Proc. Natl. Acad. Sci. USA Vol. 75, No. 7, pp. 3118-3122, July 1978
Biochemistry
Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5 (hemepeptide/proteolysis/endoplasmic reticulum/erythroid maturation)
RICHARD H. DOUGLAS* AND DONALD E. HULTQUISTt Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
Communicated by J. Lawrence Oncley, April 17, 1978
protein. Hydrolysis of human erythrocyte cytochrome b5 by trypsin yields a hemepeptide that has the same electrophoretic properties as the trypsin-solubilized liver protein (14). During erythrocyte maturation much of the cell's ultrastructure is lost as the nucleus, mitochondria, endoplasmic reticulum, and other subcellular organelles are degraded and/or extruded from the cell. For developmental and biochemical reasons it is of interest to determine the fate of proteins that are associated with the subcellular organelles of immature erythroid cells. We have hypothesized (13) that some of these proteins become solubilized during erythroid maturation and that the soluble proteins are retained and are functional in the mature erythrocyte. The similarity in the properties of human erythrocyte and liver cytochrome b5 suggested that the soluble erythrocyte protein that functions in methemoglobin reduction is derived from the microsomal protein that functions in fatty acyl coenzyme A desaturation. We have thus compared the structural similarity between the erythrocyte and liver proteins. In order to obtain the milligram quantitities of protein required for such studies, we have improved and simplified our purification procedure and have used fresh bovine erythrocytes instead of the outdated human erythrocytes we previously used. We report the isolation of homogeneous preparations of two forms of bovine erythrocyte cytochrome b5 and present evidence that the structures of these two proteins are identical to segments of the peptide chain of bovine microsomal cytochrome b5. Preliminary findings have been reported (15, 16, t). EXPERIMENTAL PROCEDURE Materials. Fresh beef blood was purchased from Kappler Packing Co., Ann Arbor, MI. Sodium dithionite, N,N'-methylenebisacrylamide, acrylamide, spectral grade pyridine, N,N,N',N'-tetramethylethylendiarine, and 20 X 20 cm silica gel plates were obtained from Eastman; Bio-Gel P-4 and P-60 (100-200, and minus 400 mesh) from Bio-Rad Laboratories; DEAE-cellulose and ammonium persulfate from Fisher; DEAE-Sephadex A-50, Tris, and cytochrome c from Sigma; 3X-recrystallized trypsin, ovalbumin, and chymotrypsinogen from Worthington; bovine serum albumin from Pentex; myoglobin from Calbiochem; bromphenol blue from Allied Chemical and Dye Corp.; and Coomassie brilliant blue from Schwarz/Mann. All other reagents were of analytical grade and were used without further purification. Purified bovine liver cytochrome bs, solubilized from microsomes with "lipase" and trypsin, was a gift from Scott Mathews. General Procedures. All solutions were prepared with dis-
ABSTRACT Homogeneous pre arations of two forms of soluble cytochrome b5 have been o tained from bovine erythrocytes by successive chromatography on DEAE-cellulose, Bio-Gel P.60, and DEAE-Sephadex. Although the two forms could be separated on disc gel electrophoresis, they appeared to have similar molecular weights of a pproximately 12,000 and identical visible absorbance spectra. The tryptic hemepeptides derived from the two forms of bovine erythrocyte cytochrome bs are electrophoretically indistinguishable from each other and from the tryptic core hemepeptide derived from liver microsomal cytochrome bs. The bovine erythrocyte tryptic hemepeptide was purified to homogeneity; its amino acid composition was shown to be identical to that of tryptic hemepeptide from liver microsomal cytochrome b5. The amino acid compositions of the two isolatable forms of erythrocyte cytochrome b5 correspond well to the compositions of the 97- and 95-residue segments of native liver microsomal cytochrome b5 that begin at the NH2 terminus. These results agree with the hypothesis that soluble erythrocyte cytochrome b5 is derived from microsomal protein by proteolysis during erythroid maturation.
