Purification and characterization of two forms of soluble NADH cytochrome b5 reductases from human erythrocytes

Comp. Biochem. Physiol. Vol. 101B,No. 1/2,pp. 235-242, 1992

0305-0491/92$5.00+ 0.00 © 1992PergamonPressplc

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PURIFICATION A N D CHARACTERIZATION OF TWO FORMS OF SOLUBLE N A D H CYTOCHROME b5 REDUCTASES FROM H U M A N ERYTHROCYTES EMEL ARINQ, TOLIN GORAV, Ur,tA,t ~APLAKOC~JLUand ORHANADALI The Department of Biology, Middle East Technical University, 06531 Ankara, Turkey (Tel: 4 223 71 00 Ext. 3105); (Fax: 90 4 223 69 45) (Received 28 May 1991)

Abstract--1. Two forms of soluble NADH cytochrome b5 reductase were purified from human erythrocytes. Two distinct fractions both having the NADH cytochrome b5 reductase activity eluted from the second DEAE-cellulose column were further purified by ultrafiltration and 5'-ADP-agarose affinity chromatography. 2. The final preparations were purified 9070- and 4808-fold, respectively, over hemolysate. Both reductases exhibited identical electrophoretic patterns when subjected to SDS-PAGE and apparent monomer M, of each reductase was determined to be 32,000 _+ 1300. 3. Vm~values ofreduetase II for the various electron aeeeptors, namely, 2,6-dichlorophenolindophenol, ferricyanide and cytochrome c through cytochrome bs were found to be 1.9, 1.8 and 2 times higher than those of reductase I. 4. Some differences were noted for reductase I and reductase II fractions. Their elution profiles from a second DEAE--cellulos¢column were quite different and that suggested that reductase II is more acidic than reductase I. Reductase II was found to be more sensitive to heat treatment than reductase I.

INTRODUCTION NADH-dependent cytochrome bs reductase is a FAD containing flavoprotein which catalyzes the reduction of cytochrome bs. It is also called N A D H cytochrome c reductase, diaphorase reductase and methemoglobin reductase. It exists as a membranebound amphipathic protein in endoplasmic reticulum of various tissues including liver (Strittmatter and Velick, 1957; Mihara and Sato, 1975), brain (Inouye and Shingawa, 1965), muscle (Leroux et al., 1975) and lung (G/iray and Arin~, 1988). Erythrocytes contain two forms of cytochrome b5 reductase; while one form exists as a membrane-bound reductase (Kitajima et aL, 1981), the second reductase is present in a soluble form (Passon and Hultquist, 1972). Microsomal cytochrome b5 reductase functions in A9-, A6-, A5-desaturation of fatty acids (Oshino and Omura, 1973; Strittmatter et aL, 1974; Lee et al., 1977; Oshino, 1980); in cholesterol biosynthesis (Reddy et al., 1977); in fatty acid elongation (Keyes et al., 1979); in desaturation of phospholipids (Pugh and Kates, 1977) and in oxidation of 4-methylsterol (Fukushima et al., 1981). Soluble erythroeyte cytochrome bs reductase has been shown to be involved in the reduction of methernoglobin (Passon and Hultquist, 1972). Amphipathic bs reductase of steer liver microsomes has M, of 34,110, contains 300 amino acids consisting of a large cytoplasmic catalytieaUy active, FAD-containing hydropliilie domain (about 275 amino adds) which occupies the C-terminal of the enzyme and a short hydrophobic membrane-binding domain, containing 1 mole of myristic acid covalently attached to N-terminal glycyl residue (Ozols et al., 1985). The

