B-matrix tubes (for bacterial cells)

FastPrep 24 5g instrument (MP Biomedicals^™) or sonicator

2. Reagents

Acetonitrile

Dimethyl sulfoxide (DMSO)

Ethyl acetate

Hemin chloride

Hydrochloric acid (HCl)

Protoporphyrin IX

3. Procedure

Different solvent combinations can be used to extract either heme or PpIX, separately, from bacterial cells. Ethyl acetate/12 M HCl (3:1, v/v) is appropriate for extracting heme, and acetonitrile/12 M HCl (82:18, v/v) for PpIX. For significant extraction of heme and PpIX together from the cell pellet, a solution composed of acetonitrile/12 M HCl/DMSO (41/9/50, v/v/v) may be used (Figure 1). Details for each of these methods are shown below and in Table 1.

Figure 1.

Schematic illustrating heme/PpIX extraction steps. (a) Empty tubes containing 0.1 mm silica spheres; (b) FastPrep 24 5g instrument, MP Biomedicals^™ used for shaking the silica beads tubes for cell lysis; (c) Lysed cells (Ethyl acetate/HCl protocol) after centrifugation; (d) Lysed cells (ACN/12M HCl, 82/18 v/v; or ACN/12M HCl/DMSO, 41/9/50, v/v) after centrifugation; (e) Heme and PpIX separation via HPLC (C18 column).

Table 1.

Methods for extracting heme and PpIX from bacterial cells

Methods	Description
Ethyl acetate/HCl for extracting heme
Step 1	Resuspend 0.1-0.2 g cell pellet in 700 μL of 20 mM Tris-HCl (pH 7.4)
Step 2	Cell lysis using a FastPrep Lysis B-matrix tubes (2 cycles: 6.0 meters/second for 40 sec)
Step 3	In a fume hood, add 300 μL of ethyl acetate and 90 μL of 12 M HCl
Step 4	Mix gently for 20 min and centrifuge the tubes at 10,000 g for 10 min at room temperature
Step 5	Collect heme from the upper layer (ethyl acetate)
Step 6	Finally, dry the sample in a vacuum centrifuge (room temperature), resuspend it in DMSO and load onto C18 column or store the dried sample at −20°C, protected from light, until analysis
Acetonitrile/HCl for extracting PpIX; Acetonitrile/HCl/DMSO for PpIX and heme
Step 1	For PpIX: Resuspend 0.1-0.2 g of cell pellet in 700 μL of acetonitrile/12 M HCl (82:18, v/v) For PpIX and heme: Resuspend cell pellet in acetonitrile/12 M HCl/DMSO (41/9/50, v/v)
Step 2	Cell lysis using a FastPrep Lysis B-matrix tubes (2 cycles: 6.0 meters/second for 40 sec)
Step 3	Centrifuge the tubes at 10,000 g for 10 min at room temperature and collect the supernatant
Step 4	Finally, for quantification, load the sample onto C18 column via HPLC and monitor the porphyrins at 400 nm

Ethyl acetate/12 M HCl protocol:

To extract heme from cell pellets while minimizing extracted proteins, it is recommended to use acidified ethyl acetate in aqueous solution, which creates two layers, facilitating the recovery of heme.

Resuspend 0.1-0.2 g of cell pellet in 700 μL of 20 mM Tris-HCl (pH 7.4) and transfer it into a 2 mL tube containing silica beads to lyse the cells: e.g. FastPrep Lysis B-matrix tubes/FastPrep 24 5g instrument, MP Biomedicals^™, run for 2 cycles: 6.0 meters/second for 40 sec. Then, add 300 μL of ethyl acetate and 90 μL of 12 M HCl to the lysed cell mixture, mix gently for 20 min and centrifuge the tubes at 10,000 g for 10 min, room temperature. Next, collect the upper layer (ethyl acetate) containing the extracted heme, dry it in a vacuum centrifuge at room temperature and resuspend it in DMSO. This protocol creates two solvent layers, the upper ethyl acetate layer and the lower aqueous layer, through which the heme is easily collected from the upper (reddish) layer and separated from the other water-soluble cell components. The extraction can be repeated up to 3 times to ensure quantitative release of heme from the cellular material, and the extracts concentrated by drying on a rotary evaporator or vacuum centrifuge (speed-vac). For safety reasons, handle HCl in a fume hood.

