ARCHIVES
OP
BIOCHEMISTRY
AND
BIOPHYSICS
Studies
172, 600-607
(1976)
on Methemoglobin
Reductase
lmmunochemical Similarity of Soluble Methemoglobin b, of Human Erythrocytes with NADH-Cytochromeb, of Rat Liver Microsome FUMIO Department
KUMA,2
RUSSELL
A. PROUGH,
OfBiochemistry,
The University School, 5323 Harry
AND
of Texas Health Hines Boulevard, Received
June
Reductase and Cytochrome Reductase and Cytochromeb,
BETTIE
SUE SILER
Science Center at Dallas, Dallas, Texas 75235
MASTERS
Southwestern
Medical
30, 1975
An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b, reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b, reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrome b, to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b, from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b, during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b, stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b, and methemoglobin. An antibody to purified, trypsin-solubilized cytochrome b, from rat liver microsomes was shown to inhibit erythrocyte cytochrome b, reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome 6, preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b, of human erythrocytes are immunochemically similar to NADH-cytochrome b, reductase and cytochrome b, of liver microsomes, respectively.
Since Gibson (1) reported the deficiency of an enzyme in erythrocytes from patients with congenital methemoglobinemia, the mechanism of reductioriof methemoglobin has been the subject of much investigation. Scott and McGraw demonstrated that the NADH-diaphorase activity in erythro-
cytes was responsible for the reduction of methemoglobin (2). This enzyme, methemoglobin reductase, has been purified from human erythrocytes by Kuma et al. (3, 41, who have demonstrated that the enzyme is a flavoprotein, having flavinadenine dinucleotide as a prosthetic group, which does not reduce methemoglobin directly but requires a cofactor to transport electrons from the reduced enzyme to methemoglobin. The similarity of its molecular properties with those of NADH-cytochrome b, reductase of liver microsomes, reported by Strittmatter and Velick (51, has been documented (3, 4). On the other hand, the nonflavoprotein nature of this enzymatic activity was reported by Sugita et al. (6) and Adachi (7), although the addi-
I This research was supported by U.S. Public Health Service Grants No. GM16488 and HL13619 (B.S.S.M.) and American Cancer Society Grant No. BC-153 (R.A.P.). Reprint requests should be addressed to Dr. B. S. S. Masters, Associate Professor of Biochemistry, The University of Texas Health Science Center at Dallas, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Tex. 75235. p Present address: Karatsu Saiseikai Hospital, Motohata-machi, Karatsu, Saga, Japan. 600 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
IMMUNOCHEMICAL
STUDIES
ON
tion of microsomal cytochrome b, increased the rate of methemoglobin reduction by their enzymes. More recently, a soluble cytochrome b, was isolated from human erythrocytes (8, 91, and Hultquist et al. (8) suggested the microsomal origin of this protein by comparing the trypsindigested cytochrome b, of erythrocytes with trypsin solubilized cytochrome b, from human liver microsomes. The purification and characterization of cytochrome b, from human erythrocytes as a cofactor of the methemoglobin reductase system have been reported by Kuma (91, the crossreactivity of human erythrocyte flavoprotein and hemoprotein with liver microsoma1 NADH-cytochrome b, reductase and cytochrome b, have been examined, and the mechanism of methemoglobin reduction in a reconstituted system has been studied.” The present study was undertaken to determine if immunochemical identity or similarity exists between the components of the methemoglobin reductase system of erythrocytes and the microsomal cytochrome b, reductase system. MATERIALS
AND
METHODS
Purification of methemoglobin reductase. Methemoglobin reductase was purified from human erythrocytes according to the method of Kuma and Inomata (4). The enzyme was homogeneous as judged by discontinuous (disc) gel electrophoresis at pH 8.9, and the absorption spectrum of the enzyme revealed no contamination by heme protein. The concentration of the enzyme was estimated from the absorbance at 460 nm with a millimolar extinction coefficient of 11.3, as determined for flavin-adenine dinucleotide by Beinert (10). Purification oferythrocyte cytochrome b,. Erythrocyte cytochrome b s was purified from human erythrocytes according to the method of Kuma (9). The procedure resulted in a homogeneous protein as judged by disc electrophoresis on 15% polyacrylamide gel at pH 8.9. The concentration of the protein was determined from the difference in absorbance between 556 and 575 nm of the reduced and oxidized forms of the protein, with the millimolar extinction coefficient of 24 (9). Purification of NADHxytochrome b, reductase from microsomes. The lysosomal-solubilized NADH-cytochrome b, reductase of rat liver microsomes was purified according to the method of Take:i Kuma, preparation.
F., and Masters,
B. S. S., manuscript
in
METHEMOGLOBIN
REDUCTION
601
sue and Omura (11). The purified enzyme was homogeneous as judged by disc gel electrophoresis. Purification of cytochrome b, from liver microsomes. The trypsin-solubilized cytochrome b, was purified according to the method of Omura and Takesue (12) from rat and pig liver microsomes to a homogeneous form. The detergent-solubilized cytochrome b,5 of rat liver microsomes was purified according to the method of Spatz and Strittmatter (13) to a specific content of 11 nmolimg by Dr. E. G. Hrycay. The cytochrome b, was devoid of NADHcytochrome c reductase and NADH-ferricyanide reductase activities. Preparation of methemoglobin. Methemoglobin was prepared from fresh hemolysates of human erythrocytes by oxidation with potassium ferricyanide and chromatography through a column of Sephadex G-75 equilibrated with 0.1 M sodium phosphate buffer, pH 7.0, to remove nonhemoglobin proteins and excess ferricyanide. Assay of methemoglobin reductase activity. The ferricyanide reductase activity of the enzyme was measured in a l-ml reaction mixture containing 1 x 10ee M potassium ferricyanide, 1 x lo-’ M NADH, and an appropriate amount of the enzyme in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was started by the addition of NADH and the rate of the oxidation of NADH was measured at 340 nm, with a coupled millimolar extinction coefficient of 6.64 for the oxido-reduction of NADH and ferricyanide.3 The cytochrome b, reductase activity of the enzyme was measured in a l-ml cuvette containing the appropriate amounts of the enzyme, cytochrome b,, and cytochrome c in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was initiated by the addition of 1 x 10mq M NADH and the rate of reduction of cytochrome c was measured at 500 nm, with a millimolar extinction coefficient of 21 (14) for the reduction of this protein. Where indicated, preimmune or immune serum which was dialyzed against 0.1 M sodium phosphate buffer, pH 7.0, substituted for an appropriate volume of the buffer. Reduction of methemoglobin. The rate of the reduction of methemoglobin was measured in a reconstituted system containing 1 x 10M4 M methemoglobin, 1 x lo-’ M NADH, 700 units of catalase, and appropriate amounts of homogeneous methemoglobin reductase and erythrocyte cytochrome b, in 1 ml of oxygen-saturated, 0.1 M sodium phosphate buffer, pH 7.0. The increase in absorbance at 577 nm was measured upon the addition of NADH, and a millimolar extinction coefficient of 11.8 for the reduction of methemoglobin to oxyhemoglobin was used. Simultaneous determination of the rate of reduction of cytochrome c and cytochrome b,. This was carried out in a l-ml reaction mixture containing appropriate amounts of methemoglobin reductase, cytochrome b,, and cytochrome c in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was initiated
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PROUGH
by the addition of 1 x lo-” M NADH and the rate of reduction of cytochrome c was measured first at 550 nm, and then the wavelength was moved quickly to 555.5 nm to measure the initial rate of the reduction of cytochrome b, after exhaustion of oxidized cytochrome c. By taking advantage of the isosbestic point of the reduced and oxidized forms of cytochrome c at 555.5 nm, a 0.5-nm shift from the a-peak of the reduced cytochrome b,, the rates of reduction were calculated. Analytical methods. Hemoglobin concentration was measured according to the method of Drabkin (15). The concentration of hemoglobin, which has four hemes per molecule, is expressed on the basis of heme concentration in this paper. Methemoglobin concentration was measured as described by Tonz (16) according to the method of Evelyn and Malloy (17). Catalase activity was measured according to the spectrophotometric method of Luck (18). Absorption spectra. These were measured in an Aminco DW-2 dual beam, dual wavelength recording spectrophotometer or in a Cary Model 14R recording spectrophotometer. Chemicals. NADH was obtained from P-L Biochemicals. Beef liver catalase and cytochrome c were the products of Sigma Chemical Co. Other chemicals were reagent grade from commercial sources. Preparation of two antisera. Antiserum to NADH-cytochrome b, reductase was prepared by injecting adult male albino rabbits with enzyme purified by lysosomal digestion of rat liver microsomes by the method of Takesue and Omura (11) and subsequently electrophoresed on polyacrylamide gel. The area of the gel containing the flavoprotein band was excised, homogenized in 0.9% saline, and injected into rabbits according to the procedure of Hrycay and Prough (19). Cytochrome b, was purified by trypsin solubilization of rat liver microsomes by the method of Omura and Takesue (12) and injected into rabbits as described by Paltauf et al. (20). RESULTS
Effects of Antibody to Rat Liver Cytochrome b, Reductase on Methemoglobin Reductase Figure 1 shows the rate of the reduction of methemoglobin measured as a function of the concentration of erythrocyte methemoglobin reductase. The rate increased, however, approximately proportional to the enzyme concentration up to 4 x 10m9M, the range in which the inhibition experiments were carried out. The data therefore suggest that the inhibition of methemoglobin reduction observed in Fig. 2 reflects directly the inhibition of methemoglobin
AND
MASTERS
FIG. 1. The rates of the reduction of methemoglobin as a function of enzyme concentration. The rate of the reduction of methemoglobin was measured in a l-ml reaction mixture containing 1 x 10e4 M methemoglobin, 2.66 x 10m6 M erythrocyte cytochrome b,, 700 units of catalase, the indicated amount of methemoglobin reductase, and 1 x lo-’ M NADH in 0.1 M sodium phosphate buffer, pH 7.0, as described in the text.