Microsomal cytochrome b5 is a membrane-bound protein that functions as a component of the fatty acyl coenzyme A desaturase system (1-3) and has been implicated in microsomal hydroxylation reactions (4). Bovine liver microsomal cytochrome b5 has been studied extensively. The amino acid sequences have been determined for the intact protein and for the hemepeptides derived from it by solubilization of bovine liver microsomes with trypsin and with "lipase" (5). The x-ray crystal structure of the trypsin-solubilized molecule has been refined to 2.0 A resolution (6). Solubilization of bovine liver microsomes with detergent yields a hydrophobic form of cytochrome b5 (7) that differs structurally from the proteasesolubilized hemepeptides in that it possesses an additional short peptide at the NH2 terminus and a longer peptide, composed primarily of hydrophobic residues, at the COOH terminus (8, 9). The oxidation-reduction reactions take place in the hydrophilic region of the molecule, whereas the hydrophobic region interacts with the endoplasmic reticulum (10). We have isolated and studied a cytochrome b5 present in the supernatant fraction of human erythrocytes (11). Erythrocyte cytochrome b5 is a hydrophilic protein which participates in the reduction of methemoglobin by transferring electrons from erythrocyte cytochrome b5 reductase to methemoglobin (12, 13). Except for being water-soluble, human erythrocyte cytochrome b5 is very similar to liver microsomal cytochrome b5. The two proteins have similar visible and electron paramagnetic resonance spectra, both have protoheme IX as their prosthetic group, and both can serve as substrate for cytochrome b5 reductase. The molecular weight of human erythrocyte cytochrome b5 is intermediate between that of the detergent-solubilized and trypsin-solubilized forms of the liver microsomal The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
Abbreviation: NaDodSO4, sodium dodecyl sulfate. * Present address: Department of Biochemistry, University of California, Berkeley, CA 94720. t To whom reprint requests should be addressed. * Hultquist, D. E. & Douglas, R. H. (1974) 168th Meeting Am. Chem. Soc. (Atlantic City, NJ), BIOL-103.
this fact.
3118
Biochemistry: Douglas and Hultquist tilled-deionized water. Phosphate buffers were prepared with potassium salts. Tris buffers were adjusted to pH with HC1. All dialysis and concentration of protein samples were performed in Amicon ultrafiltration cells equipped with UM-2 membranes. Chromatographic steps were monitored at 280 nm and 413 nm with a Gilford spectrophotometer. Visible spectra, from 400 to 600 nm, of the oxidized, reduced, and reduced pyridine hemochrome forms of the protein were determined at 250 with a Cary model 14 recording spectrophotometer. Concentrations of cytochrome b5 were determined spectrally by assuming a millimolar extinction coefficient of 115 at 413 nm (17). Throughout the procedure the ratio of absorbance at 413 nm to that at 280 nm was used as a measure of cytochrome purity. Low-temperature spectra of cytochrome b5 were taken at -196° with a Cary model 13 spectrophotometer adapted for this use. Samples were made 50% in glycerol, reduced with a few crystals of dithionite, and then frozen in liquid nitrogen. After warming to -50' samples were refrozen and spectra recorded. Disc-gel electrophoresis in 15% gels was performed by modification of the methods of Brewer and Ashworth (18). Hemeprotein bands were visualized immediately and the gels were then stained with Coomassie brilliant blue and destained with acetic acid/methanol/water (3:2:35, vol/vol). Gels were then scanned at 520 nm with a Gilford spectrophotometer equipped with a linear transport. Disc-gel electrophoresis in the presence of sodium dodecyl sulfate (NaDodSO4) was done by either the procedure of Weber and Osborn (19) or that of Brewer and Ashworth (18), with bovine serum albumin, ovalbumin, chymotrypsinogen, myoglobin, and cytochrome c as markers. Amino acid analysis on a Durham Model D-500 analyzer was performed by AAA Laboratory of Seattle, WA, on 0.2-mg