Pyridine hemochromogen

HEME QUANTITATIONS FROM PYRIDINE HEMOCHROMOGEN SPECTRA

Hemochromogen formation

• Prepare a pyridine reagent mix by adding 3mL 1M NaOH and 6mL pyridine (use glass pipet) to 19mL H₂O in a glass container (28mL total volume).
• Put 1000µL of the pyridine reagent in a cuvet
• Add 35µL sample containing ≥5µM hemin.
• Add 18µL aqueous 15mM K₃Fe(CN)₆, mix well.
• Record the oxidized spectrum at 500-650nm.  Repeat until stable.
• Add 2-5mg powdered Na₂S₂O₄ (sodium hydrosulfite, sodium dithionite).
• Mix well under the surface of the liquid mixture (minimize mixing with air).
• Record the reduced spectrum and repeat until stable.

Calculations

• For total heme (mM) subtract the absorbance readings at 540, 556 and 575nm in the oxidized spectrum from the corresponding readings in the reduced spectrum to get ∆A540, ∆A556 and ∆A575.  (For example, ∆A540 = A540 Na₂S₂O₄reduced - A540 K₃Fe(CN)₆oxidized, etc.) 
• Calculate [hemin, mM] in the mixture using the extinction coefficients:
• • 20.7/mM for ∆A540 ((∆A556-∆A540)/20.7)
  • 32.4/mM for ∆A575 ((∆A556-∆A575)/32.4)
• Factor in the dilution of the sample (30x or (1000+35+18)/35) and average the two results.
• For heme a, b, or c, multiply the difference of reduced minus oxidized absorbance at the five different wavelengths below and add the resulting five multiplication products.

Reference Information

From: Berry, E.A. and Trumpower, B.L. 1987. Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra.  Analytical Biochemistry.  161: 1-15.

Table 4

Inverse matrix of extinction coefficients of pyridine hemochromes for calculating concentration (mM) from reduced minus oxidized absorbance at five different wavelengths.

Wavelength (nm):    540                 549                 558                 588                 620  

Heme c:               -0.02778        0.04757       -0.01906         0.00084       -0.00157

Heme b:               -0.02943       -0.00088         0.04037        -0.00267       -0.00738

Heme a:               -0.02687        0.00456         0.00699          0.04353       -0.02820

Total heme may also be obtained by adding the results for hemes a, b and c for a sample.  Authentic heme solutions with known concentrations may be used to check the method and also to help check that the calculations were performed correctly to obtain expected results.