reductase by the antiserum. The inhibition of methemoglobin reduction by the antibody was further analyzed by measuring the concentration of the reduced cytochrome b, during the steady state of the reduction of methemoglobin (Table I). The absorbance change at 556 nm during the course of methemoglobin reduction in a reconstituted system showed a biphasic increase consisting of the initial rapid increase due to the reduction of cytochrome b, and a subsequent constant increase at a slower rate due to the reduction of methemoglobin. The absorbance increase due to the reduction of cytochrome b, could be measured by the extrapolation technique of Bergmeyer (21). Thus, the initial rapid rate of reduction of cytochrome b, was obtained by substracting the absorption due to the slower rate of reduction of methemoglobin. The amount of the reduced cytochrome b, was calculated from the remaining absorbance change by using a millimolar extinction coefficient of 19 for the difference in absorbance between the reduced and oxidized forms of this protein. The rate of reduction of methemoglobin was calculated by using a millimolar extinction coefficient of 5.2 for the difference in absorbance at 556 nm between oxyhemoglobin and methemoglobin. Table I indicates that the decrease in the rate of reduction of methemoglobin came from the decreased
IMMUNOCHEMICAL
STUDIES
ON
METHEMOGLOBIN
TABLE EFFECT
OF ANTIBODY
Enzyme (X lO#M)
Antise-
rum (ml)
I
TO LYSOSOMAL-SOLUBILIZED
NADPH-CYTOCHROME LIVER MICROSOMES METHEMOGLOBIN STEADY
603
REDUCTION
b, REDLJCTASE
FROM ON A RECONSTITUTED
REDUCTASE SYSTEM DURING STATE OF THE REACTION
Methemoglobin reduction (x
Relative rate (9i;)
Cytochrome bs2+ (x 106 MI
106
RAT THE
Rate/ CYtOchrome b,”
Mimin) 4 4 4
FIG. 2. Titration of methemoglobin reductase from human erythrocytes by nonimmune and immune serum to a lysosomal-solubilized NADH-cytochrome b, reductase from rat liver microsomes. All assays were performed in 0.1 M sodium phosphate buffer, pH 7.0, in a l-ml cuvette at room temperature, and activity was expressed by the activity in the presence of immune serum relative to the activity attained without the addition of serum. The enzyme was preincubated with either immune or nonimmume serum in the cuvette for 7 min. The ferricyanide reductase activity was measured in a I-ml reaction mixture containing 1 X 10m4 M potassium ferricyanide, 4 X. 10e9 M enzyme, 1 x lo-’ M NADH and the indicated amounts of immune or nonimmune serum in the buffer as described in Materials and Methods. No inhibition of ferricyanide reductase activity was obtained with either immune or nonimmune serum. The cytochrome b, reductase activity was measured in a l-ml reaction mixture containing 1 x .10e5 M cytochrome c, 1.33 x 10-fi M erythrocyte cytochrome b,, 4 x 10e9 M enzyme, 1 x lo-’ M NADH, and the indicated amounts of immune or nonimmune serum in the buffer. The rates of reduction of cytochrome c and cytochrome b, were measured separately from a single reaction mixture as described in Materials and Methods. Methemoglobin reductase activity was measured in a l-ml reaction mixture containing 1 x lOmA M methemoglobin, 4 X 10ms M methemoglobin reductase, 1.33 X lOwe M erythrocyte cytochrome b,, 700 units of catalase, 1 x 1O-4 M NADH, and the indicated amounts of immune serum as described in Materials and Methods. No inhibition of methemoglobin reductase activity was obtained with nonimmune globulin. The reductions of (0-O) cytochrome c; (&--A) cytochrome b,, (O-01 ferricyanide, and (-@) methemoglobin are shown. The dashed lines t---l indicate nonimmune serum titrations.
0 0.1
0.2
2.1 1.6 1.4
100 78 69
1.3
1.6
1.0
1.6
0.9
1.6
fl The reaction mixture contained in 1 ml, 1 x lo-” M methemoglobin, 4 x 10m9 M methemoglobin reductase, 2.66 x lo-” M erythrocyte cytochrome b,, 700 units of catalase, the indicated amounts of antiserum, and 1 x 10m4 M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The rate of increase in absorbance at 556 nm was measured at 0.1 A full scale in an Aminco DW-2 dual beam, dual wavelength spectrophotometer. Data were analyzed as described in the text.
