THE Joumn~ OF B~LOGICAL CHEMISTRY “0,. 253, No. 9, Issue of May 10, pp. 2946-2953, 1978

Pm&d I,, U S.A.

The Green Hemoproteins of Bovine Erythrocytes I. PURIFICATION AND CHARACTERIZATION*

(Received for publication, April 13, 1977)

LOUIS J. DEFILIPPIS AND DONALD E. HULTQUIST

From the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109

Two green hemoproteins. hitherto undetected in bovine tissues, have been isolated from the supernatant fraction of bovine erythrocyte hemolysates. The two hemoproteins were co-purified by a procedure which entailed hypotonic lysis, a freeze at -7o”C, removal of stromata, and chroma- tography on DEAE-cellulose, Amberlite CG-50, and Bio-Gel P-60. This procedure also yields superoxide dimutase and glutathione peroxidase. The green hemoprotein fraction was then separated on DEAE-Sephadex into two forms (I and II). Both forms appear homogeneous by electrophoresis on polyacrylamide gels at pH 6.9 and 7.6, on polyacrylamide gels at pH 8.6 in the presence of sodium dodecyl sulfate (SDS), and on cellulose acetate strips at pH 8.6. Heteroge- neity was seen upon polyacrylamide gel electrophoresis at pH 8.9 and upon isoelectric focusing. The heterogeneity of form I on gel electrophoresis at pH 8.9 appears to result from protein degradation and dissociation into apoprotein and free hemin. In all electrophoretic systems tested, the two holoproteins behaved identically.

Immunological studies give no indication of impurities. Moreover, by immunological techniques, the two forms are indistinguishable and show no structural relationship to hemoglobin.

The two forms exhibit identical molecular weights of 23,000 by SDS-polyacrylamide electrophoresis and 27,000 by gel exclusion chromatography, and both therefore exist as monomers in solution.

The reduced pyridine hemochromes of the denatured hemoproteins show absorption maxima for form I at 434, B44, and j80.5 nm and for form II at 431, 538, and 573 nm, demonstrating the heme of form I possesses substituents with greater electron-w-ithdrawing power than the substitu- ents on the heme of form II. These spectra show that the two proteins are similar to two hemoproteins detected in human erythrocytes. The hemin prosthetic groups of the two proteins have been hitherto undetected in bovine tis- sues.

* This study was supported by Research Grant AM-09250 and Training Grant GM-00187 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Section of Biochemistry, Molecular and Cell Biology, Wing Hall, Cornell University, Ithaca, N.Y. 14853.

The presence in human erythrocytes of a green hemoprotein has been reported by Morrison (1). The unique character of its hemin prosthetic group was demonstrated by the distinctive spectrum of its pyridine hemochrome derivative and by the failure to extract the hemin using the classical acid-acetone method. Further purification and characterization of the he- moprotein has been achieved (2, 3). As isolated, the ferrihem- oprotein readily binds cyanide ion. Reduction of the protein with dithionite yields the ferro form, which binds carbon monoxide. The prosthetic group of the hemoprotein has been isolated and shown to be a very labile, water-soluble hemin which is distinct from all known prosthetic groups (3). The hemin apparently possesses acetylatable functional group(s) and two electron-withdrawing groups in conjunction with the tetrapyrrol nucleus.

In this paper we report the detection of similar green hemoproteins from bovine erythrocytes and describe the reso- lution upon DEAE-Sephadex chromatography into two dis- tinct species, each of which possesses a uniquely different hemin prosthetic group.

