J. Biochent.85, 719-728 (1979)

Characterization of the Purified NADPH-Flavin Reductase

of Human Erythrocytes

Toshitsugu YUBISUI,* Takasumi MATSUKI,* Masazumi TAKESHITA,** and Yoshimasa YONEYAMA* *Department of Biochemistry , School of Medicine,and **Departmentof Medical Technology, School of Paramedicine, Kanazawa University, Kanazawa, Ishikawa 920

Receivedfor publication, August 14, 1978

The NADPH-flavin reductase of normal human erythrocytes, which has an isoelectric point of 8.1, was purified to homogeneity and the properties of the were investigated. The sedimentation coefficient of the enzyme was estimated to be 2.17S20,, by analytical ultracen trifugation. The molecular weight was estimated to be 21,500 by electrophoresis on poly acrylamide gel in the presence of sodium dodecyl sulfate, and 22,000 by gel filtration on Se phadex G-75. The purified enzyme showed an absorption maximum only at 278 nm with a distinct shoulder at 283 nm, but had no absorption in the visible region. The purified enzyme was found to have no such as flavin or heroin, judging from its absorption spectrum and fluorescence analysis. No metal ion examined could activate the enzyme activity. The stoichiometry of flavin reduction by the enzyme with NADPH as the electron donor was determined to be unity. It was found that FMN functions as an effectiveelectron carrier with the enzyme to reduce methemoglobin, especially in the absence of . In the absence of oxygen, the rate of reduction of methemoglobin through FMN was significantly faster than that observed in the presence of oxygen. This enzyme could reduce an artificial dye such as 2,6-dichlorophenolindophenol at an appreciable rate without mediating flavin, and added flavin over a wide range of concentration did not affect the initial rate of the diaphorase activity significantly. These findings appear to rule out the participation of flavin in the diaphorase activity of our enzyme.

Human erythrocytes contain two types of pyridine genase A and B. NADH-dehydrogenase I is the nucleotide dehydrogenase related to the reduction major enzyme reducing methemoglobin (2), and of methemoglobin. Scott separated (1) each of is deficient in erythrocytes of patients with heredi these further into two species, NADH tary methemoglobinemia (3). This enzyme was dehydrogenase I and II, and NADPH-dehydro recently characterized as NADH-cytochrome bb reductase (4-6). On the other hand, although NADPH-dehydrogenase (NADPH-methemoglobin Abbreviations: DTT, dithiothreitol; DCIP, 2,6-dichlo reductase) has also been studied by many workers rophenolindophenol; PCMB, p-chloromercuribenzoate; SDS, sodium dodecyl sulfate; TCA, trichloroacetic (7), the properties and physiological role of the acid. enzyme still remain to be clarified.

Vol. 85, No. 3, 1979 719 720 T. YUBISUI, T. MATSUKI, M. TAKESHITA, and Y. YONEYAMA

Previously we partially purified the NADPH (pH 7.5) or citrate-phosphate buffer (pH 4.8), dehydrogenase (NADPH-methemoglobin re 200 nmol of flavin, 200 nmol of NADPH, and an ductase) from normal human erythrocytes, and appropriate amount of the enzyme. The reaction found that the purified enzyme could reduce was started by the addition of the enzyme and methemoglobin rapidly in the presence of flavin performed at 25•Ž. The enzyme activity was as well as methylene blue in the assay mixture (8). calculated using a millimolar extinction coefficient