Advantages and Limitations of Ethyl acetate/HCl protocol: With the formation of two layers (ethyl acetate and aqueous layers), this protocol allows for a faster extraction of heme in a reduced volume, free or with little protein content, but does not allow a considerable co-recovery of PpIX.

Acetonitrile/12 M HCl (82:18, v/v) and acetonitrile/12 M HCl/DMSO (41/9/50, v/v) protocols.

Resuspend 0.1-0.2 g of cell pellet in 700 μL of acetonitrile/12 M HCl (82:18, v/v) for extracting PpIX or acetonitrile/12 M HCl/DMSO (41/9/50, v/v) for extracting heme and PpIX together. In either case, transfer the cell suspension into a 2 mL tube containing silica beads to lyse the cells (see above). After cell lysis, centrifuge the tubes at 10,000 x g for 10 min and collect the supernatant, which can be loaded onto the C18 column for analysis by HPLC. If concentration is necessary, dry the sample in a vacuum centrifuge at room temperature and resuspend it in a smaller volume of the same extraction solvent. For safety reasons, handle HCl in a fume hood.

Advantages and Limitations of Acetonitrile/HCl protocol: This protocol allows better PpIX extraction and low heme content, with little to no protein content.

Advantages and Limitations of Acetonitrile/HCl/DMSO protocol: This allows considerable extraction of heme and PpIX, with little to no protein content.

When heme or PpIX is used as a substrate in individual enzymatic reactions, such as that catalyzed by the heme-degrading HmuS (Meslé et al. 2023), the ACN/12 M HCl/DMSO solution (41/9/50, v/v) is able to solubilize both the heme and the PpIX to be quantified by HPLC, and also to denature the enzyme, interrupting the catalysis. Dilute, denatured enzymes can be precipitated by brief centrifugation.

B. Discontinuous quantification of hemes and porphyrins by HPLC

Given the differences in polarity of the porphyrins, reversed-phase high performance liquid chromatography (HPLC) is suitable for separating many of them. Figure 2 shows the separation of, for example, (1) deuteroheme, (2) heme, (3) mesoporphyrin IX, and (4) protoporphyrin IX using a C18 column. A mixture composed of equimolar concentrations of these four porphyrins was loaded onto the C18 column to monitor their separation as the concentration of acetonitrile in water was increased (see method below).

Figure 2.

Separation of the porphyrins by reversed-phase chromatography using a C18 column (Thermo Scientific, 250 x 4.6 mm) coupled to an HPLC instrument. The elution of the standards was monitored at 400 nm. The chromatography was carried out using a linear gradient between Solution A (ultrapure water + 0.1% trifluoroacetic acid, TFA) and Solution B (acetonitrile + 0.1% TFA), flow rate of 1 mL/min, oven temperature at 25 °C. The numbers from 1 to 4 represent the standard peaks and their elution time: (1) deuteroheme, eluted at 12.5 min/63% solution B; (2) heme, eluted at 14 min/71% solution B; (3) mesoporphyrin IX, eluted at 14.8 min/75% solution B; and (4) protoporphyrin IX, eluted at 16.1 min/81% solution B. Several factors can alter the elution time, including the size and type of column, column temperature and the mobile phase used.

1. Equipment

HPLC instrument (for example, Prominence-i LC-2030C 3D Plus, Shimadzu)

C18 column (for example: Thermo Scientific, 250 x 4.6 mm)

2. Reagents

Acetonitrile

DMSO

HCl

Trifluoroacetic acid (TFA)

Protoporphyrin IX

Hemin chloride

3. Procedure

Reverse phase chromatography for separating and quantifying porphyrins.

These porphyrins can be separated via reversed-phase chromatography using a linear gradient, i.e. where the stationary phase is nonpolar and the mobile phase is polar. Solution A (i.e. water) is considerably more polar than Solution B (i.e. acetonitrile or methanol). The more nonpolar the porphyrin, the stronger its interaction with the column. More weakly bound porphyrins elute first when the concentration of acetonitrile increases (Solution B). Regarding the porphyrins shown in Figure 2, the sequence from most to least polar is: deuteroheme > heme > mesoporphyrin IX > protoporphyrin IX.