EXPERIMENTAL PROCEDURE

Materials

Electrophoresis-grade acrylamide and bisacrylamide, AG l-X8 resin, and Bio-Gel P-60 (100 to 200 mesh) were obtained from Bio- Rad; Amberlite CG-50 (200 to 400 mesh) and monobasic potassium phosphate from Mallinckrodt; bovine albumin (fraction V powder) and bovine methemoglobin from Pentex; bromphenol blue and Ponceau S stain from Allied Chemical; chymotrypsinogen A and ovalbumin from Worthington; citric acid from Baker and Adamson; Coomassie brilliant blue R-250 from Mann Research; cytochrome c (horse heart, type III), DEAE-Sephadex A-50 to 120, phenylmethyl- sulfonyl fluoride, Tris base, and Tricine (N-tris(hydroxymethyl)- methylglycine) from Sigma; DEAE-cellulose, ethylenediaminetet- raacetic acid (disodium salt), and sodium lauryl sulfate (SDS, laboratory grade) from Fisher; metmyoglobin (whale skeletal mus- cle, “A” grade) from Calbiochem; Photo-flo 200, sodium dithionite (90%), and N, N, N’, N’-tetramethylethylenediamine (TEMED) from Eastman; sodium citrate from Merck; sodium thioglycollate from Difco; UM-10 ultrafiltration membranes from Amicon Corp.; and Petri dishes (plastic, sterile, 50 x 12 mm with tight lid) from Falcon/ Becton, Dickinson, and Co.

Dialysis tubing, obtained from the Food Products Division of Union Carbide, was boiled in 0.2 rnM EDTA before use. Pyridine, obtained from Fisher, was distilled from ninhydrin and stored over Linde molecular sieve 4A and dry KOH pellets.

Methods

Reduced Pyridine Hemochrone Spectra - Reduced pyridine hemo- chromes were formed by the method of Paul et al. (4). Additions were

2946

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Isolation and Study of Bovine Erythrocyte Green Hemoproteins 2947

made to 2.4 ml of hemoprotein dissolved in 50 rn~ potassium phosphate, pH 7.2, in the following order: 0.525 ml of pyridine, 0.375 mmol of NaOH (75 ~1 of 5 N NaOH), and a few grains of dithionite. The modified Thunberg cuvette was made anaerobic after the addition of pyridine. The additions and the recording of the spectra were performed as rapidly as possible. Spectra were recorded on an Aminco-Chance or Cary model 17 recording spectrophotometer.

Molecular Weight Determination by Gel Exclusion Chromatogra- phy- Gel exclusion was carried out on a column (1 x 115 cm) of Bio- Gel P-60, -400 mesh, using 0.05 M potassium phosphate buffer, pH 7.2. Cytochrome c, myoglobin, chymotrypsinogen A, pepsin, oval- bumin, and y-globulin were used as standards (1 mg each).

Molecular Weight Determination by SDS’ Electrophoresis - Poly- acrylamide gel electrophoresis in the presence of SDS and 2-mercap- toethanol was performed by two methods: that of Weber and Osborn (5) and that of Laemmli (6), the former using phosphate, and the latter, Trisiglycine, as buffers. In each method, 5 to 10 kg of bovine serum albumin, ovalbumin, lactate dehydrogenase, and myoglobin were used as standards. In order both to speed up the rate of denaturation and to avoid proteolysis (7) the protein samples were delivered directly into a solution of 1% SDS plus 1% 2-mercaptoeth- anal on a boiling water bath. After 3 min the samples were cooled and maintained at 40°C for 2 h to ensure complete reduction of any disultide bonds. In addition, the upper electrophoresis buffer was made 0.05% in 2-mercaptoethanol.

The gels were fixed in water/acetic acid/methanol (4:1:4) and stained for protein using Coomassie brilliant blue R-250 dissolved in fixing solution. The gels were destained by placing the gels in small screwcap culture tubes containing fixing solution in which also was included Bio-Rad analytical grade anion exchange resin beads, AG l-X8, 20 to 50 mesh, in the hydroxide form. These beads irreversibly bound the dye so that all excess dye could be removed in one extraction.

