Isolation and characterization of metmyoglobin reductase from yellowfin tuna (Thunnus albacares)

Comp. Biochem. Physiol. Vol. 81B, No. 4, pp. 809-814, 1985

0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd

Printed in Great Britain

ISOLATION A N D CHARACTERIZATION OF METMYOGLOBIN REDUCTASE FROM YELLOWFIN TUNA (THUNNUS ALBACARES) MARK J. LEVY,*t DAVID J. LIVINGSTON,*~ RICHARD S. CRIDDLE§ and W. DUANE BROWN*~V~ *Institute of Marine Resources and §Department of Biochemistry and Biophysics, University of California, Davis, CA 95616, USA

(Received 2 January 1985) Akstract--1. The isolation, purification, and characterization of metmyoglobin reductase from yellowfin

tuna (Thunnus albacares) is described. The enzyme has been purified 120-fold. 2. Characterization of the enzyme includes molecular weight, isoelectric point, substrate specificity, enzyme kinetics, chromatographic behavior, and sensitivity to inhibitors. 3. The physical and catalytic properties of the tuna enzyme are compared to those of bovine and blue-white dolphin metmyoglobin reductase.

INTRODUCTION

MATERIALS AND METHODS

F o r myoglobin to function as an oxygen binding protein, the heme iron must be maintained in the reduced, ferrous state. Metmyoglobin reductases, which catalyze the reduction of ferric myoglobin to the ferrous form, have been isolated from bovine heart muscle (Hagler et al., 1979) and blue-white dolphin (Stenello caeruleoalba) skeletal muscle (Matsui et al., 1975a,b). These reductases reduce myoglobin without artificial mediators such as methylene blue. As such, they are specific reductases, similar to the extensively studied methemoglobin reductases (Hegesh and Avron, 1967a,b; Leroux et aL, 1975; Hultquist and Passon, 1971; Kuma, 1981). To investigate the species distribution of this enzyme and to further characterize its function, we have studied its activity in dark muscle tissue of yellowfin tuna. This muscle contains very high concentrations of a myoglobin which autoxidizes more rapidly than mammalian myoglobins (Brown and Mebine, 1969; Livingston et al., unpublished data). The requirement for reductase in tuna dark muscle should, therefore, equal or exceed that of mammalian cardiac and skeletal muscle. In this paper we report the isolation, purification, and characterization of metmyoglobin reductase from yellowfin tuna, and compare its observed properties with those of the corresponding enzymes from bovine heart and dolphin skeletal muscle.

Yellowfin tuna metmyoglobin reductase was prepared by a modification of the method used for purification of bovine reductase (Hagler et al., 1979). All steps were carried out at 2--4°C. All chemicals were reagent grade or better. Frozen whole tuna were partially thawed and the dark muscle excised. Approximately 300 g muscle was ground, then homogenized with distilled water (1:2 w/v) using a Brinkmann Polytron for 15-30 sec at a setting of 7. The homogenate was adjusted to pH 7.5 with 2 N NH4OH, then centrifuged at 13,000 g for 20 min. The resulting supernatant solution was brought to 40% saturation with solid (NH4)2SO4, the apparent pH readjusted to 7.5, stirred for 30 rain, and centrifuged for 20 rain at 13,000g. The supernatant fraction was then brought to 70% saturation with (NH4)2SO4, adjusted to pH 7.5, stirred for 30min, and centrifuged at 13,000g for 20min. The supernatant from this final (NH4)2SO4 precipitation was set aside for the preparation of myoglobin. The precipitate was suspended in a minimum volume of water (about 100 ml) and dialyzed first against several changes of water overnight, then against 10raM sodium phosphate buffer, pH 6.0, with l m M EDTA* for 6 hr. Following dialysis, a significant amount of precipitate was removed by centrifugation at 35,000g for 20 rain. The enzyme solution was applied to a 3 x 22 cm column of carboxymethyl Sephadex C-25 equilibrated with 10mM sodium phosphate buffer, 1 mM EDTA, pH 6.0 at a flow rate of 72 ml/hr, and eluted with the same buffer. The eluant was collected until absorbance at 280 nm dropped below 0.10.D. This solution was turbid, and was clarified by centrifugation for 20 min at 35,000 g. The resulting supernatant was concentrated to 70 ml by ultrafiltration through an Amicon YM-10 membrane, then dialyzed overnight against 10 mM sodium citrate buffer, 1 mM EDTA, pH 5.5. After dialysis the solution was again centrifuged for 20 min at 35,000g and a small amount of precipitate removed. A 2.5 x 34cm column of diethylaminoethyl cellulose DE-52 (Whatman) equilibrated with 10 mM sodium citrate buffer, 1 mM EDTA, pH 5.5, was employed for further purification. Enzyme solution was applied to the column and eluted at a flow rate of 55 ml/hr using a linear gradient composed of 600 ml 10 mM sodium citrate, 1 mM EDTA, pH 5.5, and 600 ml 50 mM sodium citrate, 1 mM EDTA, 60mM NaCI, pH 5.5. Fractions (7.8ml) were collected,