The effect of flavin on the reduction of met of 6.22 at 340 nm. NAD(P)H-methemoglobin hemoglobin, however, was recognized only with reductase activity was measured by following the NADPH-dehydrogenase, and not with NADH absorbance change at 576 nm for oxyhemoglobin, . Subsequently we pre and at 555 nm for deoxyhemoglobin. The enzyme sented evidence that flavins are the effective electron activity was calculated using values of 11.9 for acceptor of the enzyme, and that the enzyme oxyhemoglobin, and 8.44 for deoxyhemoglobin, cannot transfer electrons from NADPH directly for the difference in millimolar extinction coefficient to hemoproteins such as methemoglobin and between the oxidized and reduced form of hemo cytochromes b5 and c (9). Based on these results, globin (5). Two ml of the assay mixture contained our purified enzyme, which apparently corresponds 100 lemol of phosphate buffer (pH 7.0), 200 nmol to the so-called "NADPH-methemoglobin re of NAD(P)H, FMN or methylene blue as indi ductase," was proposed to be NADPH: flavin cated, and an appropriate amount of the enzyme. . In this paper some general The concentration of methemoglobin added to properties of the purified NADPH-flavin reductase the assay mixture was 501LM (on a basis). of human erythrocytes are described. The re NAD(P)H-diaphorase activity was determined as lationship between the flavin reductase activity described by Sugita et al. (5), except that 2.0 ml and diaphorase activity of the enzyme, and the of reaction mixture containing 50 mm phosphate reduction of methemoglobin by the enzyme through buffer (pH 7.5) instead of Tris buffer was used. flavin or methylene blue are also described. In all the assays described above, the non-enzymatic reduction of the by the reduced form of

MATERIALS AND METHODS pyridine nucleotide was subtracted from the measured value. Normal Human Red Cells were obtained from Purification of the Enzyme-The NAD(P)H the central laboratory of the Tokyo Red Cross flavin ' reductase of human erythrocytes was

Blood Transfusion Service. Q-NADH, cyto purified as described previously (9) with some chrome c, FAD, FMN, glyceraldehyde-3-phosphate modifications. Preparation of hemolysates was dehydrogenase, and catalase were obtained from carried out as described previously (9), except that Boehringer Mannheim, while a-NADH and 20 leM FMN solution was replaced by 1 mm 2 NADPH were from Sigma Chemicals Co. Ultrogel mercaptoethanol. Removal of stroma from the AcA 54 was purchased from LKB, and carboxy hemolysates was achieved by centrifugation im methyl-cellulose, CM-32, from Whatman. Other mediately after adjusting the pH of the hemolysates reagents were obtained commercially. Purified to 6.4 with 6 N HCI. The pH of the supernatant cytochrome P-450 of cow adrenal mitochondria was quickly readjusted with NaOH to 7.0. The was kindly provided by Dr. N. Ashida of Kana hemolysates thus prepared were applied to DEAE zawa University. cellulose as described previously (9), and the enzyme Spectrophotometric Determinations were per was eluted from DEAE-cellulose with buffer formed with a Hitachi 124 spectrophotometer containing 0.1 mm DTT instead of 1 mm 2 fitted with a Hitachi recorder, model 056, or with mercaptoethanol. All other steps of purification a Union SM 401 recording spectrophotometer. were performed using phosphate buffer containing Enzyme Assay-NAD(P)H-flavin reductase 1 mm EDTA and 0.1 mm DTT. Other modifica activity was determined by following the decrease tions to the purification procedure were as follows; in the absorbance of NAD(P)H at 340 nm as step 3 in the previous method was replaced by described previously (9). Two ml of reaction fractionation with ammonium sulfate in this study, mixture contained 100,amol of phosphate buffer step 4 by chromatography on Ultrogel AcA 54,

J. Biochem. NADPH-FLAVIN REDUCTASE OF HUMAN ERYTHROCYTES 721 step 5 by rechromatography on Ultrogel AcA 54, The Stoichiometry of the NADPH-FMN step 6 by chromatography on CM-cellulose, step Reductase Reaction was determined by following 7 by isoelectric focusing, and step 8 by chromatog the absorbance change of NADPH at 340 nm or raphy on Ultrogel AcA 54. The final enzyme at 333 nm (the isosbestic point of the oxidized and eluted from the Ultrogel column was concentrated reduced forms of FMN), and of FMN at 445 nm with 70% ammonium sulfate, and then exhaus under anaerobic conditions. FMN and the puri tively dialyzed against 50 mm phosphate buffer fied enzyme were added to the main compartment (pH 7.5) containing 1 mm EDTA and 0.2 mm of a Thunberg tube as specified in 1.95 ml of 50 mm DTT. phosphate buffer (pH 7.5), and NADPH was