Isoelectric Focusing- Isoelectric focusing was performed on poly- acrylamide gels by a modification of the methods of Wrigley (8) and Drysdale et al. (9). LKB carrier ampholytes, pH 3 to 10, were used at a concentration of 0.3 ml/12 ml of gel solution. Gels were made from 7.5% acrylamide and 0.25% bisacrylamide using 1% ammonium persulfate as a polymerizer (0.7 ml for 12 ml of gel solution). Polymerization was complete within 1 h. The gels were pre-electro- phoresed for 10 min utilizing 0.02 M H,PO,, at the anode (top) and 0.01 M KOH at the cathode (bottom). A sucrose solution (0.05 ml of 5% sucrose) was then layered over the gels. Protein solutions, in 10% sucrose plus 2.5% ampholytes, were layered between protective 5% sucrose and the gels. The focusing was run at 4°C for 3 h during which the current dropped to zero. In order to determine the pH profile of the gels, a blank gel was run, extruded from the gel tube, frozen, and cut into l-mm sections with a Mickle slicer. The slices were left overnight at 4°C in 0.5 ml of distilled water, and the pH was read at 2°C. The sample-containing gels were washed three times for 8 h each in 200 ml 5% trichloroacetic acid (w/v) to fix proteins and remove ampholytes and then were stained overnight in 0.25% Coomassie brilliant blue R-250. The gels could not be de- stained by diffusion at room temperature; therefore, they were placed in screwcap culture tubes with 7% acetic acid and destained overnight in a shaker bath at 70-80°C.

Polyacrylamide Gel Electrophoresis ~ “Standard” polyacrylamide gels were prepared and electrophoresis performed at 4°C essentially according to the method of Brewer and Ashworth (lo), with and without a stacking gel as indicated. The stacking gel contained Tris/ phosphate buffer, pH 6.9, and the separating gel Tris-HCl, pH 8.9. Electrophoresis was performed at pH 7.6 by replacing Solution “A’ of Brewer and Ashworth with 0.5 M Tricine and TEMED (1:400, v/v) and adjusting the pH to 7.6 with KOH; the electrode buffer was 0.01 M Tricine, pH 7.6. For electrophoresis at pH 6.9, Solution A was replaced with 0.08 M KH,PO, and TEMED cl:435 v/v) which had been adjusted to pH 6.9 with KOH; the electrode buffer was 0.01 M potassium phosphate, pH 6.9. The glass tubes (8 x 0.5 cm) for electrophoresis were thoroughly cleaned and then rinsed with Photo- flo:water (1:50) in order to facilitate the eventual removal of the gels from the tubes. Samples were made 10% in sucrose and were layered under the electrode buffer on top of the gel with a Hamilton syringe. Immediately after electrophoresis, gels were scanned at both 280 and 416 nm in order to distinguish hemoprotein bands from any

’ The abbreviations used are: SDS, sodium dodecyl (lauryl) sul- fate; TEMED, N, N, N’, N’-tetramethylethylenediamine.

bands of free hemin or apoprotein that may have been generated. Cellulose Acetate Electrophoresis - Electrophoresis was performed

according to the Gelman Electrophoresis Manual (11) using Sepra- phore III strips (1 x 6 inches) and Gelman “high resolution buffer” (Trisibarbitalisodium barbital, pH 8.8; ionic strength, 0.09). After application of 60 /*g of sample, the strip was electrophoresed for 45 min at a current of 2.5 mA/strip, stained in Ponceau S (0.5 g/l00 ml of 5% aqueous trichloroacetic acid), and destained in three successive 5% trichloroacetic acid washes.

Purification and Separation of Two Forms of Green Hemoprotein - The green hemoprotein was purified from bovine erythrocytes in a fashion similar to that published for the isolation of the human hemoprotein (3). The present procedure differs from the previous method in that the second chromatography on DEAE-cellulose was replaced by a series of chromatographic purifications on DEAE- Sephadex.

Obtaining Blood- Fresh bovine blood was procured from Kappler Packing Co. The blood was collected from the steer in a container that already contained acid/citrate/dextrose as anticoagulant (4.4 g of sodium citrate, 1.6 g of citric acid, and 4.9 g of dextrose in 200 ml water/liter of whole blood).