tAuthor to whom correspondence should be addressed. :~Present address: 18-145, Chemistry Department, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA. ~[qDeceased. *Abbreviations: EDTA, ethylenediamine tetraacetic acid; Mb, myoglobin; MbO2, oxyrnyoglobin; MetMb, metmyoglobin; DCIP, 2,6-dichlorophenolindophenol; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide); FADH2, flavin adenine dinucleotide; FMNH2, flavin mononucleotide. C.B.P. 81/4B~A

809

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MARK J. LEVY et al.

assayed for reductase activity, and pooled. The pool was concentrated by ultrafiltration to 28 ml. A 2.5 x 14cm column of Affigel Blue (Bio-Rad) was equilibrated with 30 mM sodium phosphate buffer, 1 mM EDTA, pH 6.0. Five ml of the pooled sample from DE-52 was applied to the column and washed with 300 ml of the same buffer at a flow rate of 107 ml/hr. The reductase was eluted with a linear gradient composed of 350ml of the phosphate buffer and 350 ml of 30 mM sodium phosphate, 1 mM EDTA, 1M NaC1, 1 mM NADH, pH 6.0. Eight ml fractions were collected and assayed for reductase activity. Enzyme containing fractions were pooled, concentrated by ultrafiltration to 1 ml and stored at -80°C. Myoglobin was prepared from the supernatant solution following the 70% saturated ammonium sulfate precipitation. Six hundred ml of this solution was dialyzed against saturated ammonium sulfate with excess solid ammonium sulfate present. The resultant suspension was removed from dialysis and filtered through Whatman No. 1 filter paper on a Buchner funnel. The collected protein precipitate was dissolved in 25-30ml of water, and this solution was dialyzed exhaustively against water. The myoglobin solution was then centrifuged at 12,000g for 15 min. The myoglobin fraction was applied to a 1.6 x 95 cm column of Sephadex G-100 equilibrated with 50 mM Tris Buffer, pH 7.0, eluted with this buffer, and 7 9 ml fractions were collected. Myoglobin fractions were pooled and the concentration determined by absorbance at 540 nm of the met-cyano derivative (e mM = 11). This solution was then dialyzed against 30 mM sodium phosphate buffer, pH 7.0. The myoglobin solution was heated to 50c'C for 10min, cooled to 25°C, then centrifuged at 500g for 10min. Myoglobin solutions were batch washed with 1 g/10 ml dry DEAE cellulose (DE-53), followed by centrifugation at 500g for 10 min to remove DEAE and bound impurities (Hagler et al., 1979). Aliquots were then stored at -80°C until use. Metmyoglobin reductase activity was measured by its catalysis of change in absorbance at 580 nm of a yellowfin tuna metmyoglobin solution. The difference between the millimolar absorption coefficients of MetMb and MbO 2 at this wavelength is 10.3 (Livingston, 1982). Absorption was monitored in a Cary 219 spectrophotometer using a stirred 3 ml cuvette. The spectrophotometer was interfaced to an LSI-11 computer (DEC) for data collection and processing. Initial velocities were determined using a linear regression fit of the appropriate data points. Enzyme assay mixtures contained the following: 0.12 #mole MetMb in 30 mM sodium phosphate buffer, pH 7.0, plus additional phosphate buffer to a total of 600 #1; 1.5#mole disodium EDTA; 0.2/~mole potassium ferrocyanide; 15 #mole citrate buffer, pH 4.7; the sample to be assayed; and water to a total of 2.9 ml. The assay mixture was incubated for 2 min, then the reaction was started with the addition of 0.3 #mole of NADH in 100 #1 H20. Assays were carried out at room temperature (25°C) unless otherwise noted. Water was used in the reference cuvette as an assay blank. For inhibitor and cofactor studies, effector was incubated with the assay mixture for 2 min before NADH was added to initiate reduction. In the temperature dependence studies, cuvettes containing water, citrate, phosphate, and EDTA were equilibrated at the desired temperature. Ferrocyanide, metmyoglobin, and enzyme were added and incubated for 2min before addition of NADH. Temperatures in the cuvette were measured immediately before and after the reaction, and remained within 0.5°C of stated values. The reductase used in kinetic experiments was a 13-fold purified preparation unless otherwise noted. Protein concentrations were determined by dye binding (Bradford, 1976). Isoelectric focusing was performed on 7.5% polyacrylamide gels with 2% ampholytes: bands were stained for enzyme activity with DCIP and MTT (Kaplan