level of reduced cytochrome b, during the steady state of methemoglobin reduction. The rate of methemoglobin reduction per reduced cytochrome b, remained constant regardless of the addition of the antibody. These results can be interpreted to mean that the inhibition was due to the decreased rate of cytochrome b, reduction and that electron transport between the reduced cytochrome b, and methemoglobin was not affect-4 by the antibody to microsoma1 cytochrome b, reductase from rat liver. Figure 2 shows the effects of an antibody to rat liver microsomal cytochrome b, reductase on methemoglobin recluctase measured by its effects on the catalytic activity of the enzyme with potassium ferricyanicle and erythrocyte cytochrome b, as the substrates. It is clearly seen from the simultaneous determination of the rate of the reduction of cytochrome b, and cytochrome c that an antibody from rabbit injected with the homogeneous NADH-cytochrome b, recluctase of rat liver microsomes inhibits the reduction of both erythrocyte cytochrome b, and cytochrome c in the same way. This shows that the inhibition of the reduction of cytochrome c represents the inhibition of the reduction of erythrocyte
604
KUMA,
PROUGH
cytochrome b, in agreement with our previous observation that the rate of the reduction of cytochrome c strictly represents the rate of the reduction of erythrocyte cytochrome b, by the flavoprotein reductase.3 The ferricyanide reductase activity of the enzyme was not inhibited at all by the preincubation with antiserum or nonimmune serum. Since only the reduction of cytochrome b, by the enzyme was inhibited and that of ferricyanide was not inhibited by an antibody under the same experimental conditions, it is indicated that the active center of methemoglobin reductase which is involved in the interaction with erythrocyte cytochrome b, is affected, either directly or indirectly, by the antiserum to NADH-cytochrome b, reductase of rat liver microsomes. On the contrary, this experiment also indicates that the reduction of methemoglobin reductase by NADH is not affected by antibody. It should be noted, however, that the rate of the reduction of ferricyanide was measured at saturating ferricyanide concentration, whereas that of cytochrome b, reduction was measured at a concentration of one-seventy-fifth of its K, value as described in Materials and Methods. This means that the rate of reduction offerricyanide represents V, while the rate of the reduction of erythrocyte cytochrome b, is a function of V and K, values. It is concluded from the results of experiments in which inhibition of the enzyme activity was used as a criterion of interaction of methemoglobin reductase with the antibody that methemoglobin reductase of human erythrocyte is immunochemically related to NADH-cytochrome b, reductase of rat liver microsomes. The data agree with the previous observations that the molecular properties of the erythrocyte enzyme are similar to the microsomal enzyme (3, 41, and that there are cross-reactivities among the NADH-cytochrome b, reductases and cytochrome b, preparations from erythrocytes and microsomes. Figure 2 also shows the effect of an antibody to rat liver NADH-cytochrome b, reductase on a reconstituted methemoglobin reductase system consisting of the homogeneous enzyme and cytochrome b, from hu-
AND
MASTERS
man erythrocytes. It is seen that the rate of reduction of methemoglobin was inhibited by preincubation with the specific immune serum to a purified, homogeneous lysosomal-solubilized cytochrome b, reductase from rat liver microsomes to the same extent as the rate of the reduction of erythrocyte cytochrome b, was inhibited (Fig. 2). Since nonimmune serum contained detectable amounts of NADH-cytochrome b, reductase, ammonium sulfate-precipitated globulin was used in the control experiment testing the effect of nonimmune globulin and was demonstrated to have no inhibitory effect. Effect of Antibody to Rat Liver Cytochrome b, on Cytochrome b, Preparations from Various Species and Sources Interaction of purified cytochrome b, from different organs and animals with an antibody to rat liver microsomal cytochrome b, was observed by the inhibition of the enzymatic reduction of these proteins by methemoglobin reductase and NADH (Fig. 3). The inhibition by 0.2 ml of the antiserum to rat liver cytochrome b, as a function of cytochrome b, concentration showed that the degree of the inhibition was higher at the lower concentrations of erythrocyte cytochrome b,. No inhibition was seen at a 133 nM concentration of this protein (inset, Fig. 3). Thus, titrations of cytochrome b,-mediated cytochrome c reduction were performed for human erythrocyte cytochrome b, (13.3 n&, trypsin-solubilized rat liver cytochrome b, (11.2 nM), detergent-solubilized rat liver cytochrome b, (13.3 nM), and trypsin-solubilized pig liver cytochrome b, (10.5 nM) with antiserum from rabbits injected with a purified, trypsin solubilized rat liver microsomal cytochrome b,. It is seen that all cytochrome b, preparations were inhibited by the antiserum, but the degree of inhibition of erythrocyte cytochrome b,-mediated cytochrome c reduction was considerably less than the inhibition of cytochrome c reduction catalyzed by microsomal cytochrome b, preparations. The detergent-solubilized microsomal cytochrome b ,-mediated reaction was inhibited as strongly as the trypsin-solubilized hemoprotein-mediated reac-
IMMUNOCHEMICAL
STUDIES
ON
METHEMOGLOBIN
REDUCTION
605
Evidence of Cytochrome b, and Cytochrome b, Reductase in Preimmune Serum and Their Specific Inhibition in Immune Serum
FIG. 3. Inhibition of various cytochrome b, preparations by antibody to trypsin-solubilized cytochrome b, of rat liver microsomes was measured in a l-ml reaction mixture containing 4 x 16” M methemoglobin reductase, 1 x 10mfi M cytochrome c, the indicated amounts of cytochromes b,, the indicated amounts of immune and nonimmune serum, and 1 x lo+ M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The rate of the reduction of cytochrome c was measured at 550 nm and expressed as the relative activity to the activity attained without the addition of sera. (--@I, Erythrocyte cytochrome b, (13.3 nM for the complete titration curve and 26.6, 65.7, and 133 nM in the inset); (O-O), trypsin-solubilized rat liver cytochrome b, (11.2 nM); (A-A), detergent-solubilized rat liver cytochrome b, (13.3 nM); and (x--x), trypsin-solubilized pig liver cytochrome b, (10.5 nM); C---j, nonimmune serum.
tion. These data therefore indicate (i) that cytochrome b, preparations from liver microsomes of different animals are verv closely related immunochemically and ii;) that the immunochemical similarity between microsomal cytochrome b 5 and a soluble cvtochrome b, of human ervthrocvtes is less than that exhibited among liver microsomal hemoproteins from rat and pig, but it does exist. The effect of antibody to rat liver cytochrome b, on the rate of reduction of‘ methemoglobin was not amenable to study, since a higher concentration of cytochrome b, which was needed to assay the rate of the reduction of methemoglobin was not inhibited by the present antibody preparation. ”
”
”
Addition of 2.66 x 10m6 M erythrocyte cytochrome b, and 1 x 10 -4 M NADH to nonimmune serum produced &aracteristic difference spectra between the reduced and oxidized forms of cytochrome b5, while the addition of the immune serum from rabbit injected with a purified rat liver cytochrome b, reductase did not show that particular difference spectrum, suggesting the existence of NADH-cytochrome b, reductase in the nonimmune rabbit serum and its inhibition by antibody to the reductase in the immune rabbit serum. Since the rate of reduction of cytochrome b, is proportional to the enzyme concentration, the activity in the preimmune serum from rabbit was quantitated by the rate of the reduction of cytochrome b5, with methemoglobin reductase as a standard. The reaction mixture consisted of 5 X 10e6 M cytochrome c, 2.66 x lop6 M erythrocyte cytochrome b,, rabbit serum or methemoglobin reductase, and 1 x 10m4 M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The nonimmune serum or the immune serum was substituted for an appropriate volume of the buffer. As will be shown below, the nonimmune sera contained a trace of cytochrome b,-like protein, but the amount and, therefore, the contribution to the rate of reduction of cytochrome c were negligible compared to erythrocyte cytochrome ,b, added in the reaction mixture. NADHcytochrome b, reductase activities of three different nonimmune sera corresponded to 0.92, 0.92 and 0.84 nM methemoglobin reductase. On the other hand, the nonimmune serum mediated the reduction of cytochrome c by methemoglobin reductase and NADH, indicating the existence of cytochrome b,-like protein in it. On the other hand, the immune serum from rabbits injected with a purified trypsin-solubilized rat liver cytochrome b, failed to mediate electron transport between the enzyme and cytochrome c, suggesting the adsorption of the erythrocyte cytochrome b, by the antibody produced in the serum of immune rabbits. Thus the amount of cyto-
606
KUMA.