soluble erythrocyte b5 reductase has 275 amino acid residues which corresponds to catalytically active hydrophilic peptide segment of liver microsomal b5 reductase (Yubisui et al., 1986). Recently, studies carried out with the purified lung microsomal N A D H cytochrome bs reductase have indicated that lung bs reductase is very similar to its liver counterpart in terms of monomer mol. wt, molecular size of the hydrophilic peptide obtained by mild trypsin cleavage, cofactor and absolute spectrum (Giiray and Arin~, 1990; Arin~;, 1991). Hereditary methemoglobinemia is a disease in which N A D H cytochrome b5 reductase (methemoglobin reductase) is deficient. Hereditary methemoglobinemia has been classified into three types: the "erythrocyte type" (type I) in which the enzyme deficiency is restricted to erythrocytes (Scott and Griflith, 1959); the "generalized type" (type II) which shows the enzyme deficiency in almost all tissues including erythrocytes, leukocytes, muscle cells and brain cells and is associated with mental retardation or neurological disorders (Leroux et ai., 1975); and "type III" in which the enzyme defect is restricted to blood cells, such as erythrocytes, platelets and leukocytes (Tanisima et al., 1985). Recently, one species of b5 reductase mRNA was detected in rat liver cells and two m R N A species in reticulocytes, one of which was found to be different from the liver-type m R N A (Pietrini et al., 1988). Kitao et al. (1974) reported two fractions of diaphorase reductase eluted from DEAE-cellulose column, following the application of human red blood cell hemolysates. They found that only the second diaphorase reductase fraction had cytochrome b5 reductase activity.

235

EMELARINqet al.

236

During this study two distinct fractions, both having N A D H - d e p e n d e n t 2,6-dichlorophenolindophenol (DCIP) reductase activity, from the second DEAE-cellulose column chromatography of human red blood cell hemolysates, were obtained. In contrast to the previous studies (Kitao et al., 1974), both fractions contained N A D H cytochrome b s reductase activity. Further purification of the enzymes was achieved on 5'-ADP-agarose affinity chromatography and some of their properties were studied. MATERIALS AND METHODS

Chemicals 2,6-Dichlorophenolindophenol (DCIP), sodium citrate were purchased from Merck. Bovine serum albumin (BSA), NADH, E-aminocaproic acid (E-ACA), DL-dithiothreitol (DTT) were obtained from Sigma Chemical Co. Agarosehexane-adenosine 5'-diphosphate (5'-ADP-agarose), Type 2 was purchased by P.L. Biochemicals Inc. DEAE-cellulose (DE-52) was purchased from Whatmann Biochemicals Ltd. Potassium ferricyanide was obtained from Fluka A.G. Emulgen 913 was a gift from Kao-Corporation, Tokyo, Japan. All other chemicals were of the highest grade commercially available. Preparation of human red blood cell hemolysates Human blood in acid-citrate-dextrose-adenine (ACDA), within I week of its expiry date, was obtained from Kizilay blood bank, Ankara, Turkey, and used immediately. Red cells were collected from the ACDA blood after centrifugation at 300 g for 10 min. Following the removal of plasma and some of the buffy coat, red cells were washed three times at 4°C by centrifuging a suspension of cells in 4 vols of 0.9% NaC1 solution at 5000g for 10 min and then decanting the supernatant solution and buffy coat. Packed cells (510 ml) were lysed by adding 4 vols (2000 ml) of cold deionized water containing 2 mM EDTA, 0.1 mM DTT and 0.25 mM E-ACA and by freezing in liquid nitrogen and thawing. The solution was then centrifuged at 12,000g for 20rain to remove stroma. Clear supernatant solution (2100 ml) was diluted with 1.5vols (3150ml) of cold water containing 2raM EDTA, 0.1 mM DTT and 0.25mM E-ACA and adjusted to pH 7.7 with KOH solution. This hemolysate was used as a starting material to isolate NADH cytochrome b5 reductase and cytochrome bs. All subsequent steps were carried out at 4°C.