and Beutler, 1967). Chromatofocusing was performed on a 15 ml column using the method of Sluyterman and Elgersma (1978). Molecular weights were determined by gel filtration on a 0.9 × 55cm Sephadex G-100 column (Pharmacia), using bovine serum albumin (67,000), ovalbumin (45,000), chymotrypsinogen (25,000), sperm whale myoglobin (18,200), and bovine pancreatic ribonuclease (13,700) as calibration standards. Sodium dodecylsulfate polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970); protein bands were visualized by staining with Coomassie Blue R-250.

RESULTS

AND

DISCUSSION

Purification

M e t m y o g l o b i n reductase activity was first established in extracts of h o m o g e n i z e d d a r k muscle following centrifugation at 13,000g. Purification of the reducing activity employed a strategy similar to that used for bovine m e t m y o g l o b i n reductase (Hagler et al., 1979) (Fig. 1), but several changes were necessitated by differences in the ion-exchange c h r o m a t o graphic b e h a v i o r a n d stability of the enzymes a n d myoglobins of the two species. E D T A stabilizes m e t m y o g l o b i n reductase; its use in all buffers subsequent to the a m m o n i u m sulfate fractionation resulted in significant increases in recovery, particularly in the early steps of the purification (Table 2). The C M - S e p h a d e x c h r o m a t o g r a p h y step of purification was used to remove residual m y o g l o b i n from the enzyme p r e p a r a t i o n , as previously described for bovine reductase. Yellowfin t u n a m y o g l o b i n did not a d s o r b strongly to the C M - S e p h a d e x c o l u m n at p H 6.0 in 2 0 r a M sodium p h o s p h a t e buffer, the conditions used to remove bovine myoglobin, but a decrease in the buffer c o n c e n t r a t i o n to 10 m M at the same p H was effective. Presumably the lower surface charge density of tuna m y o g l o b i n (Watts et al., 1980) is responsible for this behavior. The D E A E cellulose c h r o m a t o g r a p h y conditions used for purification of bovine reductase (10-25 m M sodium phosphate, p H 7.0) were also altered for p r e p a r a t i o n of tuna enzyme, since the reductase could n o t be eluted from the DE-52 c o l u m n at p H 7.0, even in the presence of tO-

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Fig. 1. Elution of yellowfin tuna metmyoglobin reductase from DEAE cellulose column with a gradient from 10 mM sodium citrate, 1 mM EDTA, pH 5.5. to 50mM sodium citrate, 1 mM EDTA, 60 mM NaCI. A280 ( ), conductivity of eluting buffer (. . . . ), and reducing activity (-.-) (in units of absorbance change per 100sec).

Tuna metmyoglobin reductase

reductase, but differing significantly from the dolphin enzyme, which is most active at pH 5.0 (Table 1).

q.0

Isoelectric pH The isoelectric pH of yellowfin tuna metmyoglobin reductase is 5.1. Enzyme in the unstable second peak from the DEAE column has an apparent pI of 4.5. Bovine reductase has an isolectric point of 5.6-5.9, and dolphin reductase 5.0. These differences are reflected in the behavior of the reductases during ion exchange chromatography.

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Fig. 2. Dependence of tuna metmyoglobin reductase activity on pH. Assays were carried out as described in Methods and plotted as observed rate divided by the maximum rate at pH 6.5 1.0M NaC1. A pH 5.5 citrate buffer analogous to that used for dolphin reductase (Matsui et al., 1975a) was used for anion exchange chromatography (Fig. 1). The broad late peak of enzyme activity was extremely unstable, so only the first peak was used for further experiments. Affinity chromatography, using a Cibacron Blue support which is useful for pyridine nucleotide binding enzymes, significantly increased the specific activity of the reductase preparation. A 90?/00 loss of activity occurs on this column, however, which reflects the increased lability of the tuna enzyme throughout the preparation as compared to the bovine reductase. Instability of the enzyme even at the early stages of purification may account for measurements of activity in bovine heart homogenates (Hagler et al., 1979) 35-fold higher than those we found in tuna muscle homogenates. This ratio of measured levels of reducing activity is surprising, in light of the greater rate of Mb autoxidation and 5-fold higher Mb concentrations in tuna dark muscle when compared with bovine heart muscle.

pH optimum Yellowfin tuna metmyoglobin reductase has a pH optimum near 6.5 (Fig. 2), similar to that of bovine IO

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Fig. 3. Temperature dependence of tuna metmyoglobin reductase activity. Activities are plotted as observed rate divided by the maximum rate at 35°C.