Isoelectric Focusing was carried out by the placed in the side arm in 0.05 ml of H2O. After method of Svensson (10), and according to the sealing the tube with silicone grease, the air in the LKB instruction sheet using a I % (v/v) ampholine tube was evacuated and Q-gas (99.05 % helium: solution (LKB-Productor AB, Stockholm) at 0.95% isobutane) was introduced to achieve 4-6•Ž. Carrier ampholine with a pH range of anaerobiosis. The gas exchange was repeated at 3.5-10 or 7-9 was used. A maximum load of least three times. The concentration of FMN about 1 watt was applied for about 36-48 h in a was determined using a millimolar extinction glycerol gradient from 0-30% instead of a sucrose coefficient of 12.5 at 445 nm (15). gradient. DTT (0.2 mm) was added to all solu The Flavin Content of the purified enzyme tions used for the electrophoresis as a stabilizer was analyzed by treatment with 5 % TCA at 37•Ž of the enzyme. overnight (16). The FMN content in the super Electrophoresis on Polyacrylamide Gel was natant was determined fluorometrically with a performed by the method of Ornstein (11) and Hitachi MPF-4 fluorescence spectrophotometer. Davis (12). Electrophoresis was carried out at Protein was determined by the method of 4•Ž with a constant current of 2-2.5 mA per gel. Lowry et al. (17) using bovine serum albumin as a Electrophoresis on polyacrylamide gel in the standard. presence of SDS was carried out by the method of Weber and Osborn (13) at room temperature. RESULTS Proteins were pretreated with the gel electrophoresis buffer of Weber and Osborn (13) at 100•Ž for Preparation of the Enzyme-By the present 2 min. Electrophoresis was performed with a methods for purification, NADH-cytochrome bs constant current of 8 mA for 5-6 h. reductase was co-purified with NADPH-flavin reductase through the first four steps. At the Sedimentation Velocity was measured in a Hitachi 282 analytical ultracentrifuge. The 5th step (2nd chromatography on Ultrogel) sedimentation velocity experiments were carried NADH-cytochrome bb reductase was separated out with a synthetic boundary cell, and the sedi from NADPH-flavin reductase as shown in Fig. mentation coefficient was calculated from the 1. The elution patterns are very similar to those schlieren pattern and the absorbance scanning reported by Hegesh et al. (18), and the NADPH chart at 280 nm using a Hitachi recorder, model flavin reductase was further separated by chro 056. matography on a CM-cellulose column into two Estimation of the Molecular Weight of the fractions which were found to have different isoelectric points of 6.1 and 8.1. The enzyme purified enzyme by gel filtration was carried out fraction with the isoelectric point of 8.1 was further by the method of Andrews (14) on a Sephadex G-75 column equilibrated with 50 mm phosphate purified in the present studies. The enzyme was buffer (pH 7.5) containing 1 mm EDTA and purified more than 1,000-fold with respect to the 0.1 mM DTT, with or without 0.1 M KCI. Marker flavin reductase activity, with a yield of 4.3%. The purified enzyme was stable at 4•Ž, and freez proteins used were horse heart cytochrome c ing and thawing caused no appreciable loss of the (12,400), sperm whale myoglobin (17,800), rabbit enzyme activity. The specific activity of NADPH muscle glyceraldehyde-3-phosphate dehydrogenase flavin reductase of the most highly purified enzyme (36,000), ovalbumin (46,000), and bovine serum was 370 nmol/min/mg under the standard assay albumin (68,000).