The method for separating the porphyrins shown here employed a C18 column coupled to an HPLC instrument, a linear gradient with solution A (ultrapure water + 0.1% trifluoroacetic acid, TFA) and solution B (acetonitrile + 0.1% TFA), flow rate of 1 mL/min, oven temperature at 25 ºC, and monitoring at 400 nm. For absolute quantification, standards are used to prepare a standard curve (see note 2). Details for preparing a standard curve for hemin chloride and PpIX are described below:

• Prepare a 2 mM stock solution of hemin chloride dissolved in DMSO and a 2 mM solution of PpIX dissolved in acetonitrile/12 M HCl (82/18, v/v)

• Dilute each stock solution in the same solvents to get 200 μM concentration of each one (10 times dilution): 100 μL of 2 mM standards + 900 μL of DMSO for hemin chloride and acetonitrile/12 M HCl (82/18, v/v) for PpIX.

• Mix equal volumes of the 200 μM standard solutions to get 100 μM of each standard (2 times dilution): 500 μL of 200 μM hemin chloride + 500 μL of 200 μM PpIX.

• From the mixture containing both standards at 100 μM, perform dilutions using the solution acetonitrile/12 M HCl/DMSO (41/9/50, v/v) to get different concentrations from 0.1 to 100 μM standards.

• Inject these different standard mix solutions onto a C18 column coupled to an HPLC instrument. Monitor the standard elution at 400 nm.

• To construct the standard curve, plot the peak areas against the respective concentrations of hemin chloride and PpIX (from 0.1 to 100 μM). Linear regression is used to obtain the linear equation (note 3)

4. Notes

1. The elution time of these porphyrins can vary depending on a number of factors, including the size and type of column, the column temperature, and the mobile phase used. In addition, elution times tend to shift backwards (toward the more polar region) as the column undergoes prolonged use.

2. To prepare the samples for chromatography, it is recommended that they be protected from light and filtered with a 0.22 μm syringe filter before being injected onto the C18 column.

3. Calibration curves are used to determine the concentration of samples. Therefore, weigh and dilute the standards precisely. To be accurate, the experimental conditions for generating the standard curve need to be the same as for the samples, including the same chromatographic column, mobile phase, run temperature, volume of sample injected, monitoring absorbance, and solvents used to dissolve the samples.

C. Quantification of heme and porphyrins by UV-visible (UV-vis) and fluorescence emission spectroscopy

Extracted heme and porphyrins can be detected and quantified using UV-vis (heme) and fluorescence (PpIX). The focus here is on heme b (the complex of iron with protoporphyrin IX) and PpIX, though various functionalized forms of each can be treated similarly (see Milgrom et al., 1997).

1. Equipment

Thermo Scientific NanoDrop

Thermo Scientific Micro 17 Microcentrifuge

Anaerobic Chambers, Coy Laboratory Products Inc.

Cary 60 UV-Vis spectrophotometer for absorbance measurement

BioTek Synergy H1 Multimode Reader for fluorescence measurement

pH meter, Thermo Scientific

2. Reagents

Tris base

NaCl

Hydrochloric acid (HCl)

Sodium hydroxide (NaOH)

Hemin chloride

Protoporphyrin IX (PpIX)

Polysorbate 80 or polyoxyethylene sorbitan monooleate (Tween 80)

Sodium dithionite (Na₂S₂O₄)

3. Procedure

Preparation and dilution of heme, PpIX, and related porphyrins.

Limited aqueous solubility of PpIX and heme demands modification of standard biological buffers for the preparation of stock solutions, either by adding a detergent or using alkaline solution. Here, PpIX is solubilized in an experimental buffer (20 mM Tris-HCl, 250 mM NaCl (pH 7)) containing 4% Tween 80 by volume. Tween 80 is a nonionic surfactant and emulsifier often used to dissolve hydrophobic solutes in aqueous solvent.