Purification of cytochrome b5 reductase First DEAE--cellulose column chromatography. DEAEcellulose was equilibrated with 10 mM potassium phosphate buffer, pH 7.7 containing 2mM EDTA, 0.1mM DTT and 0.25 mM E-ACA (Buffer A). The clear hemolysate in Buffer A (5300 ml) was applied to the column (4.3 x 45 cm) at a flow rate of 80 ml/hr. The column was washed with approximately 11 of Buffer A. Fractions containing NADH--(DCIP) reductase activity were eluted with 20 mM Buffer A and pooled. After passing 3.21 of 20 mM Buffer A from the column, eytochrome b5 was then eluted with 0.4 M KCI in Buffer A. Second DEAE-cellulose column chromatography. The pooled NADH-DCIP reductase fractions (880 ml) were diluted with 0.5 vol (440 ml) of 2 mM EDTA, 0.1 mM DTT and 0.25mM ¢-ACA and applied to a DEAE column (4.5 x 13 cm) previously equilibrated with 30 mM Buffer A. Some of the NADH-DCIP reductase (Fraction I) was eluted during the sample application. Following the sample application, a second NADH-DCIP reductase (Fraction II) was eulted from the column with 240 ml Buffer A containing 30 mM potassium phosphate pH 7.7 and 0.3 M KC1.

5'-ADP affinity column chromatography of Fraction II. Cytochrome b5 reductase fractions eluted from a second DEAE-cellulose column with 0.3 M KC1 buffer (51 ml) were extensively dialyzed against 20 mM potassium phosphate buffer, pH 7.1, containing 1 mM EDTA, 0.1 mM DTT and applied to a 5'-ADP agarose affinity column. The affinity column (0.8 x 6.5 cm) had been previously equilibrated with this dialysis buffer. The dialyzed sample (54 ml) was applied to the column at a flow rate of 2 ml/hr. The column was washed extensively with 100 ml of equilibration buffer to elute unbound contaminating proteins from the column. Some of the NADH cytochrome b5 reductase was then eluted from the affinity column with I mM NADH in 20mM potassium phosphate buffer containing 0.1 mM DTT and I mM EDTA. Under these conditions some of the enzyme remained in the column, as will be discussed in the Results and Discussion section. The enzyme solution was concentrated by ultrafiltration using Centricon PM-10 membrane and stored in small aiiquots at -20°C. 5'-ADP affinity column chromatography of Fraction 1. Cytochrome b~ reductase fractions eluted from a second DEAE-celhilose column during sample application were applied to a 5'-ADP agarose affinity column. In order to facilitate elution of the enzyme from the column, non-ionic detergent Emulgen 913 was added both to the sample and to the column equilibration buffer. Subsequently, the sample containing 0.05% Emulgen 913 and 5% glycerol was applied to the affinity column (0.8 x 6.5) which was previously equilibrated with 20 mM phosphate buffer pH 7.1, containing 5% glycerol, 0.1raM EDTA, 0.1raM DTT, 0.1% Emulgen 913, at a flow rate of 2ml/hr. The column was washed extensively with 36 column vols of equilibration buffer containing 0.5% Emulgen 913 to elute unbound contaminating proteins from the column. In order to reduce the Emulgen 913 concentration in the effiuent to less than 0.01% (u.v. absorption to less than 0.1 at 275nm), the column was washed with about 9 column vols of 20 mM potassium phosphate buffer, pH 7.0 containing 5% glycerol, 0.1 mM DTT and 0.1 mM EDTA. NADH cytochrome b 5 reductase was then eluted from the affinity column with 1 mM NADH in 20 mM potassium phosphate buffer containing 0.1 mM DTT and 1 mM EDTA. Fractions rich in reductase activity were combined and concentrated with dry Sephadex G-25 and stored in small aliquots at -20°C. Purification of liver microsomal cytochrome b 5 Rabbit liver microsomal cytochrome b5 was solubilized in the presence of 1% Emulgen 913, 0.2% cholate and proteolyric enzyme enhibitors, PMSF and E-ACA. Solubilized microsomal cytochrome b5 was then purified by the combination of the chromatographic procedures used for the purification of cytochromes with some modifications (Strittmatter et al., 1978; Giiray and Arin(~, 1990; Adaii and Arin~, 1990). Analytical procedures Determination of protein. The protein concentration was determined by the method of Lowry et al. (1951) using crystalline BSA as standard. Determination of NADH cytochrome b 5 reductase activity. Reductase activity was measured by using either potassium ferricyanide or DCIP as a substrate or coupling the reduction of cytochrome b5 with cytochrome c. NADH-DCIP reductase activity Since ferricyanide assay cannot be used in the presence of hemoglobin for estimation of the reductase activity at early stages of isolation, only DCIP was used as an electron acceptor in the presence of NADH. The reaction was carried out at 25°C in 1 ml of 50raM Tris-HC1, pH 8.1

