QUANTITATION OF HEME-PROTEINS FROM SPECTRA OF MIXTURES

The Heme Proteins Spectra Analysis Program (HPSAP), a full-spectrum method, has been developed to quantify heme protein concentrations from spectra of mixtures. It utilizes the multiple-regression capabilities of a Lotus 1-2-3 spreadsheet. Micromolar extinction coefficients at 5-nm intervals from 500 to 640 nm are preprogrammed into the HPSAP spreadsheet for hemoglobin; oxyhemoglobinn; methemoglobin; ferrylhemoglobin; hemichrome; combined cytochromes of the mitochondria, both reduced and oxidized; and combined cytochromes of the microsomes, both reduced and oxidized. The analyst inserts experimental absorbance values at the same wavelengths into a reserved column in the spreadsheet. Multiple regression is performed to determine heme protein concentrations using the extinction coefficients of the heme proteins as independent variables and the experimental absorbance values as the dependent variable. A number of spectre have been analyzed using HPSAP with good results. Its versatility makes it a powerful method for the analysis of the complex spectra presented by oxidation in biological samples.