Temperature dependence Yellowfin tuna reductase is most active in the temperature range from 33 to 35°C, with a relatively large loss of activity above 40°C (Fig. 3). This relatively low temperature for denaturation and for optimal activity may reflect biochemical adaptation of the tuna enzyme to the (23-33°C) operating temperature of the dark muscle (Carey et al., 1971), which is significantly lower than that of mammalian muscle. Several fish muscle glycolytic enzymes have been reported to have denaturation temperatures lower than their mammalian counterparts (Feeney et al., 1972). Molecular weight The molecular weight of yellowfin tuna metmyoglobin reductase was estimated to be 30,000-40,000 by gel filtration chromatography. At least half of the enzyme activity was eluted in the void volume of columns packed with either Sephadex G-100, G-150, or G-200, suggesting a high molecular weight aggregate. Performing gel filtration in the presence of either 0.1 M NaC1 or 0.2% Triton X-100 plus 1~o glycerol did not eliminate the rapidly eluting fraction. SDS-polyacrylamide gel electrophoresis of reductase purified by either chromatofocusing or Affigel Blue chromatography resulted in three major protein bands corresponding to 32,000, 50,000 and 72,000mol. wt. It is not possible to demonstrate activity with any of the three bands because of the presence of SDS. Analysis of purified protein by polyacrylamide gel electrophoresis under nondenaturing conditions with 0-2~o Triton X-100 present to minimize aggregation again indicated three migrating protein bands. Detection of three bands in the same gel with a specific activity stain for reductase suggests that multiple forms of active enzyme exist. The reported molecular weights of bovine reductase and dolphin reductase are 32,000 and 65,000, respectively. Cofactors F A D H 2 and FMNH2 reduced metmyoglobin nonenzymatically, as did 1,2 naphthoguinone-4-sulfonic acid. The apparent Km of yellowfin tuna metmyoglobin reductase for N A D H is 2.5 × 10-tM (Fig. 4). NADPH was ineffective as a source of reducing equivalents at concentrations up to twice that used for N A D H in enzyme assays. The absolute specificity of this enzyme for N A D H vs NADPH illustrates its role as a metmyoglobin-specific reductase. This contrasts with tuna muscle diaphorases described earlier (AI-Shaibani et al., 1977), which can use either N A D H or NADPH, and reduce met-

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Fig. 5. Lineweaver-Burk double reciprocal plots of the effects of yellowfin tuna metmyoglobin ( ), and bovine metmyoglobin (.... ) concentrations on the rate of metmyogtobin reductase.

myoglobin only in the presence of an artificial mediator such as methylene blue. The inclusion of ferrocyanide in our assay system serves the same purpose as in the bovine enzyme systems, i.e. it complexes with the heme protein, forming a stoichiometric complex which is reduced by the enzyme (Hegesh and Avron, 1967a). Bovine liver cytochrome bs at stoichiometric levels could replace ferrocyanide in our assay system, in agreement with earlier reports of b5 acting as a mediator in the methemoglobin reductase system (Hultquist and Passon, 1971). Cytochrome bs may act as a physiological mediator of metmyoglobin reducing activity.

hibition increased from 10% at 10-6M PHMB to 60% at 10-5 M, and the reduction was completely inhibited at a concentration of 10 4M PHMB. Some protection against this inhibition was provided by 10-4M NADH (the concentration normally used in assays). When NADH was added before PHMB and the reduction initiated with ferrocyanide, 10 5M PHMB had no inhibitory effect. Some reduction (20% of uninhibited rate) was possible in the presence of 10-4M PHMB. These results suggest that a cysteine residue may be located in or near the pyridine nucleotide binding site. Microsomal cytochrome b5 reductase, which is a membrane-bound analog of methemoglobin reductase, has been shown to have such a sulfhydryl group (Strittmatter, 1958). NEthylmaleimide was less effective as an inhibitor of metmyoglobin reduction: at a concentration of 10 -3 M there was only 20% inhibition, and at 10 -2 M 80% inhibition of reduction. A number of other potentially inhibitory substances had no effect on the rate of the enzymatic reaction (Table 3).