Vol. 85, No. 3, 1979 722 T. YUBISUI, T. MATSUKI, M. TAKESHITA, and Y. YONEYAMA

Fig. 1. Separation of NADH and NADPH-dia phorase on an Ultrogel AcA 54 column. The dia phorase eluted from the Ultrogel column (4 x 78 cm) in Fig. 2. The absorption spectrum of the purified the first chromatography was concentrated and applied NADPH-flavin reductase. The enzyme (1.66 mg) was again to the Ultrogel column under the same conditions. dissolved in 50 mm phosphate buffer (pH 7.5) contain The enzymes were eluted from the column with 50 mm ing 1 mMEDTA and 0.1 mM DTT. Inset, an expanded phosphate buffer (pH 7.5) containing 1 mm EDTA and figure to show the shoulder at 283 nm more clearly. 0.1 mm DTT. Fractions of 5 ml were collected. purified enzyme was examined fluorometrically. conditions. The NADPH-diaphorase activity was Without any treatment the enzyme solution did 670 nmol/min/mg under the standard assay con not show any detectable fluorescence for flavin. ditions, and this value is comparable to that When 0.72 mg of the enzyme (32.7 nmol on the described by Scott et al. (1). basis of a molecular weight of 22,000) was treated Properties of the Purified Enzyme-Purity of with TCA, the amount of FMN dissociated was the enzyme: The enzyme moved as a distinct hardly detectable as shown in Fig. 3, and was band on a polyacrylamide gel in the presence of calculated to be less than 0.003 nmol. SDS as reported previously (9). In the absence Molecular weight: The molecular weight of of SDS, the enzyme also moved as a distinct band the purified enzyme was estimated to be 21,500 by on a polyacrylamide gel at pH 8.3, but during electrophoresis on polyacrylamide gel in the storage some additional minor bands appeared presence of SDS, and 22,000 by gel filtration on without loss of activity as described by Niethammer Sephadex G-75, as shown in Fig. 4. Addition of and Huennekens (19). Analytical ultracentrifuga 0.1 M KCI to the buffer did not affect the molecular tion further confirmed the purity of the enzyme. weight as determined by the gel filtration method. The enzyme sedimented as a symmetric boundary Effect of pH-The effect of pH on the in the analytical ultracentrifuge and the sedimenta NADPH-FMN reductase activity and on the tion coefficient was calculated to be 2.17 52o,w at NADPH-diaphorase activity was examined with 39,800 rpm at 1.45 mg protein per ml. various buffers. As shown in Fig. 5, the pH Absorption spectrum: The absorption spec optimum for NADPH-FMN reductase was found trum of the purified enzyme is shown in Fig. 2. at pH 4.8. Diaphorase activity was higher at The enzyme has an absorption maximum only at acidic pH than at neutral pH, as shown in Fig. 5, 278 nm with a distinct shoulder at 283 run, but but as DCIP itself changes to a red compound at has no absorption in the visible region. The acidic pH (lower than pH 6.0), the diaphorase millimolar extinction coefficient of the purified activity was determined for convenience at pH 7.5. enzyme was estimated to be 10.2 at 278 nm based Effect of Ionic Strength-The effect of ionic on a molecular weight of 22,000. The spectrum strength on the flavin reductase activity was suggests that the enzyme has no chromophore examined by changing the buffer concentration or such as flavin or hemin as a constituent. increasing the KC1 concentration in the standard Flavin content: The flavin content of the assay mixture. Increasing either phosphate buffer

J. Biochem. NADPH-FLAVIN REDUCTASE OF HUMAN ERYTHROCYTES 723

Fig. 4. Determination of the molecular weight of the NADPH-flavin reductase. (A) Electrophoresis on polyacrylamide gel in the presence of SDS using 10% gel (left line) and 5% gel (right line), (B) gel filtration on a Sephadex G-75 (2.5 x 40 cm) equilibrated with 50 mm phosphate buffer (pH 7.5) containing I mm Fig. 3. Flavin content in the purified enzyme. The EDTA and 0.1 mm DTT. Proteins were eluted from flavin content in the purified enzyme was analyzed by the column with the same buffer. Marker proteins treatment with 5% TCA as described in " MATERIALS used in both methods are a) horse heart cytochrome c, AND METHODS." ---, Authentic FMN (0.378 b) sperm whale myoglobin, c) rabbit muscle glyceral pM); , 13.1 pm purified enzyme; ...... 13.1 ,aM dehyde-3-phosphate dehydrogenase, d) ovalbumin, e) purified enzyme, ten-fold expanded. bovine serum albumin.

or KCI up to 0.4 M caused only about 10% decrease in the activity compared with those observed at low salt concentrations (10-50 mm buffer or KCI). Enzyme Activity and Enzyme Amount Added The relationship between the enzyme activity and the amount of enzyme added to the assay mixture was examined. As shown in Fig. 6, good linearity was observed between the enzyme activity and the amount of enzyme protein added. Effects of the Components in the Assay Mixture -Table I shows the effects of components in the assay mixture on the NADPH-FMN reductase and NADPH-diaphorase activity. The FMN reductase activity is highly specific for NADPH, and the activity with NADH was only 10% that with Fig. 5. Effect of pH on the NADPH-FMN reductase NADPH, while the diaphorase activity with and NADPH-diaphorase activities. The assay con NADH was 1.7 times higher than that with ditions were as described in " MATERIALS AND NADPH. NADP was found to be a potent in METHODS." Open symbols, citrate-phosphate buffer hibitor of the enzyme, but NAD was not. Both (50 trim in respect to phosphate); closed symbols, potas flavin reductase and diaphorase activity were sium phophate buffer (50 mm). Solid lines, NADPH strongly inhibited (45-60% inhibition) by 0.1 mat FMN reductase; broken lines, NADPH-diaphorase.