samples previously shown to be homogeneous by disc-gel electrophoresis. Isolation of Bovine Erythrocyte Cytochrome b5. Cytochrome b5 was isolated from very large volumes of freshly collected bovine erythrocytes by a modification of the procedure developed for isolating cytochrome b5 from outdated human erythrocytes (11, 14). The very large-scale preparations were made feasible by using DEAE-cellulose chromatography to remove the cytochrome directly from the hemolysate and by eluting the cytochrome from the DEAE-cellulose column with a combination pH and ionic strength gradient. The use of this gradient rather than only an ionic strength gradient greatly decreased the quantity of salt that subsequently had to be removed from the cytochrome preparation, thereby reducing the time required to accomplish this step. Approximately 30 liters of bovine blood were collected in a pail containing 6 liters of acid/citrate/dextrose Formula A (24.5 g of glucose, 22 g of trisodium citrate, and 8 g of citric acid per liter of water). Cells were immediately sedimented, washed, and lysed as described (11) except that hypotonic lysis was achieved with 3 vol of water and the resulting hemolysate was then stored at -60°. As needed, aliquots of the hemolysate were thawed and adjusted at 40 to pH 5.7 with dilute HCl, and the stroma was removed by centrifugation at 13,000 X g for 40 min. The supernatant fraction was adjusted to pH 7.2 with dilute KOH, centrifuged at 13,000 X g for 20 min, diluted with 4 vol of water, again adjusted to pH 7.2, and then applied to a column of DEAE-cellulose that had been equilibrated in 3 mM phosphate buffer, pH 7.2. Care was taken to remove all particulate matter and to avoid denaturation since the presence of denatured protein resulted in channeling of the column accompanied by low yields and poor resolution of proteins.
Proc. Nati. Acad. Sci. USA 75 (1978)
3119
In a typical purification, hemolysate derived from 3.5 liters of packed erythrocytes was charged onto a 6.5 X 55 cm column of DEAE-cellulose. The column was then washed with 13 liters of 3 mM potassium phosphate buffer, pH 7.2, to remove residual hemoglobin. Several contaminating proteins were then eluted by simultaneously decreasing the pH and increasing the ionic strength, using first 2.5 liters of 10 mM KH2PO4/10 mM KCI and then a gradient prepared with 4 liters of 10 mM KH2PO4/10 mM KCI in the mixer and 4 liters of 50 mM KH2PO4 in the reservoir. This gradient was followed by a wash with 4 liters of 50 mM KH2PO4. The column was then eluted with a second gradient consisting of 4 liters of 50 mM KH2PO4 in the mixer and 4 liters of 0.2 M KH2PO4 in the reservoir. After this gradient was completed, two distinct red bands could be seen on the column. They were eluted with 0.2 M KH2PO4 and were spectrally identified as cytochrome b5. Since the two red bands were incompletely resolved, the cytochrome b5-containing fractions were pooled to give 3.3 liters of solution with an A413 nm of 0.385 and an A413 nm/ A2pj nm ratio of 0.86. The pooled fractions were concentrated to a small volume and chromatographed on a Bio-Gel P-60 column (4.8 X 135 cm) in 50 mM phosphate buffer, pH 7.2. The cytochrome b5-containing fractions were pooled to give 163 ml of solution with an A413 of 0.73 and an A413 =/A2o nm ratio of 3.8. Disc-gel electrophoresis of the purer fractions indicated the presence of two hemeproteins along with several colorless proteins. The cytochrome sample from gel filtration chromatography was then concentrated and dialyzed extensively against 50 mM Tris-HCl/90 mM NaCI, pH 7.7. After deaeration, the sample was applied at a flow rate of 0.1 ml/min to a DEAE-Sephadex A-50 column (2 X 42 cm) equilibrated with the above Tris/ NaCl solution. The column was developed by the shallow concave gradient system of Nobrega et al. (20). The gradient consisted of 250 ml of 50 mM Tris-HCI/90 mM NaCl, pH 7.7, in the mixer (a 250-ml round-bottomed flask) and 250 ml of 50 mM Tris-HCl/0.25 M NaCl, pH 7.7, in the reservior (a 250-ml erlenmeyer flask). The two forms of cytochrome