Hemolysis of Cells and Removal of Strom&-The erythrocytes were washed three times at 4°C by centrifuging a suspension of cells in cold 0.15 M NaCl solution (2,000 x g for 10 min) and aspirating off the buffy coat and supernatant fraction. Packed cells (3.5 liters) were lysed by the addition of 3 volumes of cold water and the suspension was frozen at -70°C. After several days, the lysed cells were thawed in a shaker-water bath, the pH was adjusted to 5.7 to 5.8 with 1.2 M HCl, and the stromata were sedimented by centrifu- gation at 13,000 x g for 60 min. The stromata were discarded. The supernatant fluid was adjusted to pH 7.2 with 5 M KOH and centrifuged again at 13,000 x g for 30 min. The precipitate was discarded and the supernatant fraction was diluted with 4 volumes of cold water.

DEAE-cellulose Chromatography - The diluted, stromata-free hemolysate was applied to a column (8 x 70 cm) of DEAE-cellulose (equilibrated in 3 rn~ phosphate buffer, pH 7.2) at a rate of 15 ml/ min. The column was then washed successively with 15 liters of 3 mM phosphate buffer (pH 7.21, 0.5 liter of 5 mM KH,PO, containing 5 rnM KCl, and 3.5 liters of 10 rnM KH,POI containing 10 rnM KCI. A linear gradient was then set up consisting of 4 liters of 10 mM KH,PO, containing 10 rnM KC1 in the mixing chamber and 4 liters of 50 rnM KH,PO, in the reservoir. The green hemoprotein was found to be eluted toward the end of this gradient. When the gradient was complete, a wash of 50 mM KH,PO, was applied until all green hemoprotein had been eluted from the column.

The profile resulting from elution with the combined ionic strength and pH gradient is given in Fig. 1. Superoxide dismutase and glutathione peroxidase were eluted before the green hemopro- teins. Since no pink copper protein (12) was isolated by this proce- dure, the superoxide dismutase could be detected by its copper content. In order to assay for glutathione peroxidase by the method outlined by Cook and Lands (131, it was necessary to make the assay mixture 0.1 rnM in sodium azide to prevent catalase from decompos- ing the hydrogen peroxide substrate. Since superoxide dismutase and glutathione peroxidase could readily be further purified by Sephadex G-100 chromatography, the described procedure provides a simple method for isolating these enzymes from bovine erythro- cytes.

Appearance of the green hemoprotein in the effluent was heralded by a sudden drop in the pH to approximately 6.5. The green hemoproteins were detected in Fractions 171 to 330, but only after chromatographic removal of contaminating chromoproteins on Am- berlite CG-50 (see “Results and Discussion”) was it possible to detect the characteristic reduced pyridine hemochrome absorbance peaks of these hemoproteins. When the early and late fractions containing green hemoprotein were pooled separately, subsequent purification and analyses demonstrated that the early fractions were rich in form I and the late fractions rich in form II. In many preparations the green hemoprotein was pooled to give a single fraction.

Catalase is usually eluted with the green hemoprotein. Soluble cytochrome bi bound tightly to the column and was eluted with 0.2 M KH,PO, as described for human erythrocyte cytochrome b-> (14).

Detection of Green Hemoprotein- In the absence of hemoglobin, green hemoprotein was detectable visually (yellow-green appear- ance when dilute or green-brown when more concentrated), or spectrally, by its distinctive pyridine hemochrome. Due to the presence of large amounts of contaminating hemoglobin it was not

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2948 Isolation and Study of Bovine E rythrocyte Green Hemoproteins

FIG. 1. DEAE-cellulose chromatography of crude erythrocyte su- pernatant fraction. See “Methods” for details. Glutathione peroxi- dase was detected in Fractions 115 to 140, superoxide dismutase (three peaks) in Fractions 15 to 175, and the green hemoproteins in Fractions 171 to 330. Erythrocyte cytochrome b-> was detected in fractions above 1000. Fraction volume was 22 ml. dl, resistance.

possible to use a visual or spectral method with the crude fractions obtained in the DEAE-cellulose chromatography step. In order to locate the green hemoprotein, the following purification procedure was performed on small aliquots: 1) an equal volume of 50 mM phosphate buffer, pH 6.0, was added to approximately 20 ml of sample and the pH was adjusted to 6.0 with 1.2 M HCl; 2) this diluted sample was chromatographed on a column of Amberlite CG-50 (1 x 9 cm) as described under “Results and Discussion”; 3) whereas contaminating hemoproteins bound to the CG-50, the green hemo- protein did not and was subsequently washed from the column with 15 ml of 50 rnM phosphate buffer, pH 6.0; 4) the total eluate was concentrated to 10 ml or less by ultrafiltration on a UM-10 mem- brane; 5) the spectrum of the reduced pyridine hemochrome was recorded. All fractions which gave a reduced pyridine hemochrome Soret maximum at a wavelength longer than 429 nm were pooled and further purified.