237

Soluble cytochrome b~ reductases of human erythrocytes buffer containing 0.5 mM EDTA, 0.025 mM DCIP, reductase and 0.SmM NADH. Activity was assayed by measuring the rate of decrease in absorbance at 600 nm and an extinction coefficient of 21 mM -~ cm -1 was used for calculations (Hultquist, 1978). One unit of enzyme is the amount that catalyzes the reduction of 1 nmol DCIP per rain.

NADH-ferricyanide reductase activity The activity of NADH cytochrome b5 reductase with ferricyanide was assayed according to Strittmatter and Velick (1957). The assay was carried out in I ml reaction cuvettes containing 0.1 M potassium phosphate buffer, pH 7.5, 0.2raM potassium ferricyanide, reductase and 0.112raM NADH at 25°C. Decrease in absorbance at 420 nm was followed with time. A value of 1.02 raM- l cm was used for the molar extinction coefficient of ferricyanide (Schellenberg and Hellerman, 1958). One unit of reductase was defined as the amount of enzyme catalyzing the reduction of 1/~mol ferricyanide per min under the described conditions.

NADH-cytochrome b5 reductase activity by coupling to cytochrome c Cytochrome b5 reduction coupled to the reduction of cytochrome c was determined spectrophotometrically. The reduction of cytochrome c was followed at 550 mn, at 25°C, in the presence of cytochrome b~. The reaction mixture contained 0.1 M potassium phosphate buffer, pH 7.5, 0.112mM NADH, appropriate concentration of enzyme, 0.75-5.3 nmol of partially purified rabbit liver cytoehrome b~ and 36 nmol of cytoehrome c, in a final volume of 1.0 ml. The molar extinction coefficient increment for cytochrome c was taken as 19.1 mM -t cm -~ (Yonetani, 1965). One unit of reductase was defined as the amount of enzyme catalyzing the reduction of 1 nmol cytochrome c per min under the conditions described. Determination ofcytochrome b~. The cytochrome bs concentration of liver was determined from the initial dithionite-reduced minus oxidized difference spectrum using an [.

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extinction coefficient of 185 mM -1 cm -~ for the difference in absorption between 424 and 410 nm (Nishibayashi and Sato, 1968). The cytoehrome b5 concentration of column eluates of red blood cell hemolysates could not be determined by this chemical reduction method since the reduced minus oxidized spectrum of b5 contaminated with other heine proteins of hemolysate gave a peak at 430 nm instead of 424 rim. Estimation of b5 by using the difference in absorption between 430 and 410 resulted in a 3-10-fold higher apparent b5 content than the one estimated by the true enzymatic method. When hemolysate cytochrome b5 was reduced by rabbit liver b5 reductase in the presence of NADH, a peak appeared at 424 nm. The hemolysate b 5 concentration was then calculated from the enzyme-NADH reduced minus oxidized spectrum using the extinction coefficient of 185 mM -~ cm -t for the difference in absorption between 424 and 410 nm. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was employed to estimate the monomeric tool. wt of soluble erythrocyte b5 reductase fractions I and II. Eleetrophoresis was performed on 3% stacking gel and 10% separating gel in a discontinuous buffer system as described by Laemmli (1970). The gels were fixed and stained for protein with 0.25% Coomassie Blue in 50% methanol and 7% acetic acid and destained by the diffusion of unbound dye from gels by extensive washing with 30% methanol. RESULTS