b5 were resolved on this column (Fig. 1). By the nomenclature of Strittmatter and Ozols for the two forms of "lipase"-solubilized liver microsomal cytochrome b5 (21), the first band eluted was called cytochrome b5 II and the form that bound more tightly to the anion-exchange column was called cytochrome b5 I. Tubes within each peak were pooled. The fraction enriched in form II contained 0.35 jmol of cytochrome with an A413 nm/A2so nm ratio of 5.10; the fraction enriched in form I contained 0.35 ,umol of cytochrome with a ratio of 5.34. In some isolations, the elution was interrupted while the resolved bands were still on the column, and the bands were removed by the procedure of Strittmatter and Ozols (21). Because separation of the two forms of cytochrome b5 was incomplete by either procedure, fractions enriched in an individual form were pooled and rechromatographed repeatedly on DEAESephadex until the sample was assessed to be homogeneous by disc-gel electrophoresis and until the 413 nm/280 nm absorbance ratio no longer increased. The final absorbance ratio was 6.34 for cytochrome b5 I and 5.90 for cytochrome b5 II. Preparation and Isolation of Tryptic Peptides from Cytochrome bs Cytochrome b5 I, cytochrome b5 II, and mixtures of the two forms were digested with trypsin. Cytochrome b5 was dialyzed extensively against 0.1 M NH4HCO3 and concentrated to 1 mg/ml. To 1.5 ml of this solution was added 45,ug of trypsin to give a reaction mixture with a cytochrome b5 to trypsin ratio of 33.3. The mixture was incubated with shaking at 370 for 8 hr; an additional 45 ,ug of trypsin was added and the incubation was continued for an additional 8 hr.
Biochemistry: Douglas and Hultquist
3120
Proc. Natl. Acad. Sci. USA 75 (1978) Table 1. Amino acid composition (residues/heme) of bovine erythrocyte cytochrome b5 I and II
Amino acid
0~~~~~~~ .0~~~~~~~~~~~~~
Biochemistry
Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5 (hemepeptide/proteolysis/endoplasmic reticulum/erythroid maturation)
RICHARD H. DOUGLAS* AND DONALD E. HULTQUISTt Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
Communicated by J. Lawrence Oncley, April 17, 1978
protein. Hydrolysis of human erythrocyte cytochrome b5 by trypsin yields a hemepeptide that has the same electrophoretic properties as the trypsin-solubilized liver protein (14). During erythrocyte maturation much of the cell's ultrastructure is lost as the nucleus, mitochondria, endoplasmic reticulum, and other subcellular organelles are degraded and/or extruded from the cell. For developmental and biochemical reasons it is of interest to determine the fate of proteins that are associated with the subcellular organelles of immature erythroid cells. We have hypothesized (13) that some of these proteins become solubilized during erythroid maturation and that the soluble proteins are retained and are functional in the mature erythrocyte. The similarity in the properties of human erythrocyte and liver cytochrome b5 suggested that the soluble erythrocyte protein that functions in methemoglobin reduction is derived from the microsomal protein that functions in fatty acyl coenzyme A desaturation. We have thus compared the structural similarity between the erythrocyte and liver proteins. In order to obtain the milligram quantitities of protein required for such studies, we have improved and simplified our purification procedure and have used fresh bovine erythrocytes instead of the outdated human erythrocytes we previously used. We report the isolation of homogeneous preparations of two forms of bovine erythrocyte cytochrome b5 and present evidence that the structures of these two proteins are identical to segments of the peptide chain of bovine microsomal cytochrome b5. Preliminary findings have been reported (15, 16, t). EXPERIMENTAL PROCEDURE Materials. Fresh beef blood was purchased from Kappler Packing Co., Ann Arbor, MI. Sodium dithionite, N,N'-methylenebisacrylamide, acrylamide, spectral grade pyridine, N,N,N',N'-tetramethylethylendiarine, and 20 X 20 cm silica gel plates were obtained from Eastman; Bio-Gel