Vol. 85, No. 3, 1979 724 T. YUBISUI, T. MATSUKI, M. TAKESHITA, and Y. YONEYAMA

NADP at pH 7.5, but lesser inhibition (12 donor for this enzyme, as shown in Table I, a inhibition) of the flavin reductase was observed NADH was almost completely ineffective. at pH 4.8. EDTA had no effect on either enzyme Stoichiometry of the Enzyme Reaction-The activity. Boiling of the enzyme led to complete stoichiometry of the enzyme reaction was examined loss of the FMN reductase activity, but a trace of with NADPH as an electron donor and with diaphorase activity was observed. Addition of FMN as an electron acceptor under anaerobic FMN to the assay mixture for diaphorase activity conditions. As shown in Table II, the ratios of did not cause an appreciable change in this activity. the amounts of NADPH oxidized and FMN Although a-NADH was an effective electron reduced were almost unity. As the flavin perturbs

TABLE 1. Effect of components in the assay mixture. Complete assay mixture for NADPH-flavin reductase contained 0.1 mm NADPH, 50 mm citrate-phosphate buffer (pH 4.8), 0.1 mm FMN, and 28.8 pg of the puri fied enzyme in a total volume of 2.0 ml. The complete assay mixture for diaphorase activity contained 0.1 mm NADPH, 50 mm phosphate buffer (pH 7.5), 63 pm DCIP, and 14.4 pg of the purified enzyme in a total volume of 2.0 ml.

Fig. 6. Effect of the amount of enzyme added on the activity. The relationship between the NADPH-flavin reductase activity and the amount of enzyme added to the assay mixture was examined using two different batches of preparation with different purities. The assay conditions were as described in " METERIALS AND METHODS." a) Highly purified enzyme, b) less purified enzyme (before the electrofocusing step of purification).

TABLE II. Stoichiometry of the NADPH-FMN reductase reaction. The reaction mixture contained NADPH and FMN (as indicated), with 65.6 pg of the purified enzyme in 2.0 ml of 50 mm phosphate buffer (pH 7.5). Other experimental conditions were as described in " MATERIALS AND METHODS."

J. Biochem. NADPH-FLAVIN REDUCTASE OF HUMAN ERYTHROCYTES 725

the absorption of NADPH at 340 nm due to its bathocuproine sulfonate and o-phenanthroline oxidation and reduction, the isosbestic point of may be due to their structural resemblance to the oxidized and reduced forms of the flavin, FMN, because other metal chelators such as 8 333 nm, was chosen to determine the change of hydroxyquinoline, diethyldithiocarbamate, and NADPH during the reaction and eliminate error. KCN showed only weak (20%) or no inhibition. The millimolar extinction coefficient of 6.02 at Moreover, as described below, addition of Feet, 333 nm for NADPH was determined based on a Feat or Cult (1-10 pM) to the assay mixture did millimolar extinction coefficient of 6.22 at 340 nm. not have any stimulative effect on the enzyme In experiment 1 the amount of NADPH oxidized activity. In contrast to the sensitive response of was followed at 340 nm, and in experiment 2 at the flavin reductase activity to various reagents, 333 nm. To calculate the amount of NADPH the diaphorase activity was resistant to various oxidized from the data at 340 nm, a correction reagents, and only proflavin and bathocuproine was made by subtracting the absorbance at 340 nm inhibited the diaphorase activity significantly. due to the reduction of FMN. It should be noted that PCMB and HgCl2 inhibited Effects of Various Reagents-Table III shows the flavin reductase activity strongly, but did not the effects of various reagents on the NADPH inhibit the diaphorase activity at all. Fifty per FMN reductase and NADPH-diaphorase ac cent inhibition by superoxide dismutase of the tivities. The flavin reductase activity was in flavin reductase activity was observed at pH 4.8, hibited by proflavin hemisulfate, acrinol, batho but no significant inhibition was observed at cuproine sulfonate, o-phenanthroline, PCMB, pH 7.5. Diphosphoglycerate, an allosteric effector HgC12, and 2,3-diphosphoglycerate. Inhibition by of hemoglobin, inhibited only the flavin reductase