The low solubility of heme in water is addressed by dissolving solid hemin chloride in 1M NaOH solution and then diluting the stock to a desired concentration using the experimental buffer. The alkaline water deprotonates the propionic acid side chains, thus making the heme macrocycle ionic and more readily dissolved. Stocks of PpIX and hemin can be conveniently generated at 0.2 mM and 5 mM concentrations in these solvents.

Determining the concentrations of heme and PpIX.

The concentrations of heme and PpIX are measured on the Cary 60 UV-Vis spectrophotometer:

• The PpIX stock solution is prepared in 20 mM Tris-HCl, 250 mM NaCl (pH 7) containing 4% Tween 80. 50 μL of the stock PpIX is added to 2 mL of the same buffer (2050 μL total volume) to record the spectrum.

• The concentration of PpIX is determined from Beer’s Law (absorbance = ε x concentration x path length) using ε = 262 mM⁻¹cm⁻¹ at 405 nm. The pH of the heme solution is adjusted to 8 before further use.

• The heme stock solution is prepared in 1M NaOH and 10 μL of the stock heme is diluted to 2 mL using the experimental buffer above (2010 μL total volume).

• The concentration of heme is measured from the recorded spectrum using ε = 58.44 mM⁻¹cm⁻¹ at 385 nm. (See Mukherjee et al., 2014; Pal et al., 2021; Yi and Ragsdale, 2007).

Absorption properties of protoporphyrin IX (PpIX) and heme.

Porphyrin macrocycles, both metal-free and metal-bound, exhibit characteristic absorption properties due to transitions between porphyrin π and π* orbitals (Gouterman, 1978). The most intense absorption band, known as the Soret, is in the near UV region. Less intense bands in the visible region are known as the Q-bands. A major difference in the absorbance properties of metal-free and metal-bound porphyrins is in the number of Q-bands. The unbound porphyrins have four distinct Q-bands. Incorporation of a metal ion into the cavity of the macrocycle reduces the number of Q-bands to two (called α and β) because of the increased symmetry of the ring resulting from the deprotonation of the pyrrole nitrogen atoms (Milgrom, 1997). Additional metal-to-ligand or ligand-to-metal charge transfer bands may be present if the metal and porphyrin have available orbitals (occupied and unoccupied by electrons, respectively) with complementary symmetry and energies.

The reddish-purple solution of PpIX in pH 7 buffer containing Tween 80 has a Soret band at 405 nm and four Q-bands at 505, 540, 575, and 630 nm (Figure 1, purple spectrum). However, absorption properties of the Fe(III) bound PpIX are completely different from that of PpIX with a broad Soret at 385 nm and α and β bands at 496 and 620 nm (green spectrum).

4. Notes

1. It is always recommended to use the protoporphyrin IX (PpIX) solution in the dark to avoid photoexcitation, which can lead to further reaction with O₂. Solutions under N₂ atmosphere will remain stable for longer times.

2. For absorption spectroscopy it is recommended to use the heme and protoporphyrin solutions within 3 hours after preparation.

Reduction of ferric heme (Fe(III)) to ferrous heme (Fe(II)) by sodium dithionite (Na₂S₂O₄) and measurement of the absorbance spectrum.

• Before the reduction process, the oxidized heme solution is degassed using a slow purge of inert N₂ gas over the surface of the solution in a septum-sealed vial or round bottom flask, taking care not to dry the solution.

• The reduction may be carried out inside the septum-sealed vessel using a gas-tight syringe to introduce the degassed reductant, or by bringing degassed solutions into a glove box, maintaining an atmosphere of 2.5% H₂/97.5% N₂.

• 450 μM Na₂S₂O₄ (prepared using degassed buffer inside the glove box) is added to the degassed 150 μM heme stock solution (three-fold excess compared to heme concentration).

• 20 mM Tris-HCl, 250 mM NaCl (pH 7) is measured as a blank in a screw cap sealed quartz cuvette with 1 cm path length.

• The spectrum of the reduced heme is measured in a screw cap or septum sealed quartz cuvette with 1 cm path length.

Absorption properties of oxidized and reduced heme.