P-4 and P-60 (100-200, and minus 400 mesh) from Bio-Rad Laboratories; DEAE-cellulose and ammonium persulfate from Fisher; DEAE-Sephadex A-50, Tris, and cytochrome c from Sigma; 3X-recrystallized trypsin, ovalbumin, and chymotrypsinogen from Worthington; bovine serum albumin from Pentex; myoglobin from Calbiochem; bromphenol blue from Allied Chemical and Dye Corp.; and Coomassie brilliant blue from Schwarz/Mann. All other reagents were of analytical grade and were used without further purification. Purified bovine liver cytochrome bs, solubilized from microsomes with "lipase" and trypsin, was a gift from Scott Mathews. General Procedures. All solutions were prepared with dis-
ABSTRACT Homogeneous pre arations of two forms of soluble cytochrome b5 have been o tained from bovine erythrocytes by successive chromatography on DEAE-cellulose, Bio-Gel P.60, and DEAE-Sephadex. Although the two forms could be separated on disc gel electrophoresis, they appeared to have similar molecular weights of a pproximately 12,000 and identical visible absorbance spectra. The tryptic hemepeptides derived from the two forms of bovine erythrocyte cytochrome bs are electrophoretically indistinguishable from each other and from the tryptic core hemepeptide derived from liver microsomal cytochrome bs. The bovine erythrocyte tryptic hemepeptide was purified to homogeneity; its amino acid composition was shown to be identical to that of tryptic hemepeptide from liver microsomal cytochrome b5. The amino acid compositions of the two isolatable forms of erythrocyte cytochrome b5 correspond well to the compositions of the 97- and 95-residue segments of native liver microsomal cytochrome b5 that begin at the NH2 terminus. These results agree with the hypothesis that soluble erythrocyte cytochrome b5 is derived from microsomal protein by proteolysis during erythroid maturation.
Microsomal cytochrome b5 is a membrane-bound protein that functions as a component of the fatty acyl coenzyme A desaturase system (1-3) and has been implicated in microsomal hydroxylation reactions (4). Bovine liver microsomal cytochrome b5 has been studied extensively. The amino acid sequences have been determined for the intact protein and for the hemepeptides derived from it by solubilization of bovine liver microsomes with trypsin and with "lipase" (5). The x-ray crystal structure of the trypsin-solubilized molecule has been refined to 2.0 A resolution (6). Solubilization of bovine liver microsomes with detergent yields a hydrophobic form of cytochrome b5 (7) that differs structurally from the proteasesolubilized hemepeptides in that it possesses an additional short peptide at the NH2 terminus and a longer peptide, composed primarily of hydrophobic residues, at the COOH terminus (8, 9). The oxidation-reduction reactions take place in the hydrophilic region of the molecule, whereas the hydrophobic region interacts with the endoplasmic reticulum (10). We have isolated and studied a cytochrome b5 present in the supernatant fraction of human erythrocytes (11). Erythrocyte cytochrome b5 is a hydrophilic protein which participates in the reduction of methemoglobin by transferring electrons from erythrocyte cytochrome b5 reductase to methemoglobin (12, 13). Except for being water-soluble, human erythrocyte cytochrome b5 is very similar to liver microsomal cytochrome b5. The two proteins have similar visible and electron paramagnetic resonance spectra, both have protoheme IX as their prosthetic group, and both can serve as substrate for cytochrome b5 reductase. The molecular weight of human erythrocyte cytochrome b5 is intermediate between that of the detergent-solubilized and trypsin-solubilized forms of the liver microsomal The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
Abbreviation: NaDodSO4, sodium dodecyl sulfate. * Present address: Department of Biochemistry, University of California, Berkeley, CA 94720. t To whom reprint requests should be addressed. * Hultquist, D. E. & Douglas, R. H. (1974) 168th Meeting Am. Chem. Soc. (Atlantic City, NJ), BIOL-103.
this fact.
