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A rapid, new method to measure hemoglobin-oxygen equilibrium curves is described using the protocatechuic acid/protocatechuic acid 3,4-dioxygenase system [C. Bull and D.P. Ballou (1981) J. Biol. Chem. 256, 12673-12680] to deoxygenate hemoglobin solutions enzymatically. The reaction is followed by simultaneous measurements of hemoglobin spectra using a diode array spectrophotometer and oxygen tensions using a polarographic O2 microelectrode. Multicomponent analysis allows the determination of fractions of oxyhemoglobin, deoxyhemoglobin, and high-spin and low-spin methemoglobins in each spectrum collected as the reaction proceeds. Fractional saturation as a function of oxygen partial pressure is calculated as the ratio of oxyhemoglobin to oxy- plus deoxyhemoglobin. Several advantages are offered by this method: (i) Hemoglobin-O2 binding curves are obtained rapidly and reproducibly; (ii) the speed of the reaction limits methemoglobin formation by autooxidation; (iii) there is no gas-liquid interface, eliminating protein denaturation at the surface; and (iv) direct calculations of fractional saturation are made using spectral analysis, thus avoiding the assumption of a linear transition between deoxy- and oxyhemoglobin.