PROUGH
chrome b, in nonimmune rabbit serum was quantitated by using erythrocyte b, as a standard. The reaction mixture consisted of 5 x 10e6 M cytochrome c, 4 x 10mR M methemoglobin reductase, rabbit serum or erythrocyte cytochrome b5, and 1 x 10e4 M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The nonimmune or immune serum was substituted for an appropriate volume of the buffer. The rate was proportional to the amount of cytochrome b,, and the content of cytochrome b,-like protein in the nonimmune serum corresponded to 1.8 nM erythrocyte cytochrome b,. These data indicate (i) that trace amounts of cytochrome b, reductase and cytochrome b, derived from lysed erythrocytes or other tissue cells exist in the serum of nonimmune rabbit and (ii) that these are specifically absorbed out of the serum of immune rabbit injected with rat liver cytochrome b, reductase and cytochrome b5, respectively. DISCUSSION
Evidence has been presented in this paper that a soluble methemoglobin reductase and cytochrome b, of human erythrocytes are immunochemically similar to microsomal NADH-cytochrome b, reductase and cytochrome b, of rat liver, respectively. The results are in agreement with the reports of Kuma and Inomata (4, 9), who have purified homogeneous methemoglobin reductase and cytochrome b5 from human erythrocytes and demonstrated that the molecular properties of the enzyme and cytochrome b, are similar to those of the reductase and cytochrome b, of liver microsomes, respectively, and that both reductases can catalyze the rapid reduction of either erythrocyte cytochrome b, or microsomal cytochrome b, by NADH. The close relationship, therefore, between the methemoglobin reductase system and the microsomal cytochrome b, reductase system has been established from three different viewpoints. The physiological role of cytochrome b, of microsomes is not completely understood. The only recognized roles of this protein are its function as an electron carrier in the fatty acid desaturase system (22, 23) and in plasmalogen biosynthesis
AND
MASTERS
(20). In addition, cytochrome b, has been implicated in the synergism observed in in vitro drug metabolism reactions as catalyzed by both NADH and NADPH compared to either pyridine nucleotide alone (24, 25). Another role has been established for a soluble cytoplasmic cytochrome b, (8, 9) in the transport of electrons to methemoglobin to maintain hemoglobin in the reduced state in erythrocytes. Since NADH-methemoglobin reductase and cytochrome b, exist freely soluble in the cytoplasm of erythrocytes where, being different from microsomal proteins, no organization has yet been recognized, the enzyme seems to catalyze the rapid reduction of cytochrome b, in the presence of NADH. The redox state of NADH-NAD, therefore, should be represented by that of cytochrome b,, and so this protein which is originally an electron carrier, seems also to act as a distributor of the oxidationreduction potential in the erythrocyte cytoplasm. The redox state of hemoglobin in erythrocytes is, therefore, one of the expressions of the redox state of the cytoplasm, where more than 99% of hemoglobin is in the reduced state. It is suspected that some mechanism should exist to protect the removal of electrons from free cytochrome b, in the erythrocyte cytoplasm. It has been observed that about half of the electrons were auto-oxidized from cytochrome b, in a reconstituted methemoglobin reductase system under aerobic conditions, which may be the case in erythrocytes. It is interesting to know whether methemoglobin reductase is responsible only for the reduction of erythrocyte cytochrome b, or whether it is also related to electron transport between the reduced cytochrome b, and methemoglobin. It has been shown in this paper that the antibody to cytochrome b, reductase inhibited only the reduction of cytochrome b, by the enzyme without affecting the electron transport between cytochrome b, and methemoglobin, suggesting that the enzyme is not concerned with the second electron transfer reaction. This has also been proved by the kinetic study of the mechanism of methemoglobin reduction3
IMMUNOCHEMICAL
STUDIES
ON
It should in addition be pointed out that considerable immunochemical differences were also found between a soluble cytochrome b, of erythrocytes and microsomal cytochrome b, of rat and pig livers. Amino acid sequences of liver microsomal cytochrome 6, from human, monkey, calf, pig, rabbit, and chicken have been reported by Tsugita et al. (261, and Nobrega and 0~01s (27) to be similar among mammals. In addition, Hultquist et al. (8) suggested the similarity of the structures of erythrocyte cytochrome b, and liver microsomal cytochrome b, of human from the electrophoretie mobility of trypsin-digested fragments of these proteins. The present results cannot be interpreted directly in the light of these reports. The difference in the primary structure could be the reason for the functional and immunochemical differences observed, but it might also be possible that sol.ubilization procedures, either tryptic digestion or detergent solubilization, could cause unique effects on the tertiary structure of the resulting solubilized microsomal protein and on its properties, including functional activities and interaction with antibody, whereas erythrocyte cytochrome b, is a soluble protein and retains its native properties. However, the recognition by antibodies to proteolytically solubilized cytochrome b, and NADH-cytochrome b, reductase of the respective microsome-bound antigens suggests that gross changes in tertiary structure of the solubilized antigens have not taken place. Finally, the evolution and/or differentiation of such related flavoproteins and hemoproteins is of real interest. The fact that similar proteins exist within the same species in different tissues in both membranebound and soluble forms is significant to those interested in the biogenesis of membrane systems. REFERENCES B&hem. J. 42, 13-23. 2. SCOTT, E. M., AND MCGRAW, J. C. (1962)J. Biol. Chem. 23’7, 249-252. 3. KUMA, F., HIRAYAMA, K., ISHIZAWA, S., AND NAKAJIMA, H. (1972) J. Biol. Chem. 247,5501. GIBSON,
Q. H. (1948)
METHEMOGLOBIN
REDUCTION
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555. F., AND INOMATA, H. (1972) J. Biol. 4. KUMA, Chem. 247, 556-560. P., AND VELICK, S. F. (1957) J. 5. STRITTMATTER, Biol. Chem. 228. 785-799. 6. SUGITA, Y., NOMURA, S., AND YONEYAMA, Y. (1971) J. Biol. Chem. 246. 6072-6078. 7. ADACHI, K. 11972) Biochim. Biophys. Acta 289, 262-268. 8. HULTQUIST, D. E., DEAN, R. T., AND DOUGLAS, R. H. (1974)Biochem. Biophys. Res. Commun. 60, 28-34. 9. KUMA, F. (1974) Fed. Proc. 33. 1370. 10. BEINERT, H. (1960) in The Enzymes (Bayer, P. D., ed.), 2nd ed., Vol. 2, p. 339, Academic Press, New York. 11. TAKESUE, S., AND OMLJRA, T. (1970)5. B&hem. (Tokyo) 67, 267-276. 12. OMURA, T., AND TAKESUE, S. (1970) J. Biochem. (Tokyo) 67, 249-257. 13. SPATZ, L., AND STRITTMATTER, P. (1971) Proc. Nut. Acad. Sci. USA 68, 1042-1046. 14. MASSEY, V. (1959) Biochim. Biophys. Acta 34, 255-256. D. L. (1932) J. Biol. Chem. 98, 71915. DRABKIN, 733. 16. T~Nz, 0. (1968)BibI. Haematol. No. 28.47-49. 17. EVELYN, K. A., AND MALLOY, H. T. (19381 J. Biol. Chem. 126, 655-662. 18. LUCK, H. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.1, pp. 885-894, Verlag Chemie and Academic Press, New York. 19. HRYCAY, E. G., AND PROUGH, R. A. (1974)Arch. Biochem. Biophys. 165, 331-339. 20. PALTAUF, F., PROUGH, R. A., MASTERS, B. S. S., AND JOHNSON, J. M. (1974) J. Biol. Chem. 249, 2661-2662. 21. BERGMEYER, H. U. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 14-42, Verlag Chemie, Weinheim; Academic Press, New York. 22. OSHINO, N., AND OMURA, T. (1973) Arch. Biothem. Biophys. 157, 395-404. 23. OSHINO, N., IMAI, Y., AND SATO, R. (1971) J. Biochem. (Tokyo) 69, 155-167. 24. HILDEBRANDT, A., AND ESTABROOK, R. W. (1971) Arch. Biochem. Biophys. 143, 66-79. 25. MANNERING, G. J., KUWAKARA, S., AND OMURA, T. (1974) Biochem. Biophys. Res. Commun. 57, 476-481. 26. TSUGITA, A., KOBAYASHI, M., TANI, S., KYO, S., RASHID, M. A., YOSHIDA, Y., KAJIHARA, Y., AND HAGIHARA, B. (1970) Proc. Nat. Acad. Sci. USA 67,442-447. 27. NOBREGA, F. G., AND OZOLS, J. (1971) J. Biol. Chem. 246, 1706-1717.
