The preparation and some physical properties of sulfhemoglobin.

Sulfhemoglobins were prepared from hemoglobin A and carboxypeptidase A-treated hemoglobin. The synthesis was achieved by oxidation of the ferric hemoglobins with H,O, followed by reaction with excess hydrosulfide. ERR spectra of these ferric sulfhemoglobins at high pH were well resolved from the resonances of contaminating ferric hemoglobins. Thus, quantitative ERR measurements provided a convenient method for estimating the purity of the sulfhemoglobin preparations. Results of such measurements were used to calculate the optical absorption spectrum of pure sulfhemoglobin. The intensity of the absorption band near 620 nm, an often used optical marker for sulfhemoglobin, is about 2 times greater than reported previously (Beutler, E. (1972) in Hematology (Williams, W. J., Beutler, E., Ersley, A. J., and Rundles, R. W., eds) p. 1381, McGraw-Hill Book Co., New York). Furthermore, the extinction coefficient and position of this band varied with oxygen tension. Under anaerobic conditions, it is at 619 nm CC mlr, = 21.4) and at 1 atm it shifts to 623 nm (E,,,~ = 25.3). Addition of CO to sulfhemoglobin shifts the absorption maximum from 619 to 613 nm and the extinction coefficient increases by 26%. The infrared stretching frequency of bound CO is 10 cm~’ lower than that for CO bound to hemoglobin A. Acid-base titration of high spin ferric sulfhemoglobin to the low spin form has a pK of 7.8 compared to 8.1 for ferric hemoglobin titrated under the same conditions.