Comp. Biochem. Physiol. Vol. 101B,No. 1/2,pp. 235-242, 1992
0305-0491/92$5.00+ 0.00 © 1992PergamonPressplc
Printed in Great Britain
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 [.
SAMPLE
"T 3
~ _,_
WASH
~,
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
20.mM BUFFER A
5~).4.
0.4 M KCt in BUFFER A
H e m o g l o b i ~ '~ CytochrOme b5
"z E
,J
c E o
T 6 ~
eO0c,=e
0 0 2 - I00~ ,¢
o
u
\
-75 ~
4~
j
.0
I
==
¢Z
Q.
o
-50 o
u o
2~ -25
0
\\ J
t I0 O0
,~
i
q 6000
I
I 700 0
J I
" ' q h - - - ~ ~e, 800 0
I0000
llOl~
Volume (m[) Fig. 1. E ] u t i o n profile o f h u m a n red b l o o d cell hemo]ysates f r o m the first D E A E - c e l l u l o s e
column
(4.3 x 48 cra). The fraction between 6250 and 7130 ml which contained NADH-
0305-0491/92$5.00+ 0.00 © 1992PergamonPressplc
Printed in Great Britain
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 [.
SAMPLE
"T 3
~ _,_
WASH
~,
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
20.mM BUFFER A
5~).4.
0.4 M KCt in BUFFER A
H e m o g l o b i ~ '~ CytochrOme b5
"z E
,J
c E o
T 6 ~
eO0c,=e
0 0 2 - I00~ ,¢
o
u
\
-75 ~
4~
j
.0
I
==
¢Z
Q.
o
-50 o
u o
2~ -25
0
\\ J
t I0 O0
,~
i
q 6000
I
I 700 0
J I
" ' q h - - - ~ ~e, 800 0
I0000
llOl~
Volume (m[) Fig. 1. E ] u t i o n profile o f h u m a n red b l o o d cell hemo]ysates f r o m the first D E A E - c e l l u l o s e
column
(4.3 x 48 cra). The fraction between 6250 and 7130 ml which contained NADH-