TABLE III. Effects of various reagents on the enzyme activity. Assay conditions for FMN reductase and diaphorase were as in Table I except that the concentration of FMN used for the FMN reductase was 216 pM. The reaction was started by addition of the enzyme.

a Diethyldithiocarbamate reduced DCIP non-enzymatically.

Vol. 85, No. 3, 1979 726 T. YUBISUI, T. MATSUKI, M. TAKESHITA, and Y. YONEYAMA activity, but inositolhexaphosphate did not affect concentrations were significantly faster than those either enzyme activity. Addition of metal ions observed in the presence of oxygen. These results (1-10 pM) such as Fe2+, Fe3+, Mg2+, and Ca2+ did apparently suggest that some FMNH2 reacts not affect the enzyme activity, but Cue+, Zn2+, and rapidly with oxygen, and the efficiency of FMN SeO32- were significantly inhibitory. as an electron carrier to reduce methemoglobin is Reduction of Methemoglobin-Although this poor in the presence of oxygen, in other words, enzyme has a high Km value for flavin (9), the that this enzyme can reduce methemoglobin concentration of flavin in human erythrocytes is effectively even at low concentrations of flavin in known to be low. Thus, to understand the role the absence of oxygen. of this enzyme in the reduction of methemoglobin in human erythrocytes, the effect of FMN on he DISCUSSION reduction of methemoglobin was examined at various concentrations of FMN lower than the We have previously presented evidence that the Km value. The experiments were performed both so-called "NADPH-dehydrogenase" (NADPH in the presence and absence of oxygen, because methemoglobin reductase) in human erythrocytes the reduced form of FMN reacts rapidly with is an enzyme which catalyzes the transfer of oxygen. The rate of reduction of methemoglobin electrons specifically from NADPH to flavin. by the enzyme was affected by the concentration We therefore named the enzyme NADPH-flavin of FMN as well as by oxygen, as shown in Table reductase temporarily. The overall reduction of IV. In the absence of oxygen the rates of reduc methemoglobin by the enzyme in the presence of tion of methemoglobin through FMN at various flavin was explained in terms of the enzymatic reduction of flavin and subsequent non-enzymatic reduction of methemoglobin by the reduced flavin TABLE IV. Reduction of methemoglobin through (9) (Fig. 7). Further evidence to support the identi FMN by NADPH-flavin reductase under various con ty of our enzyme, NADPH-flavin reductase, with ditions. The reaction was performed in an assay mix the "NADPH-methemoglobin reductase" is now ture of 2.0 ml containing 50 mm phosphate buffer (pH presented in Fig. 1. Our enzyme is apparently 7.0), 0.1 mm NADPH, 50 ps methemoglobin (on a identical with Scott's NADPH-diaphorase heme basis), 26.4 pg, 14.5 sg, and 16.4 isg of the purified (NADPH-methemoglobin reductase), as described enzyme from different batches of preparation for experi above. The NADPH-flavin reductase with the ments 1, 2, and 3, respectively, and FMN as indicated. isoelectric point of 8.1 purified in the present The reduction of methemoglobin through FMN was studies seems to correspond to Scott's diaphorase determined by following the increase of oxyhemoglobin or deoxyhemoglobin at 576 nm or at 555 nm, respectively, as described in " MATERIALS AND METHODS."

Fig. 7. Electron transfer sequences from NADPH and NADH to methemoglobin. The electron transfer sequences for the NADPH-flavin reductase and the NADH-cytochrome b5 reductase systems (4, 5) in human erythrocytes are shown. The vertical arrow between a The concentration of methemoglobin was fixed at NADH-cytochrome b5 reductase and cytochrome b5 50 pM, assuming that the concentration of methemo indicates blockage of the electron transfer due to the globin and total hemoglobin in human erythrocytes are lack of NADH-cytochrome b5 reductase in the case of 1% of the total hemoglobin and 5 mm, respectively. hereditary methemoglobinemia.