Anzberlitc~ CG-50 Chromatograph,y- The pooled crude fractions of green hemoprotein were made 0.1 mm in phenylmethylsulfonyl fluoride by the addition of an appropriate amount of a 0.1 M solution dissolved in 1-propanol. Phenylmethylsulfonyl fluoride was added to inhibit bacterial proteases even though erythrocyte proteases are notoriously stable toward such standard protease inhibitors (15). After adjusting the pH of the solution to 6.0 with 1.2 M HCl, the solution was passed down a column (4 x 15 cm) of Amberlite CG-50 which had been equilibrated with 50 rnM potassium phosphate buffer, pH 6.0. The column was then washed with 350 ml of the same buffer. The green hemoprotein passed through the resin whereas hemoglobin and other contaminating proteins remained bound to the top of the column.

Gc4 Filtration Chromatography- After making the eluate from the CG-50 column 0.1 mM in phenylmethylsulfonyl fluoride, the pH was adjusted to 7.2. The total eluate from the CG-50 was concen- trated by ultrafiltration on a UM-10 membrane to a volume of 15 ml, applied to a column (6.5 x 175 cm) of Bio-Gel P-60, and eluted using 50 rnM phosphate buffer, pH 7.2. All fractions with an absorbance ratio of A 1,11 ,,,,, /A. LHll ,,,,, greater than 0.9 were pooled.

DEAE-Sephadm Chromatography: Separation into Two Forms- The pooled eluate from the P-60 column was concentrated by ultrafiltration on a UM-10 membrane to a volume of 7 ml. This concentrate was dialyzed for 6 h against two changes of 500 ml of 10 rnM phosphate buffer, pH 7.5, containing 20 rnM KCl, and then applied to a DEAE-Sephadex column (2.6 x 31 cm) equilibrated in the same buffer. The hemoprotein was eluted with a linear gradient composed of 250 ml of 10 mM potassium phosphate buffer, pH 7.5, containing 20 rnM KCl, in the mixing chamber and 250 ml of 10 mM potassium phosphate buffer, pH 6.5, containing 50 mM KCl, in the reservoir. Two hemoproteins were eluted from the column, the first designated form I and the second form II (Fig. 2).

Furthrr Purification of Form I-The two forms were further purified by repeated chromatography on DEAE-Sephadex. Form I was dialyzed in 20 rnM Tris-HCI, pH 7.8, containing 30 mM KC1 and then applied in a small volume to a DEAE-Sephadex column (2.6 x 31 cm) (equilibrated in the same buffer). The protein was eluted

1 I I I I , ,

1.25 -

.25-

40 80 120 160 200 240 280 FRACTION *

FIG. 2. First DEAE-Sephadex chromatography of the green he- moproteins. The column was eluted with a linear gradient composed of 250 ml of 10 mM potassium phosphate buffer plus 20 rnM KC1 in the mixing chamber and 250 ml of 10 mM potassium phosphate buffer plus 50 mM KC1 in the reservoir. See “Methods” for details. The fractions were pooled according to the similarity of their reduced pyridine hemochrome spectra. Fractions 175 to 202 (with reduced pyridine hemochrome absorption maxima at 580, 544, and 434 nm), were pooled and designated form I; Fractions 210 to 285 (with reduced pyridine hemochrome absorption maxima at 573, 538, and 431 nm) were pooled and designated form II. Fraction volume was 8 ml.

with a linear gradient of 350 ml of the above buffer in the mixing chamber and 350 ml of 20 mM Tris-HCl plus 70 mM KCl, pH 7.8, in the reservoir. Chromatography on a third DEAE-Sephadex column gave additional purification. When forms I and II were not com- pletely separated on the first DEAE-Sephadex column, both forms were further purified as described in the following section for form II.