Inhibitors

Increasing amounts of quinacrine dihydrochloride progressively inhibited metmyoglobin reduction. This result suggests that a flavin may be present in this enzyme, as in erythrocyte soluble cytochrome b5 reductase (Passon and Hultquist, 1972). Since both FADH 2 and FMNH2 nonenzymatically reduce metmyoglobin, it is impossible to determine their effect on the enzymatic reaction. The enzymatic reduction of metmyoglobin was also inhibited by p-hydroxymercuribenzoic acid (PHMB). The degree of in-

Kinetics

The apparent Km of yellowfin tuna reductase for metmyoglobin is 4.6 × 10 6 M , and for bovine met-

Table 1. General properties of metmyoglobin reductases

pI

pH optimum

Temperature of maximum activity (~C)

5.1 5.6-5.9 5.0

6.5 6.5 5.0

35 37 25

Molecular weight

Source Yellowfin tuna Bovine heart* Blue-white dolphint

~ 30,000 32,000 64,000

*Data from Hagler et al. (1979). %Data from Matsui et al. (1975a, b). Table 2. Purification and yield of metmyoglobin reductase from yellowfin tuna Procedure Crude supernatant Ammonium sulphate precipitation 40-70% CM-Sephadex C-25 DEAE cellulose DE-52 Affi-gel Blue

Volume (ml)

Total units (pmol/min)

Spec. act. (units/mg protein)

Yield (%)

615

26,500

2.3

100

1.00

ll0 70 25 5

14,600 5770 2560 300

2.65 2.84 30.4 274

55 22 10 1

1.15 1.23 13.2 119

Purif.

813

Tuna metmyoglobin reductase Table 3. Effect of various inhibitors on the rate of enzymatic metmyoglobin reduction

Inhibitor p -Hydroxymercuribenzoic acid Quinacrine dihydrochloride

Concentration (raM) 0.001 0.01 0.1 0.25 1.0

10.0 0.25

N-Ethylmaleimide

1.0

ct,ct'-Dipyridyl Phenylmethylsulfonyl fluoride N-Acetylimidazole Iodoacetamide Hydroxylamine hydrochloride

10.0 0.25 10.0 0.25 0.25 0.25 0.25

Activity remaining (%) 90 40 0 100 80 25 100 80 20 100 100 100 100 100 100

reduction of the one-electron acceptors cytochrome b5 and the ferrocyanide-metmyoglobin complex. The much higher specific activities for metmyoglobin reduction by this enzyme in bovine heart and yellowfin tuna, as opposed to the non-specific diaphorases, accentuates the importance of these enymes in maintaining myoglobin in its physiologically active form. Acknowledgements--We thank Star Kist Foods, Terminal Island, California for generous donations of yellowfin tuna and Daniel A. Watts for his advice and technical assistance. This work was supported in part by NOAA Office of Sea Grant, Department of Commerce under Grant No. NA80AA-D-00120, Project No. R/F-68. The US Government is authorized to produce and distribute reprints for governmental purposes, notwithstanding any copyright notations that may appear hereon. REFERENCES

myoglobin is 5.8 x l0 6M. The Lineweaver-Burk plots of the bovine and tuna enzymes with metmyoglobin from each species are shown in Fig. 5. This apparent lack of species specificity suggests that there may not be any significant difference in the sites of interaction between reductase and substrate in these reactions. It should be noted that the assay system used in these experiments, with metmyoglobin-ferrocyanide complex as acceptor, may not be a proper model for the physiological interaction. This explanation is supported by evidence that methemoglobin reductase actually catalyzes N A D H reduction of cytochrome bs, with a subsequent non-enzymatic reaction of reduced cytochrome b5 with methemoglobin (Kuma, 1981). We have obtained evidence which supports this pathway in the bovine metmyoglobin reductase system (Livingston, 1982). Since the amount of reductase obtained from yellowfin tuna was insufficient for mechanistic studies, we could not determine the Km for cytochrome b 5. We did find that bovine liver cytochrome b 5 could replace ferrocyanide in our assays, and equimolar quantities of either cofactor gave similar activities. A correct physiological interpretation must await the identification of an in situ moderator in muscle tissue. CONCLUSION We have isolated and purified 120-fold a metmyoglobin reducing activity from the dark muscle of yellowfin tuna (Thunnus albacares). Activity levels in crude homogenate are significantly lower than those detected in bovine heart muscle preparations; however, this may be a result of the much lower stability of the tuna enzyme. A purification strategy similar to the bovine system has been used to purify the tuna enzyme with modifications made to accommodate the lower pI of the latter. The tuna enzyme has a p H optimum similar to the bovine enzyme, but the molecular weights and isoelectric p H ' s of the two differ significantly. Kinetic and inhibitor studies of the tuna enzyme imply mechanistic features similar to the bovine heart enzyme, as well as the methemoglobin reductase system of erythrocytes. These enzymes are all specific for N A D H , may contain flavin, and can mediate

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