J. Biochem. NADPH-FLAVIN REDUCTASE OF HUMAN ERYTHROCYTES 727

A (1) or to the methemoglobin reductase I of and 1.1 mg of the the purified enzyme was ob Niethammer and Huennekens (19), based on its tained, corresponding to a yield of 4.5% of the chromatographic behavior and the absence of total activity from 5 liters of packed red cells. flavin in the enzyme. The yield of the enzyme was almost the same as Participation of flavin in the reduction of that reported by Sugita et al. (5). By a simple methemoglobin by NADH and NADPH calculation from the yields of the preparations, the methemoglobin reductases in human erythrocytes original contents of both NADPH-flavin reductase has been suggested by some workers (6, 20, 21). and NADH-cytochrome b5 reductase in red cells However, conflicting results have been reported were roughly estimated to be 165-200 mg and (1, 5, 22). Our enzymes purified in the previous 4.4-4.8 mg per 1 liter of the packed cells, respec (9) and the present studies were judged to have no tively. Based on molecular weights of 22,000 for flavin content, based on the absorption spectrum the NADPH-flavin reductase, and 33,000 for the and analysis of the flavin content in the enzyme. NADH-cytochrome b5 reductase (20), the contents In the present study the flavin content in our of both enzymes correspond to 7.5-9.1 pmol of enzyme was found to be less than 1 mol per NADPH-flavin reductase, and 0.133-0.145 ttmol 3.27 x 10" mol of the enzyme (7.2 x 108 g of of NADH-cytochrome b5 reductase per 1 liter of protein). The flavin content in Scott's NADPH the packed cells. In contrast to these differences diaphorase A was also found to be similar, 1 mol in content of the enzymes, the specific activities per 6.8 x 108 g of protein (1). W ma.) of the enzymes are 1 ymol/min/mg for the The effects of various metal ions on both NADPH-flavin reductase and 160 umol/min/mg flavin reductase and diaphorase activities of our for the NADH-cytochrome b5 reductase. Based enzyme were examined, but no metal ion which on these data the assumption can be made that stimulates the enzyme activity was found. Purified NADPH-flavin reductase and NADH-cytochrome cytochrome P-450 from cow adrenal mitochondria, b5 reductase in I liter of the packed cells can and pyridoxal-5'-phosphate and its derivatives reduce 200 pmol and 770 umol of methemoglobin were examined as electron acceptors of the enzyme, per min, respectively, under optimal conditions, but none of these substances were reduced. i.e., if the electron carriers are saturated. Reduc Recently Morelli and De Flora (23) described tion of methemoglobin by NADPH-flavin re the existence of an NADP(H) binding protein in ductase, however, is affected not only by the con human erythrocytes which has no enzyme activity, centration of flavin, but also by that of oxygen, as and has a molecular weight of 68,000, composed shown in Table IV. The effect of flavin on the of two polypeptide chains of 33,000. Based on reduction of methemoglobin was also studied in its high affinity for NADPH (dissociation con in vitro experiments in our laboratory using normal stant, 1 x 10-8 M) the protein may participate in and methemoglobinemic red cells, and a distinct the regulation of the NADPH-flavin reductase stimulation of the reduction of methemoglobin activity in human erythrocytes. upon addition of flavin to the incubation mixture In the present studies, starting from 5 liters of red cells was observed (24). These results are of packed red cells, 17 mg of purified NADPH considered to suggest that some of the flavin added flavin reductase with an isoelectric point of 8.1 to the incubation mixture directly or indirectly was obtained, a yield of 4.3 % of the total activity. participated in the activation of the NADPH As a nearly equivalent amount of enzyme with an flavin reductase, as methylene blue does (7). isoelectric point of 6.1 was separated on a CM It has been generally accepted that enzymes cellulose column, about 35 mg of purified NADPH which can catalyzes the reduction of DCIP are flavin reductase was obtained from 5 liters of flavoproteins (25). Although our enzyme requires packed red cells. In another batch of the prep flavin to reduce methemoglobin, it should be aration, a yield of about 48 mg of the purified noted that the reduction of DCIP by our enzyme enzyme was similarly estimated. On the other occurred without mediating flavin. Addition of hand, the NADH-cytochrome b5 reductase co flavin up to 0.3 mm to the assay mixture did not purified with the NADPH-flavin reductase through affect the initial rate of the diaphorase activity. the first four steps was also exhaustively purified, It is not likely that only 1 mol of enzyme per