Further Purification of Form II- Form II was purified further by repeated DEAE-Sephadex chromatography. The DEAE-Sephadex was equilibrated in 10 mM potassium phosphate buffer, pH 7.2, containing 20 rnM KCl. After dialyzing the protein in the equilibra- tion buffer, the dialysate was applied to the column. Form II was eluted with a linear gradient consisting of 500 ml of 10 mM potassium phosphate buffer, pH 7.0, containing 20 rnM KCl, in the mixing chamber and 500 ml of 20 mM potassium phosphate buffer, pH 7.0, containing 20 mM KCl, in the reservoir. A third DEAE-Sephadex column gave additional purification (see Table I for details).

Storage of Two Forms - The purified proteins were frozen in small aliquots and stored at -70°C. When stored under these conditions there was no observable change in spectral properties, and no interconversion between the two forms. No interconversion between forms was observed after storage for a month at 4°C.

Elicitation of Antibodies- Male, New Zealand White rabbits, weighing 2 to 4 kg, were used for immunization. Purified hemopro- tein (mainly form I with some form II) was obtained by either DEAE-Sephadex chromatography or preparative polyacrylamide gel electrophoresis, concentrated to approximately 4 mg/ml, and mixed with an equal amount of complete Freund’s adjuvant to give a total volume of 1.0 ml. Crude hemoprotein (the Amberlite CG-50 flow- through) plus Freund’s adjuvant was injected into a third rabbit. The immunogen was first injected into both of the rear footpads but on successive administrations was injected subcutaneously at sev- eral sites on the rabbit’s back. Booster injections were given after 2 weeks. The animals were allowed to rest for 4 weeks and then were bled.

Purification of Inmunoglobulins- Approximately 35 to 40 ml of blood were collected from each rabbit from the marginal vein of an ear. The clot was allowed to form at room temperature for 0.5 h and then at 4” C for 3 h. The resultant sera were decanted and centri- fuged at 4000 x g for 10 min to remove any unclotted cells or other debris.

Hemoglobin was removed by ammonium sulfate precipitation of the IgG fraction (16). To the sera was added 0.6 volume of saturated (NH&SO, (0°C) that had been neutralized with ammonium hydrox- ide. The immunoglobulin-containing precipitate was obtained by centrifugation, washed with 2 ml of saturated (NH&SO,/H,O (6:10, v/v), dissolved in 20 ml of H,O, and reprecipitated by addition of 0.6 volume of saturated (NH&SO,. The precipitate was then dissolved in 10 rnM potassium phosphate, pH 8.0.

Agar Diffusion and Immunoelectrophoresis - Plates for agar-gel

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Isolation and Study of Bovine Erythrocyte Green Hemoproteins

diffusion were prepared according to the procedure of Goldstein and So (17) using a 1% solution of Noble agar in 0.85% saline buffered with 0.1 M phosphate, pH 7.2, but containing no azide.

Agar-gel electrophoresis was carried out on slides (1 x 3 inch) according to the Gelman manual (11) and to Clausen (18). After electrophoresis the appropriate solutes were allowed to diffuse for 24 to 36 h at 4°C.

Protein Visualization-The plate or slide was placed on a desicca- tor insert in a bucket and was washed in a large excess of 0.9% NaCl followed by three water washes for 8 h each. Slow mixing of the solution was effected by a magnetic stirrer. The proteins were stained with 0.05% aqueous Coomassie brilliant blue and nonspecif- ically bound dye was removed by three washes with large amounts of water.

Amino Acid Analysis- Lyophilized, desalted protein samples were hydrolyzed in 0.5 ml of constant boiling HCl. The hemin was not removed prior to hydrolysis. Duplicate samples were frozen, degassed, sealed under nitrogen, and hydrolyzed for 20 and 71 h at 110°C. A modified Phoenix model K82003 automated amino acid analyzer was used to quantitate the amino acids. Average values of amino acid residues (excluding tryptophan) were summed in order to obtain a value for protein.

