Background

G.G. Stokes was the first to prepare what we now refer to as hemochromagen. As early as 1863, he was monitoring changes in the hemoglobin absorbance spectrum upon reduction of the heme to the Fe(II) form Stokes (1863). Stokes had reduced blood in the presence of ammonia; what he was seeing were the intense α and β peaks of Fe(II) heme b from hemoglobin in complex with ammonia. Later authors (Anson and Mirsky, 1928) were able to show that the hemochromagen, as it had been called by Christian Bohr (Edsall, 1972) , was heme in complex with some nitrogenous ligand. It can be formed as well by simply reducing myoglobin under denaturing conditions, in which case histidines serve as the axial ligands. Hill demonstrated that the pyridine hemochromagen is formed from the nitrogen of two pyridine molecules binding to the axial position of the reduced heme (Hill, 1926; Hill, 1929); this was confirmed by Smith (1959) and Gallagher and Elliot (Gallagher et al., 1965; Gallagher et al., 1968).

With advances in spectroscopic techniques, later workers were able to use hemochromagen for an important analytical purpose (Hill, 1929). The regularity of the α peak of reduced pyridine hemochromagen, its high extinction coefficient, and the fact that it follows Beer's Law over a wide range allows its use for determining the total heme composition of a sample. De Duve (1948) published one of the first protocols for this, and determined the extinction coefficient (ε) at 557 nm to be 32 mM^-1 cm^-1. The method used relied on gravimetric determination of the standard heme samples, which could lead to an underestimate of the true heme concentration if the sample is impure. Paul et al. (1953) re-determined the extinction coefficient for pyridine hemochromogen from recrystallized heme b and myoglobin, using the iron content as an internal control. They found a value (34.7 mM^-1 cm^-1) significantly higher than the previous value of 32 mM^-1 cm^-1, representing a difference of roughly 9%. Both values have been in use comparatively recently: e.g. (Scott and Lecomte, 2000; Fushitani and Riggs, 1988; Miyoshi et al., 1997; Yachie et al., 1999; Lee et al., 2000; Huche et al., 2006), which use 32 mM^-1 cm^-1, and (Sono et al., 1984; Senturia et al., 2012; Sinclair et al., 2001; Berry and Trumpower, 1987), which use 34.7 mM^-1 cm^-1. In this assay we have taken the value given by Paul et al. (1953) to be most accurate.

[Principle of action] The heme-containing protein solution is first mixed with Solution I (see Recipes) containing pyridine, NaOH and potassium ferricyanide. Pyridine serves as a ligand for heme in the Fe(II) state. NaOH keeps sodium dithionite stable. Potassium ferricyanide ensures that all dissolved heme is in the Fe(III) state at first. Then, excess sodium dithionite in solution is added to reduce the heme from Fe(III) to Fe(II). Another option is to use a pipette tip to add a few grains of solid dithionite to the cuvette, and allow it to dissolve. Recipes similar to the current one have been recommended by Sinclair et al. (2001); Berry and Trumpower (1987); Antonini and Brunori (1971). The concentration of heme in the sample can be determined from the absorbance of the reduced sample; the extinction coefficient for reduced pyridine hemochromagen is 34.7 mM^-1 cm^-1 at 557 nm for heme b. It can also be done using the difference spectrum between the reduced and oxidized samples. Don't forget, in either case, to take your dilution factor into consideration. This protocol is written for heme b; however, other types of heme form hemochromagens as well and can be quantified using the same technique. See Berry and Trumpower (1987) or Table 1 for extinction coefficients for hemes a and c.