OP
BIOCHEMISTRY
AND
BIOPHYSICS
Studies
172, 600-607
(1976)
on Methemoglobin
Reductase
lmmunochemical Similarity of Soluble Methemoglobin b, of Human Erythrocytes with NADH-Cytochromeb, of Rat Liver Microsome FUMIO Department
KUMA,2
RUSSELL
A. PROUGH,
OfBiochemistry,
The University School, 5323 Harry
AND
of Texas Health Hines Boulevard, Received
June
Reductase and Cytochrome Reductase and Cytochromeb,
BETTIE
SUE SILER
Science Center at Dallas, Dallas, Texas 75235
MASTERS
Southwestern
Medical
30, 1975
An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b, reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b, reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrome b, to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b, from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b, during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b, stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b, and methemoglobin. An antibody to purified, trypsin-solubilized cytochrome b, from rat liver microsomes was shown to inhibit erythrocyte cytochrome b, reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome 6, preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b, of human erythrocytes are immunochemically similar to NADH-cytochrome b, reductase and cytochrome b, of liver microsomes, respectively.
Since Gibson (1) reported the deficiency of an enzyme in erythrocytes from patients with congenital methemoglobinemia, the mechanism of reductioriof methemoglobin has been the subject of much investigation. Scott and McGraw demonstrated that the NADH-diaphorase activity in erythro-
cytes was responsible for the reduction of methemoglobin (2). This enzyme, methemoglobin reductase, has been purified from human erythrocytes by Kuma et al. (3, 41, who have demonstrated that the enzyme is a flavoprotein, having flavinadenine dinucleotide as a prosthetic group, which does not reduce methemoglobin directly but requires a cofactor to transport electrons from the reduced enzyme to methemoglobin. The similarity of its molecular properties with those of NADH-cytochrome b, reductase of liver microsomes, reported by Strittmatter and Velick (51, has been documented (3, 4). On the other hand, the nonflavoprotein nature of this enzymatic activity was reported by Sugita et al. (6) and Adachi (7), although the addi-
I This research was supported by U.S. Public Health Service Grants No. GM16488 and HL13619 (B.S.S.M.) and American Cancer Society Grant No. BC-153 (R.A.P.). Reprint requests should be addressed to Dr. B. S. S. Masters, Associate Professor of Biochemistry, The University of Texas Health Science Center at Dallas, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Tex. 75235. p Present address: Karatsu Saiseikai Hospital, Motohata-machi, Karatsu, Saga, Japan. 600 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
IMMUNOCHEMICAL
STUDIES
ON
tion of microsomal cytochrome b, increased the rate of methemoglobin reduction by their enzymes. More recently, a soluble cytochrome b, was isolated from human erythrocytes (8, 91, and Hultquist et al. (8) suggested the microsomal origin of this protein by comparing the trypsindigested cytochrome b, of erythrocytes with trypsin solubilized cytochrome b, from human liver microsomes. The purification and characterization of cytochrome b, from human erythrocytes as a cofactor of the methemoglobin reductase system have been reported by Kuma (91, the crossreactivity of human erythrocyte flavoprotein and hemoprotein with liver microsoma1 NADH-cytochrome b, reductase and cytochrome b, have been examined, and the mechanism of methemoglobin reduction in a reconstituted system has been studied.” The present study was undertaken to determine if immunochemical identity or similarity exists between the components of the methemoglobin reductase system of erythrocytes and the microsomal cytochrome b, reductase system. MATERIALS
AND
METHODS
Purification of methemoglobin reductase. Methemoglobin reductase was purified from human erythrocytes according to the method of Kuma and Inomata (4). The enzyme was homogeneous as judged by discontinuous (disc) gel electrophoresis at pH 8.9, and the absorption spectrum of the enzyme revealed no contamination by heme protein. The concentration of the enzyme was estimated from the absorbance at 460 nm with a millimolar extinction coefficient of 11.3, as determined for flavin-adenine dinucleotide by Beinert (10). Purification oferythrocyte cytochrome b,. Erythrocyte cytochrome b s was purified from human erythrocytes according to the method of Kuma (9). The procedure resulted in a homogeneous protein as judged by disc electrophoresis on 15% polyacrylamide gel at pH 8.9. The concentration of the protein was determined from the difference in absorbance between 556 and 575 nm of the reduced and oxidized forms of the protein, with the millimolar extinction coefficient of 24 (9). Purification of NADHxytochrome b, reductase from microsomes. The lysosomal-solubilized NADH-cytochrome b, reductase of rat liver microsomes was purified according to the method of Take:i Kuma, preparation.
F., and Masters,
B. S. S., manuscript
in
METHEMOGLOBIN
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sue and Omura (11). The purified enzyme was homogeneous as judged by disc gel electrophoresis. Purification of cytochrome b, from liver microsomes. The trypsin-solubilized cytochrome b, was purified according to the method of Omura and Takesue (12) from rat and pig liver microsomes to a homogeneous form. The detergent-solubilized cytochrome b,5 of rat liver microsomes was purified according to the method of Spatz and Strittmatter (13) to a specific content of 11 nmolimg by Dr. E. G. Hrycay. The cytochrome b, was devoid of NADHcytochrome c reductase and NADH-ferricyanide reductase activities. Preparation of methemoglobin. Methemoglobin was prepared from fresh hemolysates of human erythrocytes by oxidation with potassium ferricyanide and chromatography through a column of Sephadex G-75 equilibrated with 0.1 M sodium phosphate buffer, pH 7.0, to remove nonhemoglobin proteins and excess ferricyanide. Assay of methemoglobin reductase activity. The ferricyanide reductase activity of the enzyme was measured in a l-ml reaction mixture containing 1 x 10ee M potassium ferricyanide, 1 x lo-’ M NADH, and an appropriate amount of the enzyme in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was started by the addition of NADH and the rate of the oxidation of NADH was measured at 340 nm, with a coupled millimolar extinction coefficient of 6.64 for the oxido-reduction of NADH and ferricyanide.3 The cytochrome b, reductase activity of the enzyme was measured in a l-ml cuvette containing the appropriate amounts of the enzyme, cytochrome b,, and cytochrome c in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was initiated by the addition of 1 x 10mq M NADH and the rate of reduction of cytochrome c was measured at 500 nm, with a millimolar extinction coefficient of 21 (14) for the reduction of this protein. Where indicated, preimmune or immune serum which was dialyzed against 0.1 M sodium phosphate buffer, pH 7.0, substituted for an appropriate volume of the buffer. Reduction of methemoglobin. The rate of the reduction of methemoglobin was measured in a reconstituted system containing 1 x 10M4 M methemoglobin, 1 x lo-’ M NADH, 700 units of catalase, and appropriate amounts of homogeneous methemoglobin reductase and erythrocyte cytochrome b, in 1 ml of oxygen-saturated, 0.1 M sodium phosphate buffer, pH 7.0. The increase in absorbance at 577 nm was measured upon the addition of NADH, and a millimolar extinction coefficient of 11.8 for the reduction of methemoglobin to oxyhemoglobin was used. Simultaneous determination of the rate of reduction of cytochrome c and cytochrome b,. This was carried out in a l-ml reaction mixture containing appropriate amounts of methemoglobin reductase, cytochrome b,, and cytochrome c in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was initiated
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by the addition of 1 x lo-” M NADH and the rate of reduction of cytochrome c was measured first at 550 nm, and then the wavelength was moved quickly to 555.5 nm to measure the initial rate of the reduction of cytochrome b, after exhaustion of oxidized cytochrome c. By taking advantage of the isosbestic point of the reduced and oxidized forms of cytochrome c at 555.5 nm, a 0.5-nm shift from the a-peak of the reduced cytochrome b,, the rates of reduction were calculated. Analytical methods. Hemoglobin concentration was measured according to the method of Drabkin (15). The concentration of hemoglobin, which has four hemes per molecule, is expressed on the basis of heme concentration in this paper. Methemoglobin concentration was measured as described by Tonz (16) according to the method of Evelyn and Malloy (17). Catalase activity was measured according to the spectrophotometric method of Luck (18). Absorption spectra. These were measured in an Aminco DW-2 dual beam, dual wavelength recording spectrophotometer or in a Cary Model 14R recording spectrophotometer. Chemicals. NADH was obtained from P-L Biochemicals. Beef liver catalase and cytochrome c were the products of Sigma Chemical Co. Other chemicals were reagent grade from commercial sources. Preparation of two antisera. Antiserum to NADH-cytochrome b, reductase was prepared by injecting adult male albino rabbits with enzyme purified by lysosomal digestion of rat liver microsomes by the method of Takesue and Omura (11) and subsequently electrophoresed on polyacrylamide gel. The area of the gel containing the flavoprotein band was excised, homogenized in 0.9% saline, and injected into rabbits according to the procedure of Hrycay and Prough (19). Cytochrome b, was purified by trypsin solubilization of rat liver microsomes by the method of Omura and Takesue (12) and injected into rabbits as described by Paltauf et al. (20). RESULTS
Effects of Antibody to Rat Liver Cytochrome b, Reductase on Methemoglobin Reductase Figure 1 shows the rate of the reduction of methemoglobin measured as a function of the concentration of erythrocyte methemoglobin reductase. The rate increased, however, approximately proportional to the enzyme concentration up to 4 x 10m9M, the range in which the inhibition experiments were carried out. The data therefore suggest that the inhibition of methemoglobin reduction observed in Fig. 2 reflects directly the inhibition of methemoglobin
AND
MASTERS
FIG. 1. The rates of the reduction of methemoglobin as a function of enzyme concentration. The rate of the reduction of methemoglobin was measured in a l-ml reaction mixture containing 1 x 10e4 M methemoglobin, 2.66 x 10m6 M erythrocyte cytochrome b,, 700 units of catalase, the indicated amount of methemoglobin reductase, and 1 x lo-’ M NADH in 0.1 M sodium phosphate buffer, pH 7.0, as described in the text.
