Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions

Materials and Reagents

1. Heme-containing sample
   Note: The concentration of heme in your stock solution should be at least 10 μM and not much greater than 80 μM. Dithiothreitol (DTT) and other reducing agents can reduce potassium ferricyanide and may interfere with the oxidized sample, but not with the reduced sample. Any common biological buffer should be compatible with this procedure so long as it has no significant absorbance in the 500 nm to 600 nm range and does not form a complex with heme, as should be the case with all Good's buffers. We have personally used phosphate, tris, HEPES, EPPS, MES, and CHES without issue.
   
2. 0.5 M NaOH
   
3. Pyridine (Sigma-Aldrich, catalog number: 360570 )
   
4. Potassium ferricyanide(III) (Sigma-Aldrich, catalog number: 702587 )
   
5. Sodium dithionite (Sigma-Aldrich, catalog number: 157953-5 G )
   
6. Deionized water
   
7. 0.2 M NaOH, 40% (v/v) pyridine, 500 μM potassium ferricyanide (see Recipes)
   
8. 0.1 M potassium ferricyanide (K₃[Fe(CN)₆]) (see Recipes)
   
9. 0.5 M sodium dithionite in 0.5 M NaOH (see Recipes)
   

Equipment

1. Spectrophotometer with a bandwidth ≤ 2 nm
   
2. Fume hood
   
3. Quartz or glass cuvette, 1 cm length
   
4. Pipettes
   Note: It is generally good to have a spectrophotometer capable of relatively low spectral bandwidth (SBW) in order to get highly accurate measurements. It is suggested in the literature that the ratio of SBW to the natural bandwidth (NBW) should be 0.1 or lower in order to get an error of less than 0.5% (Surles and Erickson, 1974; Brodersen, 1954). The α band of reduced pyridine hemochromagen has a NBW of around 20 nm, hence the recommendation of 2 nm or less for SBW. Check with your manufacturer if you are unsure of your SBW. Most modern spectrophotometers have SBW less than 4 nm, corresponding to an error of less than 2%. It is also important to note that this error is not random, but results in an underestimate of the true absorbance; having a higher SBW/NBW ratio leads to a 'flattening' of absorbance peaks. If possible, use a scanning spectrophotometer. This allows you to verify that your spectrum looks similar to the spectrum shown in Figure 1.
   
   
   Figure 1. Example spectrum of reduced and oxidized pyridine hemochromagen (heme b). The bandwidth is set to 1 nm, with data interval 1 nm.
   

Procedure

The volumes shown below can be scaled up or down as desired, as long as the concentrations stay the same.

1. Set the spectrophotometer to ≤ 2 nm SBW (if adjustable) and data interval at 1 nm between 500 and 600 nm (at least). The assay is not strongly temperature sensitive; room temperature works well.
   
2. Blank the spectrophotometer with Solution I mixed 1:1 with whichever buffer your protein sample is in.
   
3. Empty cuvette. Add 0.5 ml Solution I to 0.5 ml of your heme-containing sample and mix well.
   
4. Scan this mixture; this is your oxidized sample.
   
5. Add 10 μl Solution III to your oxidized sample and mix well. The sample should turn a reddish color.
   
6. Scan immediately and again every minute until the absorbance peak no longer increases; this should take no more than 5 min. The scan with the highest peak is the reduced spectrum.
   
7. Using the extinction coefficients in Table 1, and taking into account your dilution factors, calculate the concentration of heme in your original sample using Beer's law, A = ε c l (Absorbance = extinction coefficient x concentration x pathlength). You can use the absolute absorbance of the reduced sample, or the difference spectrum.
   
   Table 1. Extinction coefficients for pyridine hemochromogens

   Compound	Wavelength	ε (mM-1 cm-1)	Solvent	Ref.
   Pyr2-heme b Reduced	557 nm	34.7	NaOH, 10-40% pyr.	Paul et al., 1953
   Pyr2-heme b, Red. - Oxid.	557 nm min 540 nm	23.98	NaOH, 10-40% pyr.	Berry and Trumpower, 1987
   Pyr2-heme a, Red. - Oxid.	587 nm min 620 nm	23.98	NaOH, 10-40% pyr.	Berry and Trumpower, 1987
   Pyr2-heme c Reduced	550 nm	30.27	NaOH, 10-40% pyr.	Berry and Trumpower, 1987
   Pyr2-heme c, Red. - Oxid.	550 nm min 535 nm	23.97	NaOH, 10-40% pyr.	Berry and Trumpower, 1987

   
   
8. To determine the extinction coefficient of the heme when it is bound to your native protein, you will also need a high quality scan of the protein sample in a suitable buffer. Using Beer's law and the sample heme concentration, calculate ε for the heme absorbance peaks of your native protein.
   

Recipes

1. Solution I [0.2 M NaOH, 40% (v/v) pyridine, 500 μM potassium ferricyanide]
   Add 2/5 volume 0.5 M NaOH to 2/5 volume pyridine in fume hood or well-ventilated space and dilute to final volume with water
   Add 1/200 volume 0.1 M potassium ferricyanide (Solution II)
   Paul et al. (1953) reported that NaOH concentrations between 0.02 and 0.5 M and pyridine concentrations between 10% and 50% make no difference to the molar absorptivities of the hemochromagen.
   Hazards: Pyridine is toxic, flammable, and has what the Merck Index calls a "characteristic disagreeable odor." Mix solutions in a fume hood or well-ventilated area.
   Warnings: Pyridine eventually oxidizes; when this happens it turns yellow. Do not use if yellow, and seal the pyridine bottle after use.
   
2. Solution II {0.1 M potassium ferricyanide (K₃[Fe(CN)₆])}
   Hazards: Releases cyanide if mixed with strong acids.
   Warnings: Potassium ferricyanide must be made fresh and used within 24 h. Dispose of in a separate container.
   
3. Solution III (0.5 M sodium dithionite in 0.5 M NaOH)
   Sodium dithionite, off the shelf, is usually no more than 85% pure, no matter the supplier (McKenna et al., 1991).
   Note: It should always be dissolved and diluted in neutral or basic solution; acidic conditions cause it to release hydrogen sulfide, a poisonous gas. It must be made fresh every time.
   Storage: Sodium dithionite should be kept away from water. Fresh sodium dithionite is powdery and free-flowing. It is best to purchase it in small amounts and open a fresh bottle periodically, due to degradation upon exposure to air (McKenna et al., 1991). Sodium dithionite has a redox potential of -0.66 V at pH 7.0, 25 °C (Mayhew, 1978), and doesn’t absorb strongly in the > 400 nm region.
   

Acknowledgments

This work was funded in part by a grant from the NIH (GM080563) to F. G. and a UCLA Dissertation Year Fellowship to I. B. We would also like to thank Aaron T. Smith and Judith N. Burstyn for advice.

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