Reduction of the oxidized heme yields an absorbance spectrum with a sharpened Soret band at 385 nm, a prominent visible band at 580 nm, with a shoulder at 550 nm (Figure 3b).

Figure 3.

Absorption spectra of (a) protoporphyrin IX (PpIX) (purple, 8.5 μM) and heme (green, 25 μM) and (b) oxidized (Fe(III), green) (6 μM) and reduced heme (Fe(II), red) (6 μM) measured in 20 mM Tris-HCl, 250 mM NaCl (pH 7) (4% Tween 80 added to PpIX).

Fluorescence spectroscopy of Protoporphyrin (PpIX).

• The PpIX stock solution is prepared in 20 mM Tris-HCl, 250 mM NaCl (pH 7) containing 4% Tween 80. 50 μL of the stock PpIX is added in 2 mL of the buffer to record the spectrum.

• The concentration of PpIX in the stock is confirmed by measuring its UV-vis spectrum in the Cary 60 UV-Vis spectrophotometer using ε = 262 mM⁻¹cm⁻¹ at 405 nm.

• The PpIX stock solution is serially diluted in the working buffer above to different final concentrations from 0.1 μM up to 20 μM.

• The fluorescence spectra of the diluted stocks are measured using a BioTek Synergy H1 Multimode Reader. The emission spectra are recorded with the excitation wavelength at 400 nm.

Fluorescence properties of Protoporphyrin (PpIX).

The extended conjugation of porphyrins makes them strongly fluorescent. Metallated porphyrins with available d-orbitals can re-absorb the emitted photons, thereby quenching the fluorescence spectrum. Heme is consequently not fluorescent.

Fluorescence emission from PpIX is recorded with the excitation wavelength at 400 nm. The emission spectrum has a maximum intensity band (λ_max) at 640 nm with another low-intensity band at 703 nm (not shown) (Figure 4). The emission spectrum changes for natural precursors or variants of PpIX with different functional groups around the periphery of the macrocycle or with N-alkylation. The intensity of PpIX emission likewise increases with an increase in PpIX concentration up to 5 μM under the conditions used here (Figure 4); however, the intensity is found to decrease at higher PpIX concentration (from 7 to 20 μM), possibly due to PpIX aggregation.

Figure 4.

Fluorescence emission spectra PpIX at concentrations from 0.1 - 20 μM (pH 7). The intensity of PpIX fluorescence emission increases from 0.1 μM to 7 μM as indicated by the ascending lines from black to gray. PpIX emission spectra at 10 and 20 μM concentrations are demonstrated by purple lines (light to dark gradient color).

D. Distinguishing porphyrins with catabolic and anabolic origins using stable isotopes and LC-MS

Using radioactive labels is a classic way of tracing the metabolism of a compound from its initial introduction into a biological system to its ultimate fate. Stable isotopic labeling offers a safer and more ecological alternative that moreover opens the door to spectroscopic approaches that specifically detect the labeled atoms. Magnetically active nuclei can be specifically monitored using proton (¹H), carbon (¹³C), and nitrogen (¹⁵N) nuclear magnetic resonance (NMR). NMR can then be used to determine the structures of the endpoint metabolites. Similarly, mass-shifted metabolites can be identified in liquid chromatography mass spectrometric (LC-MS) measurements and their structures predicted based on exact masses and fragmentation patterns.

To investigate heme metabolism by bacteria, stable-isotope labeled heme can be used as a component of a chemically defined culture medium, which allows the analysis of heme metabolites by NMR and LC-MS. Isotopically-labeled heme can be obtained from recombinant heme proteins, like the soluble fragment of cytochrome b5, expressed in Escherichia coli strains cultivated in a medium containing isotopically-labeled ¹⁵N or ¹³C components, such as ¹⁵N ammonium chloride or ¹³C glucose (used as sole sources of carbon and nitrogen, respectively). Alternatively, ¹³C labeled 5-aminolevulinic acid (ALA), a heme biosynthetic precursor, can be added to the growth medium at the time of induction of protein expression (Rivera and Walker, 1995; Rodríguez-Marañón, 1996). This method is especially useful if it is desirable to label only specific locations on the tetrapyrrole. Labeled heme must be extracted from the cytochrome b5 or other protein carrier, and then introduced into the bacterial culture, mouse chow, enzymatic reaction, etc. Details of the production (based on the methods of Rivera and Walker) and extraction of labeled heme are given below.