reductase by the antiserum. The inhibition of methemoglobin reduction by the antibody was further analyzed by measuring the concentration of the reduced cytochrome b, during the steady state of the reduction of methemoglobin (Table I). The absorbance change at 556 nm during the course of methemoglobin reduction in a reconstituted system showed a biphasic increase consisting of the initial rapid increase due to the reduction of cytochrome b, and a subsequent constant increase at a slower rate due to the reduction of methemoglobin. The absorbance increase due to the reduction of cytochrome b, could be measured by the extrapolation technique of Bergmeyer (21). Thus, the initial rapid rate of reduction of cytochrome b, was obtained by substracting the absorption due to the slower rate of reduction of methemoglobin. The amount of the reduced cytochrome b, was calculated from the remaining absorbance change by using a millimolar extinction coefficient of 19 for the difference in absorbance between the reduced and oxidized forms of this protein. The rate of reduction of methemoglobin was calculated by using a millimolar extinction coefficient of 5.2 for the difference in absorbance at 556 nm between oxyhemoglobin and methemoglobin. Table I indicates that the decrease in the rate of reduction of methemoglobin came from the decreased
IMMUNOCHEMICAL
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ON
METHEMOGLOBIN
TABLE EFFECT
OF ANTIBODY
Enzyme (X lO#M)
Antise-
rum (ml)
I
TO LYSOSOMAL-SOLUBILIZED
NADPH-CYTOCHROME LIVER MICROSOMES METHEMOGLOBIN STEADY
603
REDUCTION
b, REDLJCTASE
FROM ON A RECONSTITUTED
REDUCTASE SYSTEM DURING STATE OF THE REACTION
Methemoglobin reduction (x
Relative rate (9i;)
Cytochrome bs2+ (x 106 MI
106
RAT THE
Rate/ CYtOchrome b,”
Mimin) 4 4 4
FIG. 2. Titration of methemoglobin reductase from human erythrocytes by nonimmune and immune serum to a lysosomal-solubilized NADH-cytochrome b, reductase from rat liver microsomes. All assays were performed in 0.1 M sodium phosphate buffer, pH 7.0, in a l-ml cuvette at room temperature, and activity was expressed by the activity in the presence of immune serum relative to the activity attained without the addition of serum. The enzyme was preincubated with either immune or nonimmume serum in the cuvette for 7 min. The ferricyanide reductase activity was measured in a I-ml reaction mixture containing 1 X 10m4 M potassium ferricyanide, 4 X. 10e9 M enzyme, 1 x lo-’ M NADH and the indicated amounts of immune or nonimmune serum in the buffer as described in Materials and Methods. No inhibition of ferricyanide reductase activity was obtained with either immune or nonimmune serum. The cytochrome b, reductase activity was measured in a l-ml reaction mixture containing 1 x .10e5 M cytochrome c, 1.33 x 10-fi M erythrocyte cytochrome b,, 4 x 10e9 M enzyme, 1 x lo-’ M NADH, and the indicated amounts of immune or nonimmune serum in the buffer. The rates of reduction of cytochrome c and cytochrome b, were measured separately from a single reaction mixture as described in Materials and Methods. Methemoglobin reductase activity was measured in a l-ml reaction mixture containing 1 x lOmA M methemoglobin, 4 X 10ms M methemoglobin reductase, 1.33 X lOwe M erythrocyte cytochrome b,, 700 units of catalase, 1 x 1O-4 M NADH, and the indicated amounts of immune serum as described in Materials and Methods. No inhibition of methemoglobin reductase activity was obtained with nonimmune globulin. The reductions of (0-O) cytochrome c; (&--A) cytochrome b,, (O-01 ferricyanide, and (-@) methemoglobin are shown. The dashed lines t---l indicate nonimmune serum titrations.
0 0.1
0.2
2.1 1.6 1.4
100 78 69
1.3
1.6
1.0
1.6
0.9
1.6
fl The reaction mixture contained in 1 ml, 1 x lo-” M methemoglobin, 4 x 10m9 M methemoglobin reductase, 2.66 x lo-” M erythrocyte cytochrome b,, 700 units of catalase, the indicated amounts of antiserum, and 1 x 10m4 M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The rate of increase in absorbance at 556 nm was measured at 0.1 A full scale in an Aminco DW-2 dual beam, dual wavelength spectrophotometer. Data were analyzed as described in the text.
