Simultaneous Determination of Hemoglobin and Myoglobin Oxygen Binding Curves by Spectral Curve Fitting

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Abstract
A completely optical method is described for the simultaneous determination of hemoglobin and myoglobin oxygen saturation. Solution oxygen concentrations were computed from measurements of phosphorescence decay of a soluble palladium porphyrin using the Stern-Volmer quenching relationship. Visible absorption spectra were recorded of hemoglobin/myoglobin mixtures progressively deoxygenated by bacterial aerobic metabolism. Separate hemoglobin and myoglobin oxygenation curves were resolved from the spectra of solutions containing both proteins by curve fitting, with the use of singular value decomposition of the spectra vs. oxygen concentration matrix. The method yielded a P50 of 1.2 Torr for horse heart myoglobin at 24 ± 1°C, while the P50 for sheep hemoglobin was 23 Torr at the same temperature. These values, obtained from mixtures, compare favorably with literature values determined for hemoglobin and myoglobin separately.