1. Equipment

Autoclave

Shaker incubator

Electrophoresis system

Bruker 600 MHz Avance III solution NMR spectrometer

Agilent 1290 infinity UHPLC coupled to an Agilent 6538 high-resolution Q-TOF mass analyzer

2. Reagents

Ampicillin

Ethyl acetate

HCl

KH₂PO₄

MgSO₄

NaCl

Na₂HPO₄

CaCl₂

FeCl₃

Glucose or ¹³C-glucose

Ammonium chloride or ¹⁵N-ammonium chloride

Luria-Bertani medium (LB medium)

Isopropyl β-D-1-thiogalactopyranoside (IPTG)

DEAE resin

3. Procedure

Preparation of Labeled Minimal Medium and Generation of Labeled Heme.

A minimal medium typically comprises salts such as Na₂HPO₄, KH₂PO₄, NH₄Cl, and NaCl, supplemented with a carbon source. M9 salts are a commonly used medium for bacterial growth in molecular biology experiments. The following is a recipe for preparing 1 liter of M9 medium and the general steps (not detailed) for obtaining isotopically-labeled heme.

• Weigh: 6 g of Na₂HPO₄, 3 g of KH₂PO₄, 1 g of NH₄Cl and 0.5 g of NaCl

• Mix these salts in distilled, deionized water and adjust the pH to 7.0. Final volume 1 liter.

• Sterilize the solution by autoclaving at 121°C for 15 minutes, 1 atm.

• Add to the 1 liter autoclaved solution: 20 mL of 20% ¹³C-labeled glucose (w/v), 2 mL of 1 M MgSO₄, 0.1 mL of 1 M CaCl₂, 0.5 mL of 100 mM ferric chloride, and 1 mL of 100 mg/mL ampicillin (used as a selective marker, see note 1). Prior to mixing with the 1 liter autoclaved M9 salts, these solutions should be filtered using a 0.22 μm pore filter. If ¹⁵N-labeled ammonium chloride was used to produce ¹⁵N-labeled heme, add non-labeled glucose. Alternatively, the given amounts of labeled carbon or nitrogen source can be added at the time of protein induction, in which case the medium should contain half of the amount of the unlabeled C/N source indicated above.

• From a growth on a selective LB agar (containing a selective antibiotic, like ampicillin), select a colony of the E. coli competent cells containing a plasmid that harbors the gene encoding the soluble portion of cytochrome b5. Inoculate a single colong into LB liquid medium and incubate overnight at 37 °C, 180 rpm; it is the starter culture. Next, transfer 1/100 (v/v) of the starter culture to the 1 liter of labeled minimal medium and incubate in a shaker incubator at 37 °C, 180 rpm.

• At the mid-log phase of the E. coli growth, induce the expression of cytochrome b5 by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside), incubate the culture at 25 °C and determine the best time to collect the bacterial cells (see note 2). The labeled compound could be added at the point of protein expression induction, as stated above.

• Harvest the cells by centrifugation and lyse them (by sonication) to obtain the cell free soluble proteins.

• Partially purify the labeled cytochrome b5 using an anion exchange resin (DEAE-FF) and 20 mM Tris-HCl buffer, pH 8.0. This step ensures the isolation of labeled cytochrome b5 from other cellular components.

• Assess the purity of the labeled protein using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (12% SDS-PAGE) and determine the concentration of the purified labeled cytochrome b5 using a quantitative assay such as the Bradford assay (Bradford, 1976).

• Extract the labeled heme from the partially purified cytochrome b5 using a suitable method, like the ethyl acetate protocol described in section A.

• Check the purity of the labeled-heme through reversed-phase chromatography, as described in section B.

• Now, the extracted labeled heme is used to feed the bacteria as part of the growth medium. After bacterial growth, the cell pellets are harvested by centrifugation, resuspended in acetonitrile/12 M HCl (82/18, v/v) and lysed to obtain heme metabolites for LC-MS (Figure 5) or NMR analyses (Figure 6).