3118
Biochemistry: Douglas and Hultquist tilled-deionized water. Phosphate buffers were prepared with potassium salts. Tris buffers were adjusted to pH with HC1. All dialysis and concentration of protein samples were performed in Amicon ultrafiltration cells equipped with UM-2 membranes. Chromatographic steps were monitored at 280 nm and 413 nm with a Gilford spectrophotometer. Visible spectra, from 400 to 600 nm, of the oxidized, reduced, and reduced pyridine hemochrome forms of the protein were determined at 250 with a Cary model 14 recording spectrophotometer. Concentrations of cytochrome b5 were determined spectrally by assuming a millimolar extinction coefficient of 115 at 413 nm (17). Throughout the procedure the ratio of absorbance at 413 nm to that at 280 nm was used as a measure of cytochrome purity. Low-temperature spectra of cytochrome b5 were taken at -196° with a Cary model 13 spectrophotometer adapted for this use. Samples were made 50% in glycerol, reduced with a few crystals of dithionite, and then frozen in liquid nitrogen. After warming to -50' samples were refrozen and spectra recorded. Disc-gel electrophoresis in 15% gels was performed by modification of the methods of Brewer and Ashworth (18). Hemeprotein bands were visualized immediately and the gels were then stained with Coomassie brilliant blue and destained with acetic acid/methanol/water (3:2:35, vol/vol). Gels were then scanned at 520 nm with a Gilford spectrophotometer equipped with a linear transport. Disc-gel electrophoresis in the presence of sodium dodecyl sulfate (NaDodSO4) was done by either the procedure of Weber and Osborn (19) or that of Brewer and Ashworth (18), with bovine serum albumin, ovalbumin, chymotrypsinogen, myoglobin, and cytochrome c as markers. Amino acid analysis on a Durham Model D-500 analyzer was performed by AAA Laboratory of Seattle, WA, on 0.2-mg samples previously shown to be homogeneous by disc-gel electrophoresis. Isolation of Bovine Erythrocyte Cytochrome b5. Cytochrome b5 was isolated from very large volumes of freshly collected bovine erythrocytes by a modification of the procedure developed for isolating cytochrome b5 from outdated human erythrocytes (11, 14). The very large-scale preparations were made feasible by using DEAE-cellulose chromatography to remove the cytochrome directly from the hemolysate and by eluting the cytochrome from the DEAE-cellulose column with a combination pH and ionic strength gradient. The use of this gradient rather than only an ionic strength gradient greatly decreased the quantity of salt that subsequently had to be removed from the cytochrome preparation, thereby reducing the time required to accomplish this step. Approximately 30 liters of bovine blood were collected in a pail containing 6 liters of acid/citrate/dextrose Formula A (24.5 g of glucose, 22 g of trisodium citrate, and 8 g of citric acid per liter of water). Cells were immediately sedimented, washed, and lysed as described (11) except that hypotonic lysis was achieved with 3 vol of water and the resulting hemolysate was then stored at -60°. As needed, aliquots of the hemolysate were thawed and adjusted at 40 to pH 5.7 with dilute HCl, and the stroma was removed by centrifugation at 13,000 X g for 40 min. The supernatant fraction was adjusted to pH 7.2 with dilute KOH, centrifuged at 13,000 X g for 20 min, diluted with 4 vol of water, again adjusted to pH 7.2, and then applied to a column of DEAE-cellulose that had been equilibrated in 3 mM phosphate buffer, pH 7.2. Care was taken to remove all particulate matter and to avoid denaturation since the presence of denatured protein resulted in channeling of the column accompanied by low yields and poor resolution of proteins.
