Multicomponent analysis of heme protein spectra in biological materials.

Absorption spectra of heme proteins in a tissue homogenate contain information about the oxidation status of the biological sample. Deconvolution of absorption spectra can be used to quantitate the amount of individual heme proteins present in the mixture. The heme spectral analysis program (HSAP), a computer spreadsheet program, was used to quantitatively calculate values for heme proteins measured spectrally (390 to 450 and 500 to 640 nm) in tissue homogenates undergoing oxidation. The amount of oxidized heme proteins obtained by HSAP can be compared to other measurements of tissue oxidation. Precise quantitation of the amount of heme proteins present in a homogenate sample provided accurate assessment of the oxidized heme proteins calculated by HSAP. This quantitation was achieved through modification of existing pyridine hemochrome methods. Input into HSAP of the total heme protein content via the pyridine hemochrome value generated reproducible values for oxidized heme proteins. The program has broad potential as a multicomponent analysis tool. Modification of HSAP led to the development of a difference spectra analysis program (DSAP) which was used to quantitate the type and amount of heme proteins observed in mitochondrial difference spectra. In the present application, HSAP and DSAP provide methods for interpreting complex spectral information of multicomponent biological samples that undergo oxidation.