AND

DISCUSSION

Figure 1 shows a n elution profile o f h u m a n red b l o o d cell hemolysates from the first D E A E column. D u r i n g the sample application a n d the washing of the column, the fractions containing D C I P - r e d u c t a s e activity were eluted. These fractions reduced D C I P directly w i t h o u t the addition of N A D H a n d w h e n N A D H was added, the rate o f D C I P reduction was n o t affected. F r a c t i o n s containing N A D H - d e p e n d e n t

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Table 1. Purificationof two forms of solubleNADH cytochromebs reductasefrom red blood cell hemolysates Total Specific Total reductase reductase Volume protein activity activity Recovery (%) Fractions (ml) (mg) (units*) (units/mg protein) Step Total Purification Hemolysate 2400 240,170 408,289 1.7 100 100 1 First DEAE-celluloseeluate 880 2068 70,857 34.3 17.3 17.3 20.2 Second DEAE Fraction I 260 361 19,748 54.7 27.9 4.8 32.2 PM 30 concentration 23 323 15,800 48.9 80.0 3.9 28.8 5'-ADP affinitychromatography, dry Sephadex G-25 concentration 6 0.47 2260 4808 14.3 0.55 2828 Second DEAE FractionII 51 303 11,475 37.9 16.2 2.8 22.3 5'-ADP affinitychromatography and PM 10 concentration 0.8 0.2 1814 9070 15.8 0.44 5335 *One unit of reductase is definedas the amountof enzymecatalyzingthe reductionof I nmoiDCIP per minutein the presenceof NADH under the describedconditions.

DCIP reductase activity were eluted just after this fraction with 20 mM Buffer A. Soluble cytochrome b5 was duted from the same DEAE-cellulose column with 0.4 M KCI in Buffer A. At this step, although reductase was purified 20-fold with respect to hemolysates, the yield was only 17.3% (Table 1). As seen in Fig. 2, chromatography of NADHdependent DCIP-reductase obtained from the first DEAE-cellulose column, yielded two distinct NADH-dependent DCIP reductase fractions on the second DEAE-cellulose column. Fraction I reductase was eluted during the sample application with 30 mM Buffer A, Fraction II reductase was duted from the column with 30 mM Buffer A containing 0.3 M KCI. Since Fraction II reductase eluted as a sharp peak from the second DEAE column, this fraction was first applied to a 5'-ADP-agarose affinity column equilibrated with 20 mM phosphate buffer, pH 7.1, containing 0.1 mM DTT and 1 mM EDTA. As can

be seen in Fig. 3, during the sample application and the washing procedure, contaminating proteins were eluted. Then N A D H - D C I P reductase was eluted from the affinity column as a sharp peak with the column equilibration buffer containing 1 mM NADH. After the elution of this reductase, the column still remained yellow; in order to elute the remaining reductase activity from the column, the equilibration buffer having the following composition passed through the column: B: 1 mM NADH in 0.3 M KC1 (17 ml), C: 5 mM NADH in 0.3 M KCI (9 ml), D: I mM NADH in 0.2% chelate and 0.3 M KC1 (17 ml), E: 1 mM NADH in 0.2% chelate, 0.2% Emulgen and 0.3M KCI (35ml) and F: l m M NADH in 0.2% chelate, 0.2% Emulgen and 0.7 M NaC1 (50 ml). As seen in Fig. 3, NADH-dependent reductase was further eluted with Buffer D containing 1 mM NADH in 0.2% cholate and 0.3 M KCI and with Buffer F containing I mM NADH in 0.2% 0.3 M KC~

