A spectrophotometric method for estimating hemin in biological systems

Hemin chlorides exhibit two absorption maxima in the Soret region, one at about 360-380 nm (S′ band) and the other between 400 and 430 nm (S band). We present here a simple and fast spectrophotometric assay to determine concentration of hemin between 1.15 and 9.20 μM employing the Soret region (S′ band) as a reference. In this method the hemin is quantitatively extracted from biological materials by acidified chloroform. By recording the absorbance of the chloroform extract at its maximum peak at 388, 450, and 330 nm and applying the correction formula Ac = 2A388 − (A450 + A330), a very good linear correlation between the Ac and the concentration of hemin is attained. The method can be used to estimate hemin in the presence of protein (0.06-5.00 mg/ml) and porphyrin (0.19-2.97 μM). Compared with the pyridine hemochromogen method, the assay reported here is highly reproducible, with 15- to 30-fold more sensitivity, and it allows the quantification of four times lower hemin concentrations.