Proc. Nati. Acad. Sci. USA 75 (1978)
3119
In a typical purification, hemolysate derived from 3.5 liters of packed erythrocytes was charged onto a 6.5 X 55 cm column of DEAE-cellulose. The column was then washed with 13 liters of 3 mM potassium phosphate buffer, pH 7.2, to remove residual hemoglobin. Several contaminating proteins were then eluted by simultaneously decreasing the pH and increasing the ionic strength, using first 2.5 liters of 10 mM KH2PO4/10 mM KCI and then a gradient prepared with 4 liters of 10 mM KH2PO4/10 mM KCI in the mixer and 4 liters of 50 mM KH2PO4 in the reservoir. This gradient was followed by a wash with 4 liters of 50 mM KH2PO4. The column was then eluted with a second gradient consisting of 4 liters of 50 mM KH2PO4 in the mixer and 4 liters of 0.2 M KH2PO4 in the reservoir. After this gradient was completed, two distinct red bands could be seen on the column. They were eluted with 0.2 M KH2PO4 and were spectrally identified as cytochrome b5. Since the two red bands were incompletely resolved, the cytochrome b5-containing fractions were pooled to give 3.3 liters of solution with an A413 nm of 0.385 and an A413 nm/ A2pj nm ratio of 0.86. The pooled fractions were concentrated to a small volume and chromatographed on a Bio-Gel P-60 column (4.8 X 135 cm) in 50 mM phosphate buffer, pH 7.2. The cytochrome b5-containing fractions were pooled to give 163 ml of solution with an A413 of 0.73 and an A413 =/A2o nm ratio of 3.8. Disc-gel electrophoresis of the purer fractions indicated the presence of two hemeproteins along with several colorless proteins. The cytochrome sample from gel filtration chromatography was then concentrated and dialyzed extensively against 50 mM Tris-HCl/90 mM NaCI, pH 7.7. After deaeration, the sample was applied at a flow rate of 0.1 ml/min to a DEAE-Sephadex A-50 column (2 X 42 cm) equilibrated with the above Tris/ NaCl solution. The column was developed by the shallow concave gradient system of Nobrega et al. (20). The gradient consisted of 250 ml of 50 mM Tris-HCI/90 mM NaCl, pH 7.7, in the mixer (a 250-ml round-bottomed flask) and 250 ml of 50 mM Tris-HCl/0.25 M NaCl, pH 7.7, in the reservior (a 250-ml erlenmeyer flask). The two forms of cytochrome b5 were resolved on this column (Fig. 1). By the nomenclature of Strittmatter and Ozols for the two forms of "lipase"-solubilized liver microsomal cytochrome b5 (21), the first band eluted was called cytochrome b5 II and the form that bound more tightly to the anion-exchange column was called cytochrome b5 I. Tubes within each peak were pooled. The fraction enriched in form II contained 0.35 jmol of cytochrome with an A413 nm/A2so nm ratio of 5.10; the fraction enriched in form I contained 0.35 ,umol of cytochrome with a ratio of 5.34. In some isolations, the elution was interrupted while the resolved bands were still on the column, and the bands were removed by the procedure of Strittmatter and Ozols (21). Because separation of the two forms of cytochrome b5 was incomplete by either procedure, fractions enriched in an individual form were pooled and rechromatographed repeatedly on DEAESephadex until the sample was assessed to be homogeneous by disc-gel electrophoresis and until the 413 nm/280 nm absorbance ratio no longer increased. The final absorbance ratio was 6.34 for cytochrome b5 I and 5.90 for cytochrome b5 II. Preparation and Isolation of Tryptic Peptides from Cytochrome bs Cytochrome b5 I, cytochrome b5 II, and mixtures of the two forms were digested with trypsin. Cytochrome b5 was dialyzed extensively against 0.1 M NH4HCO3 and concentrated to 1 mg/ml. To 1.5 ml of this solution was added 45,ug of trypsin to give a reaction mixture with a cytochrome b5 to trypsin ratio of 33.3. The mixture was incubated with shaking at 370 for 8 hr; an additional 45 ,ug of trypsin was added and the incubation was continued for an additional 8 hr.
Biochemistry: Douglas and Hultquist
3120
Proc. Natl. Acad. Sci. USA 75 (1978) Table 1. Amino acid composition (residues/heme) of bovine erythrocyte cytochrome b5 I and II
Amino acid
0~~~~~~~ .0~~~~~~~~~~~~~