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Fig. 3. 5'-ADP-agarose column chromatography (0.8 x 6.5 era) of Fraction II reductase of the second DEAE--cellulose column eluate. Elution: (A): I mM NADH, (B): I mM NADH, 0.3 M KC1, (C): 5 mM NADH, 0.3 M KC1, (D): I mM NADH, 0.3 M KC1, 0.2% chelate, (E): 1 mM NADH, 0.3 M KC1, 0.2% chelate, 0.2% Emulgen 913, (F): 1 mM NADH, 0.3 M KC1, 0.2% chelate, 0.2% Emulgen 913, 0.7 M NaC1. chelate, 0.2% Emulgen and 0.7M NaC1. These reductase fractions could not be characterized further because of the low content of the enzymes. Fraction I reductase eluted from the second DEAE-cellulose column, was concentrated from 260ml down to 23 ml by ultrafiltration using an Amicon PM-30 filter. During this step about 20% of the reductase activity was lost (Table 1). When compared with lung microsomal cytochrome bs reductase, erythrocyte reductase was found to be more stable than the lung enzyme. Ultrafiltration using the PM-30 filter could not be applied for the concentration of lung b5 reductase, since the lung enzyme lost about 60% of its activity during the ultrafiltration step (Giiray and Arin(;, 1990). Concentrated Fraction I reductase was then applied to 5'-ADPagarose column equilibrated with 20 mM phosphate buffer, pH 7.1, containing 5% glycerol, 0.1 mM EDTA, 0.1 mM DTT and 0.1% Emulgen 913. As seen in Fig. 4, contaminating proteins and denatured cytochrome b5 reductase were eluted during the application of the sample. Following the extensive wash of the column, cytochrome bs reductase was then eluted with 1 mM NADH. The amount of the denatured protein eluted from the 5'-ADP-agarose affinity column was varied from one preparation to another depending on the time which elapsed between the second DEAE-cellulose column and the 5'-ADPagarose alTmity column applications. In this particular case (Fig. 4), there was about 2 weeks between the two procedures. The results of a typical experiment for the purification of reductases from red blood cell hemolysates are shown in Table 1. The data indicate that Fraction CBPB IOIII.2--P

I and Fraction II reductases from the hemolysates were purified approximately 2228- and 5335-fold, respectively. Although the specific content of Fraction II reductase was 1.9 times higher than that of Fraction I reductase (9070 vs 4808), SDS--PAGE protein patterns (not shown) gave similar results. The apparent monomer M, of reductase I and reductase II was calculated to be 32,000 +_ 1300 by SDS-PAGE. In addition, a second minor protein band corresponding to M, 50,000 was observed in SDS-PAGE protein profiles of both reductases. In another set of the purified reduetase I and II preparations a faster moving band corresponding to M, 29,000 + 1000 was observed about 2 weeks after the purification of the enzymes. A 29,000 M, reductase probably represents the proteolytically cleaved form of the native b5 reductase as in the case of microsomal P-450 reductase of lung (Serabjit-Singh et aL, 1979; I~can and Aring, 1986, 1988) and liver (I~can and Arinq, 1988). Figure 5 shows the effect of cytochrome bs concentration on N A D H cytoehrome c reductase activities of Fraction I and II reductases. As seen in the figure, in the presence of fixed amounts of Fractions I and II reductase and cytochrome c, the rate of NADH-dependent cytoehrome c reduction increased by the addition of increasing amounts of cytochrome b s to the reconstitution medium. The reaction rates were initially proportional to the increase in added cytochrome bs. With the additional increase in bs concentrations, the reactions' rates become constant. Vn~ values of Fraction I and Fraction II reductase were calculated as 660 and 1330 umol cytochrome c reduced min-t nag reductase -~, respectively, from the

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Fig. 4. 5'-Agarose-affinity column chromatography (0.8 x 6.5 cra) of Fraction I reductase of the second DEAE-cellulose column chromatography. The column was washed extensively as described in Materials and Methods. NADH cytochrome b5 reductase was then eluted from the column with I mM NADH in 20 mM potassium phosphate buffer, pH 7.1, containing 5% glycerol. 0.1 mM DTT and 0.1 mM EDTA. Lineweaver-Burk plot (not Km values for Fractions I determined to be 2.1 and respectively, from the same

shown). The apparent and II reductase were 1 . 8 # M cytochrome bs, graph.

