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I. An enzyme that catalyzes the reduction of erythrocyte cytochrome b 5 has been isolated from the supernatant fraction of erythrocyte hemolysates by chromatography on DEAE-cellulose, Amberlite CG-5 o, and Bio-Gel P-6o. 2. Erythrocyte cytochrome b 5 reductase has been shown to contain FAD. Incubation of the reductase in the absence of EDTA results in both the appearance of flavin fluorescence and the loss of enzymatic activity with time. 3. The reductase catalyzes the reduction of erythrocyte cytochrome b 5 5o times faster with NADH than with NADPH. The apparent Km of NADH was calculated to be 1.6. IO-~ M and the turnover number is 128o moles of cytochrome b 5 per min per mole of flavin. The reduction of electron acceptors proceeded in the following decreasing order of rate: K~Fe(CN)6, 2,6-dichlorophenolindophenol (DCIP), cytochrome b 5, methylene blue, cytochrome c, 03, oxidized glutathione, and methemoglobin. 4-The FAD prosthetic group, the substrate specificity, and the effect of ionic strength, pH, and EDTA on activity suggest that the reductase is the same enzyme as NADH dehydrogenase I, the enzyme lacking in many cases of congenital methemoglobinemia. The properties of the reductase, including its molecular weight, are very similar to those of the cytochrome b 5 reductases solubilized from microsomes and mitochondria of other tissues.

I. A hemeprotein with properties similar to microsomal cytochrome b 5 has been detected in the supernatant fraction of hemolysates of human, beef, and rabbit erythrocytes. A method has been developed for determining the amount of this soluble cytochrome in small volumes of blood. The amount of the protein decreases during cell storage at 4 °C. Blood cells rich in retieulocytes contain more of the protein than do mature cells. 2. The cytochrome has been purified from human erythrocytes by a procedure which employs chromatography on Amberlite CG-5o and DEAE-cellulose, ultrafiltration, and gel filtration. The purified protein sedimented in the ultracentrifuge as a single peak with an Szo,w of 1.4o. However, minor impurities were detected by polyacrylamide disc electrophoresis. 3-The molecular weight of the purified protein has been calculated to be 14600 from sedimentation and diffusion measurements and 18400 as determined by gel filtration. The prosthetic group has been identified as protoheme IX. The spectral properties of the hemeprotein are those of a low spin heine complex. The EPR spectrum of the oxidized form shows g values of 3.03, 2.21, and 1.39 and the visible spectrum has a Soret absorbance maximum at 413 nm. The protein is reducible by dithionite or NADH plus cytochrome b 5 reductase and the reduced form shows absorbance maxima at 423,527, and 556 nm with a shoulder at 560 nm. 4. The cytochrome b 5 differs from the other B-type cytochrome of erythrocyte, S-protein (hemeprotein 559), and is not derived from this protein. The erythrocyte cytochrome b 5 is similar to the cytochrome b 5 solubilized from liver microsomes in terms of spectral properties, molecular weight, prosthetic group, and reactivity.

I. A procedure employing mild conditions has been developed for purifying Sprotein (hemoprotein 559, erythrocyte), a protein present in the stromal fraction of human red blood cells. S-protein, solubilized by extracting the stroma of red blood cells with pH 8.2 buffer, was purified by chromatography on DEAE-cellulose and Bio-Gel P-6o.2. The oxidized and reduced forms of this protein show visible absorption spectra typical of b-type cytochromes. The oxidized form of the protein shows absorbance maxima at 412, 533, and 565 m#, and the dithionite-reduced spectrum shows maxima at 426, 530, and 559 m/z. The prosthetic group has been isolated and identified as protoheme IX by spectral and chromatographic techniques. The spectral properties of this protein differ from those of cytochrome b~.3. The reduced form of the protein binds CO, and the resulting complex shows absorbance maxima at 421, 54 o, and 568 m#. This spectrum, the CO-reduced minus reduced difference spectrum, and the spectra of the oxidized and reduced forms of the free protein are all indistinguishable from the corresponding spectra of hemoprotein P-42o, the altered form of the microsomal hydroxylase, hemoprotein P-45 o. The spectral properties, together with other physical properties, suggest that S-protein is P-42o from erythrocytes.