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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.

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