level of reduced cytochrome b, during the steady state of methemoglobin reduction. The rate of methemoglobin reduction per reduced cytochrome b, remained constant regardless of the addition of the antibody. These results can be interpreted to mean that the inhibition was due to the decreased rate of cytochrome b, reduction and that electron transport between the reduced cytochrome b, and methemoglobin was not affect-4 by the antibody to microsoma1 cytochrome b, reductase from rat liver. Figure 2 shows the effects of an antibody to rat liver microsomal cytochrome b, reductase on methemoglobin recluctase measured by its effects on the catalytic activity of the enzyme with potassium ferricyanicle and erythrocyte cytochrome b, as the substrates. It is clearly seen from the simultaneous determination of the rate of the reduction of cytochrome b, and cytochrome c that an antibody from rabbit injected with the homogeneous NADH-cytochrome b, recluctase of rat liver microsomes inhibits the reduction of both erythrocyte cytochrome b, and cytochrome c in the same way. This shows that the inhibition of the reduction of cytochrome c represents the inhibition of the reduction of erythrocyte
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cytochrome b, in agreement with our previous observation that the rate of the reduction of cytochrome c strictly represents the rate of the reduction of erythrocyte cytochrome b, by the flavoprotein reductase.3 The ferricyanide reductase activity of the enzyme was not inhibited at all by the preincubation with antiserum or nonimmune serum. Since only the reduction of cytochrome b, by the enzyme was inhibited and that of ferricyanide was not inhibited by an antibody under the same experimental conditions, it is indicated that the active center of methemoglobin reductase which is involved in the interaction with erythrocyte cytochrome b, is affected, either directly or indirectly, by the antiserum to NADH-cytochrome b, reductase of rat liver microsomes. On the contrary, this experiment also indicates that the reduction of methemoglobin reductase by NADH is not affected by antibody. It should be noted, however, that the rate of the reduction of ferricyanide was measured at saturating ferricyanide concentration, whereas that of cytochrome b, reduction was measured at a concentration of one-seventy-fifth of its K, value as described in Materials and Methods. This means that the rate of reduction offerricyanide represents V, while the rate of the reduction of erythrocyte cytochrome b, is a function of V and K, values. It is concluded from the results of experiments in which inhibition of the enzyme activity was used as a criterion of interaction of methemoglobin reductase with the antibody that methemoglobin reductase of human erythrocyte is immunochemically related to NADH-cytochrome b, reductase of rat liver microsomes. The data agree with the previous observations that the molecular properties of the erythrocyte enzyme are similar to the microsomal enzyme (3, 41, and that there are cross-reactivities among the NADH-cytochrome b, reductases and cytochrome b, preparations from erythrocytes and microsomes. Figure 2 also shows the effect of an antibody to rat liver NADH-cytochrome b, reductase on a reconstituted methemoglobin reductase system consisting of the homogeneous enzyme and cytochrome b, from hu-
AND
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man erythrocytes. It is seen that the rate of reduction of methemoglobin was inhibited by preincubation with the specific immune serum to a purified, homogeneous lysosomal-solubilized cytochrome b, reductase from rat liver microsomes to the same extent as the rate of the reduction of erythrocyte cytochrome b, was inhibited (Fig. 2). Since nonimmune serum contained detectable amounts of NADH-cytochrome b, reductase, ammonium sulfate-precipitated globulin was used in the control experiment testing the effect of nonimmune globulin and was demonstrated to have no inhibitory effect. Effect of Antibody to Rat Liver Cytochrome b, on Cytochrome b, Preparations from Various Species and Sources Interaction of purified cytochrome b, from different organs and animals with an antibody to rat liver microsomal cytochrome b, was observed by the inhibition of the enzymatic reduction of these proteins by methemoglobin reductase and NADH (Fig. 3). The inhibition by 0.2 ml of the antiserum to rat liver cytochrome b, as a function of cytochrome b, concentration showed that the degree of the inhibition was higher at the lower concentrations of erythrocyte cytochrome b,. No inhibition was seen at a 133 nM concentration of this protein (inset, Fig. 3). Thus, titrations of cytochrome b,-mediated cytochrome c reduction were performed for human erythrocyte cytochrome b, (13.3 n&, trypsin-solubilized rat liver cytochrome b, (11.2 nM), detergent-solubilized rat liver cytochrome b, (13.3 nM), and trypsin-solubilized pig liver cytochrome b, (10.5 nM) with antiserum from rabbits injected with a purified, trypsin solubilized rat liver microsomal cytochrome b,. It is seen that all cytochrome b, preparations were inhibited by the antiserum, but the degree of inhibition of erythrocyte cytochrome b,-mediated cytochrome c reduction was considerably less than the inhibition of cytochrome c reduction catalyzed by microsomal cytochrome b, preparations. The detergent-solubilized microsomal cytochrome b ,-mediated reaction was inhibited as strongly as the trypsin-solubilized hemoprotein-mediated reac-
IMMUNOCHEMICAL
STUDIES
ON
METHEMOGLOBIN
REDUCTION
605
Evidence of Cytochrome b, and Cytochrome b, Reductase in Preimmune Serum and Their Specific Inhibition in Immune Serum
FIG. 3. Inhibition of various cytochrome b, preparations by antibody to trypsin-solubilized cytochrome b, of rat liver microsomes was measured in a l-ml reaction mixture containing 4 x 16” M methemoglobin reductase, 1 x 10mfi M cytochrome c, the indicated amounts of cytochromes b,, the indicated amounts of immune and nonimmune serum, and 1 x lo+ M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The rate of the reduction of cytochrome c was measured at 550 nm and expressed as the relative activity to the activity attained without the addition of sera. (--@I, Erythrocyte cytochrome b, (13.3 nM for the complete titration curve and 26.6, 65.7, and 133 nM in the inset); (O-O), trypsin-solubilized rat liver cytochrome b, (11.2 nM); (A-A), detergent-solubilized rat liver cytochrome b, (13.3 nM); and (x--x), trypsin-solubilized pig liver cytochrome b, (10.5 nM); C---j, nonimmune serum.
tion. These data therefore indicate (i) that cytochrome b, preparations from liver microsomes of different animals are verv closely related immunochemically and ii;) that the immunochemical similarity between microsomal cytochrome b 5 and a soluble cvtochrome b, of human ervthrocvtes is less than that exhibited among liver microsomal hemoproteins from rat and pig, but it does exist. The effect of antibody to rat liver cytochrome b, on the rate of reduction of‘ methemoglobin was not amenable to study, since a higher concentration of cytochrome b, which was needed to assay the rate of the reduction of methemoglobin was not inhibited by the present antibody preparation. ”
”
”
Addition of 2.66 x 10m6 M erythrocyte cytochrome b, and 1 x 10 -4 M NADH to nonimmune serum produced &aracteristic difference spectra between the reduced and oxidized forms of cytochrome b5, while the addition of the immune serum from rabbit injected with a purified rat liver cytochrome b, reductase did not show that particular difference spectrum, suggesting the existence of NADH-cytochrome b, reductase in the nonimmune rabbit serum and its inhibition by antibody to the reductase in the immune rabbit serum. Since the rate of reduction of cytochrome b, is proportional to the enzyme concentration, the activity in the preimmune serum from rabbit was quantitated by the rate of the reduction of cytochrome b5, with methemoglobin reductase as a standard. The reaction mixture consisted of 5 X 10e6 M cytochrome c, 2.66 x lop6 M erythrocyte cytochrome b,, rabbit serum or methemoglobin reductase, and 1 x 10m4 M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The nonimmune serum or the immune serum was substituted for an appropriate volume of the buffer. As will be shown below, the nonimmune sera contained a trace of cytochrome b,-like protein, but the amount and, therefore, the contribution to the rate of reduction of cytochrome c were negligible compared to erythrocyte cytochrome ,b, added in the reaction mixture. NADHcytochrome b, reductase activities of three different nonimmune sera corresponded to 0.92, 0.92 and 0.84 nM methemoglobin reductase. On the other hand, the nonimmune serum mediated the reduction of cytochrome c by methemoglobin reductase and NADH, indicating the existence of cytochrome b,-like protein in it. On the other hand, the immune serum from rabbits injected with a purified trypsin-solubilized rat liver cytochrome b, failed to mediate electron transport between the enzyme and cytochrome c, suggesting the adsorption of the erythrocyte cytochrome b, by the antibody produced in the serum of immune rabbits. Thus the amount of cyto-
606
KUMA.