Subjecting the reductase I and II fractions to a temperature of 50°C for 2 min resulted in a 14% loss of activity in reductas¢ II, while the treatment did not effect the activity o f reductase I (Fig. 6). The effects of incubation for 5, 15 and 30 min at 50°C on the activities of the fractions were also investigated; the

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Soluble cytochrome b5 reductases of human erythrocytes

241

Table 2. Catalytic activities of purified Fractions I and II reductases with various electron aceeptors Enzymeactivity* Concentration Electron acceptors (/~M) Reductase I Reductase II DCIP 25 4808 9064 Ferricyanide 200 128 230 Cytochrome bs 5.3 plus cytochrome c 36 620 1280 *Enzymeactivitiesweredeterminedin the presenceof NADHas describedin Materials and Methods and expressed as nanomolesof DCIP or cytochrome c reduced per minuteper milligramor micromoleof ferricyanidereducedper minuteper milligram.

reductase II preparation was found to be more sensitive to the 50°C treatment. In one part of the experiment, purified reductase I and II fractions were subjected to hydroxylapatite column chromatography to get rid of the minor 50,000 M, contaminating protein. As seen in Fig. 6, reductase I and II eluted from hydroxlapatite columns became more sensitive to heat treatment and they both lost their activities completely in 30 min. However, the enzymatic activity loss pattern of reductase I was again found to be different from that of reductase II. Reductases eluted from the hydroxylapatite also lost their activities completely when stored at - 2 0 ° C for 3 days and the addition of BSA, 1 mg/ml of enzyme did not protect the enzymes from denaturation. Some properties of NADH-dependent reductase I and reductase II fractions of human red blood cell bemolysates were found to be similar. Both reductases exhibited identical electrophoretic patterns when subjected to SDS-PAGE and the apparent monomer Mr of each reductase was found to be 32,000. The ability of Fraction I and II reductases to transfer electrons to the electron acceptors such as DCIP, ferricyanide and cytochrome e through cytochrome bs, were found to be similar. However, as shown in Table 2, Vn~xvalues of Fraction II reductase for these electron acceptors were found to be 1.9, 1.8 and 2 times higher than those of Fraction I reductase. Some differences were noted for Fraction I and Fraction II cytochrome b5 reductases. Their elution profiles from the second DEAE-cellulose column were quite different. Fraction I reductase was eluted during the sample application. On the other hand Fraction II reductase could only be eluted in the presence of 0.3 M KC1 (Fig. 2). This pattern of elution suggests that Fraction II reductase is more acidic than the Fraction I reductase. A further difference was observed when both reductases were subjected to 50°C for varying periods of time (Fig. 6). Reductase II was found to be more sensitive to heat treatment than the reductase I fraction. Recently one species of cytochrome bs reductase mRNA was detected in rat liver cells and two mRNA species in reticulocytes, one of which was found to be different from the liver type mRNA (Pietrini et al., 1988). Thus, the existence of two forms of b5 reductase in erythrocytes is possible. Although there seem to be some differences between the two purified red blood cell cytochrome b5 reductases, the absolute identity of these reductases remains to be established by amino acid analysis, comparison of peptides, by

the identification of essential amino acids and by microsequencing. Acknowledgements--The financial support provided by a grant from the Middle East Technical University AFP 91-07-01-01 is gratefully acknowledged. We also thank the kind personnel of the Red Crescent Blood Bank from which the blood samples were obtained.

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