Vol. 85, No. 3, 1979 728 T. YUBISUI, T. MATSUKI, M. TAKESHITA, and Y. YONEYAMA

3.27 x 104 mol is responsible for the diaphorase 9. Yubisui, T., Matsuki, T., Tanishima, K., Takeshita, activity. Therefore, it is reasonable to conclude M., & Yoneyama, Y. (1977) Biochem. Biophys. Res. that flavin does not function catalytically in the Commun. 76, 174-182 diaphorase activity of our enzyme. 10. Svensson, H. (1972) Arch. Biochem. Biophys. Suppl. The relationship between the flavin reductase 1,132-138 11. Ornstein, L. (1964) Ann. New York Acad. Sci. 121, and diaphorase activities of our enzyme is not yet Art. 2, 321-349 clear. Although some differences can be seen in 12. Davis, B.J. (1964) Ann. New York Acad. Sci. 121, Table III between the responses of flavin reductase Art. 2, 404-427 and diaphorase to various reagents, data obtained 13. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, in this study are not sufficient to indicate whether 4406-4412 FMN and DCIP may be bound to the same site 14. Andrews, P. (1964) Biochem. J. 91, 222-233 on the enzyme or not. The obscure nature of 15. Koziol, J. (1971) in Methods in Enzymology (McCor the electron donor specificity of our enzyme for mick, D.B. & Wright, L., eds.) Vol. 18B, pp. 257 diaphorase activity also requires further study. 260, Academic Press, New York Similar properties of methemoglobin reductase 16. Burch, H.B., Bessey, O.A., & Lowry, O.H. (1948) have been described by other workers (7, 19, 22) J. Biol. Chem. 175, 457-470 17. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & as regards methemoglobin and dye reduction. Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 18. Hegesh, E., Calmanovicci, N., Lupo, M., & Boch REFERENCES kowsky, R. (1971) J. Lab. Clin. Med. 77, 859-866 1. Scott, E.M., Duncan, LW., & Ekstrand, V. (1965) 19. Niethammer, D. & Huennekens, F.M. (1971) Arch. J. Biol. Chem. 240, 481-485 Biochem. Biophys. 146, 564-573 2. Scott, E.M. & McGraw, J.C. (1962) J. Biol. Chem. 20. Kuma, F. & Inomata, H. (1972) J. Biol. Chem. 237,249-252 247,556-560 3. Scott, E.M. (1960) J. Clin. Invest. 39, 1176-1179 21. Beutler, E. (1969) Experientia 25, 805 4. Hultquist, D.E. & Passon, P.G. (1971) Nature (New 22. Kajita, A., Kewar, G.K., & Huennekens, F.M. Biol.) 229, 252-254 (1969) Arch. Biochem. Biophys. 130, 662-673 5. Sugita, Y., Nomura, S., & Yoneyama, Y. (1971) 23. Morelli, A. & De Flora, A. (1977) Arch. Biochem. J. Biol. Chem. 246, 6072-6078 Biophys. 179, 698-705 6. Passon, P.G. & Hultquist, D.E. (1972) Biochim. 24. Matsuki, T., Yubisui, T., Tomoda, A., Yoneyama, Biophys. Acta 275, 62-73 Y., Takeshita, M., Hirano, M., Kobayashi, K., & 7. Kiese, M. (1974) in Methemoglobinemia: A Com Tani, Y. (1978) British J. Haematol. 39, 523-528 prehensive Treatise pp. 14-25, CRC Press, Cleve 25. Massey, V. & Veeger, C. (1963) Ann. Rev. Biochem. land, Ohio 32,579-638 8. Yubisui, T., Okamoto, H., Tanishima, K., Take shita, M., & Yoneyama, Y. (1976) The 16th Inter national Congress of Hematology Abst 85 (Kyoto)

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