Figure 5.

Electrospray ionization (ESI) mass spectrometry of metabolites extracted from the human gut symbiont bacterium Bacteroides thetaiotaomicron. (a) Total Ion Chromatogram (TIC) from metabolites, arrows indicate heme and PpIX. (b and c) ESI spectrum of heme (616.1794 m/z) and PpIX (563.2674 m/z). The bacterium was cultivated in a minimal medium containing 15 μM hemin chloride (pH 7.1), under an anoxic atmosphere (2.5% H₂/97.5% N₂) at 37 °C. Cells were collected at mid-log phase growth and resuspended in ACN/12 M HCl to be lysed, see section A.

Figure 6.

¹H NMR spectrum of standard PpIX dissolved in ACN/12 M HCl (82/18, v/v).

Detection and quantification of heme and PpIX by LC-MS.

The same procedure as described in sections A-C to generate both heme and PpIX standards of varying concentrations and bacterial cell extracts suitable for LC-MS analysis. Because HPLC detects only chromophoric compounds, it tends to underestimate the diversity of compounds retained in cellular extracts. By contrast, LC-MS only requires that the compounds can be successfully ionized and detected. The LC-MS total ion chromatogram (TIC) consequently reveals several additional compounds. Quantification is achieved by measuring standards of varying concentrations and integrating their peaks in the ion chromatograms. These standard curves are then compared with integrated peak intensities from the extracted ion chromatograms (EICs) for heme and PpIX (and additional porphyrins if required), respectively. EICs are extracted based on the expected exact theoretical masses of the metabolites of interest. The mass spectra measured for the heme and PpIX confirm the compounds’ identities.

To measure LC-MS, porphyrins separation was carried out using the column Agilent Eclipse C18 (2.1 x 50 mm) coupled to an HPLC instrument, linear gradient with the mobile phase composed of water + 0.1% formic acid (solution A) and acetonitrile + 0.1% formic acid (Solution B) and a flow rate of 0.6 mL/min. Mass Spectrometer (detector) Agilent 6538 Q-TOF MS, (+) electrospray ionization, m/z 50-1700, 4Hz scan speed, collision energy 25, top 2 and the Agilent MassHunter Acquisition 10.0 software (Figure 5).

Detection of PpIX by NMR.

Ferric heme and low-spin ferrous heme are paramagnetic and consequently difficult to characterize by NMR. However, PpIX and its metabolites can be readily identified by NMR. If the heme is ¹³C or ¹⁵N labeled, then these heteroatoms can also be identified among the downstream metabolites of PpIX and the compounds containing them can be structurally characterized using multinuclear/multidimensional NMR methods.

Depending on the scientific question being asked, cellular extracts may be prepared as described above. These extraction procedures are intended to isolate either heme and relatively polar compounds, or PpIX and less polar compounds. Alternatively, both heme and PpIX can be coextracted. In instances where PpIX, its more polar breakdown products, and potentially other cellular metabolites are required, an extraction procedure that retains a broad spectrum of most of the cell’s small molecules is desirable. However, it may be essential first to remove paramagnetic heme, for example, by binding it to an affinity column l-histidine-immobilized sepharose (HIS) resin (Asher and Bren, 2010). Alternatively, the cellular lysates may be treated with a mixture of commercially available heme oxygenase and biliverdin reductase in order to convert the heme to bilirubin and free iron.

As an illustration, a PpIX spectrum (PpIX concentration > 10 μM) was recorded on a Bruker 600 MHz (¹H Larmor frequency) AVANCE III solution NMR spectrometer equipped with a 5 mm triple resonance (¹H, ¹³C, ¹⁵N), liquid helium-cooled cryoProbe, automatic sample loading system (SampleJet), and Topspin software (Billerica, MA, USA, Bruker version 3.2). The Bruker lc1pngpf2 (double solvent suppression, spoil gradient) pulse sequence was used for the acquisition of 1D ¹H NMR spectra, which was recorded at 300 K with 512 scans, 32K data points, and a ¹H spectral window of 30 ppm (Figure 6).