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chrome b, in nonimmune rabbit serum was quantitated by using erythrocyte b, as a standard. The reaction mixture consisted of 5 x 10e6 M cytochrome c, 4 x 10mR M methemoglobin reductase, rabbit serum or erythrocyte cytochrome b5, and 1 x 10e4 M NADH in 0.1 M sodium phosphate buffer, pH 7.0. The nonimmune or immune serum was substituted for an appropriate volume of the buffer. The rate was proportional to the amount of cytochrome b,, and the content of cytochrome b,-like protein in the nonimmune serum corresponded to 1.8 nM erythrocyte cytochrome b,. These data indicate (i) that trace amounts of cytochrome b, reductase and cytochrome b, derived from lysed erythrocytes or other tissue cells exist in the serum of nonimmune rabbit and (ii) that these are specifically absorbed out of the serum of immune rabbit injected with rat liver cytochrome b, reductase and cytochrome b5, respectively. DISCUSSION
Evidence has been presented in this paper that a soluble methemoglobin reductase and cytochrome b, of human erythrocytes are immunochemically similar to microsomal NADH-cytochrome b, reductase and cytochrome b, of rat liver, respectively. The results are in agreement with the reports of Kuma and Inomata (4, 9), who have purified homogeneous methemoglobin reductase and cytochrome b5 from human erythrocytes and demonstrated that the molecular properties of the enzyme and cytochrome b, are similar to those of the reductase and cytochrome b, of liver microsomes, respectively, and that both reductases can catalyze the rapid reduction of either erythrocyte cytochrome b, or microsomal cytochrome b, by NADH. The close relationship, therefore, between the methemoglobin reductase system and the microsomal cytochrome b, reductase system has been established from three different viewpoints. The physiological role of cytochrome b, of microsomes is not completely understood. The only recognized roles of this protein are its function as an electron carrier in the fatty acid desaturase system (22, 23) and in plasmalogen biosynthesis
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MASTERS
(20). In addition, cytochrome b, has been implicated in the synergism observed in in vitro drug metabolism reactions as catalyzed by both NADH and NADPH compared to either pyridine nucleotide alone (24, 25). Another role has been established for a soluble cytoplasmic cytochrome b, (8, 9) in the transport of electrons to methemoglobin to maintain hemoglobin in the reduced state in erythrocytes. Since NADH-methemoglobin reductase and cytochrome b, exist freely soluble in the cytoplasm of erythrocytes where, being different from microsomal proteins, no organization has yet been recognized, the enzyme seems to catalyze the rapid reduction of cytochrome b, in the presence of NADH. The redox state of NADH-NAD, therefore, should be represented by that of cytochrome b,, and so this protein which is originally an electron carrier, seems also to act as a distributor of the oxidationreduction potential in the erythrocyte cytoplasm. The redox state of hemoglobin in erythrocytes is, therefore, one of the expressions of the redox state of the cytoplasm, where more than 99% of hemoglobin is in the reduced state. It is suspected that some mechanism should exist to protect the removal of electrons from free cytochrome b, in the erythrocyte cytoplasm. It has been observed that about half of the electrons were auto-oxidized from cytochrome b, in a reconstituted methemoglobin reductase system under aerobic conditions, which may be the case in erythrocytes. It is interesting to know whether methemoglobin reductase is responsible only for the reduction of erythrocyte cytochrome b, or whether it is also related to electron transport between the reduced cytochrome b, and methemoglobin. It has been shown in this paper that the antibody to cytochrome b, reductase inhibited only the reduction of cytochrome b, by the enzyme without affecting the electron transport between cytochrome b, and methemoglobin, suggesting that the enzyme is not concerned with the second electron transfer reaction. This has also been proved by the kinetic study of the mechanism of methemoglobin reduction3
IMMUNOCHEMICAL
STUDIES
ON
It should in addition be pointed out that considerable immunochemical differences were also found between a soluble cytochrome b, of erythrocytes and microsomal cytochrome b, of rat and pig livers. Amino acid sequences of liver microsomal cytochrome 6, from human, monkey, calf, pig, rabbit, and chicken have been reported by Tsugita et al. (261, and Nobrega and 0~01s (27) to be similar among mammals. In addition, Hultquist et al. (8) suggested the similarity of the structures of erythrocyte cytochrome b, and liver microsomal cytochrome b, of human from the electrophoretie mobility of trypsin-digested fragments of these proteins. The present results cannot be interpreted directly in the light of these reports. The difference in the primary structure could be the reason for the functional and immunochemical differences observed, but it might also be possible that sol.ubilization procedures, either tryptic digestion or detergent solubilization, could cause unique effects on the tertiary structure of the resulting solubilized microsomal protein and on its properties, including functional activities and interaction with antibody, whereas erythrocyte cytochrome b, is a soluble protein and retains its native properties. However, the recognition by antibodies to proteolytically solubilized cytochrome b, and NADH-cytochrome b, reductase of the respective microsome-bound antigens suggests that gross changes in tertiary structure of the solubilized antigens have not taken place. Finally, the evolution and/or differentiation of such related flavoproteins and hemoproteins is of real interest. The fact that similar proteins exist within the same species in different tissues in both membranebound and soluble forms is significant to those interested in the biogenesis of membrane systems. REFERENCES B&hem. J. 42, 13-23. 2. SCOTT, E. M., AND MCGRAW, J. C. (1962)J. Biol. Chem. 23’7, 249-252. 3. KUMA, F., HIRAYAMA, K., ISHIZAWA, S., AND NAKAJIMA, H. (1972) J. Biol. Chem. 247,5501. GIBSON,
Q. H. (1948)
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555. F., AND INOMATA, H. (1972) J. Biol. 4. KUMA, Chem. 247, 556-560. P., AND VELICK, S. F. (1957) J. 5. STRITTMATTER, Biol. Chem. 228. 785-799. 6. SUGITA, Y., NOMURA, S., AND YONEYAMA, Y. (1971) J. Biol. Chem. 246. 6072-6078. 7. ADACHI, K. 11972) Biochim. Biophys. Acta 289, 262-268. 8. HULTQUIST, D. E., DEAN, R. T., AND DOUGLAS, R. H. (1974)Biochem. Biophys. Res. Commun. 60, 28-34. 9. KUMA, F. (1974) Fed. Proc. 33. 1370. 10. BEINERT, H. (1960) in The Enzymes (Bayer, P. D., ed.), 2nd ed., Vol. 2, p. 339, Academic Press, New York. 11. TAKESUE, S., AND OMLJRA, T. (1970)5. B&hem. (Tokyo) 67, 267-276. 12. OMURA, T., AND TAKESUE, S. (1970) J. Biochem. (Tokyo) 67, 249-257. 13. SPATZ, L., AND STRITTMATTER, P. (1971) Proc. Nut. Acad. Sci. USA 68, 1042-1046. 14. MASSEY, V. (1959) Biochim. Biophys. Acta 34, 255-256. D. L. (1932) J. Biol. Chem. 98, 71915. DRABKIN, 733. 16. T~Nz, 0. (1968)BibI. Haematol. No. 28.47-49. 17. EVELYN, K. A., AND MALLOY, H. T. (19381 J. Biol. Chem. 126, 655-662. 18. LUCK, H. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.1, pp. 885-894, Verlag Chemie and Academic Press, New York. 19. HRYCAY, E. G., AND PROUGH, R. A. (1974)Arch. Biochem. Biophys. 165, 331-339. 20. PALTAUF, F., PROUGH, R. A., MASTERS, B. S. S., AND JOHNSON, J. M. (1974) J. Biol. Chem. 249, 2661-2662. 21. BERGMEYER, H. U. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 14-42, Verlag Chemie, Weinheim; Academic Press, New York. 22. OSHINO, N., AND OMURA, T. (1973) Arch. Biothem. Biophys. 157, 395-404. 23. OSHINO, N., IMAI, Y., AND SATO, R. (1971) J. Biochem. (Tokyo) 69, 155-167. 24. HILDEBRANDT, A., AND ESTABROOK, R. W. (1971) Arch. Biochem. Biophys. 143, 66-79. 25. MANNERING, G. J., KUWAKARA, S., AND OMURA, T. (1974) Biochem. Biophys. Res. Commun. 57, 476-481. 26. TSUGITA, A., KOBAYASHI, M., TANI, S., KYO, S., RASHID, M. A., YOSHIDA, Y., KAJIHARA, Y., AND HAGIHARA, B. (1970) Proc. Nat. Acad. Sci. USA 67,442-447. 27. NOBREGA, F. G., AND OZOLS, J. (1971) J. Biol. Chem. 246, 1706-1717.