RESULTS AND DISCUSSION

Purification of Two Green Hemoproteins- Two previously undetected green hemoproteins have been isolated from the erythrocytes of freshly drawn bovine blood. The proteins are present in the soluble fraction of hemolysates obtained by hypotonic lysislfreeze-thaw. The proteins were purified by a sequence of column chromatographic procedures involving DEAE-cellulose, Amberlite CG-50, Bio-Gel P-60, and DEAE- Sephadex. Table I summarizes the data for each step in the purification of the proteins from 3.5 liters of washed, packed cells.

The green hemoprotein was resolved into two distinct forms upon chromatography on DEAE-Sephadex using potassium phosphate buffer. The first green hemoprotein to be eluted was designated form I and the second, form II. Approximately

7.5 mg of form I and 10 mg of form II were obtained after twice repeating the chromatography on DEAE-Sephadex. These calculations were made using a molecular weight of 27,000 and the extinctions presented in the heading to Table I. The two forms could be clearly distinguished by differences in the spectra of their reduced pyridine hemochrome derivatives (Fig. 3).

Surprisingly, we could detect no charge differences between forms I and II. We observed (see below) that the holoproteins

The proteins were isolated from 3.5 liters of packed bovine erythrocytes. In order to estimate per cent yield, the yield from the DEAE-cellulose column was assumed to be 40% and the yield from the CG-50 column was assumed to be 90%, both based on per cent yield where known amounts of sample were applied to columns. Since the amount of the green hemoprotein at the initial steps of the purification could not be determined, the following assumptions were made in order to estimate the extent of purification. 1) The amount of protein (977 g) in the hemolysate was estimated from the amount of hemoglobin present (880 g), using the assumption that, in the supernatant fraction of the hemolysate, hemoglobin comprised 90% of the protein present. 2) The concentration of protein in subsequent stages was estimated using the assumption that a 1 mg/

step

TABLE I

Purification of green hemoprotein forms I and II

Diluted red cell supernatant fraction applied to DEAE-cellulose

DEAE-cellulose Amberlite CG-50 and concentration Bio-Gel P-60 1st DEAE-Sephadex:

Form I Form II

2nd DEAE-Sephadex: form I 3rd DEAE-Sephadex: form I 2nd DEAE-Sephadex: form II 3rd DEAE-Sephadex: form II

0 1 I I6 4 I I I I I I. 400 450 500 550 600 650

0

WAVELENGTHlnm)

FIG. 3. Reduced pyridine hemochrome spectra of form I and form II. The hemochromes were formed under anaerobic conditions as described under “Methods.” The absorbance scale at the right ordinate applies to wavelengths longer than 485 nm and that on the left to wavelengths shorter than 510 nm. The concentrations of form I and form II were 4.39 and 4.83 pM, respectively.

36.5

0.946 1.04 1.10 3.5 x 103 7.5 40.0 40 120 120.0 3.39 3.67 1.08 171 6.72 36.0 90 1180 9.8 1.41 2.10 1.49 224 5.04 27.0 75 1650 1.4

0.405 0.762 1.49 185 1.51 8.1 61 3620 2.2 0.153 0.284 1.86 520 1.71 9.1 61 3790 2.3 0.270 0.567 2.10 121 0.74 4.0 49 5070 1.4 0.183 0.465 2.54 78 0.38 2.0 51 6080 1.2 0.412 0.901 2.19 58 0.60 3.2 35 4540 1.2 0.110 0.268 2.44 89 0.28 1.5 46 5000 1.1

ml of protein solution has an A,,,, = 1.0. 3) After Amberlite CG-50 chromatography no other chromophores were present, and so the ratio of absorbance at 416 nm to the absorption at 280 nm was taken as an index or purity. 4) The -fold purification by the first DEAE- Sephadex column was adjusted to account for the separation of the two forms from each other. For example, even though there was little increase in purity of form I over extraneous protein, the separation of 1.71 pmol of form II from 1.51 pmol of form I yielded a 2.2-fold increase of purity of form I. 5) A millimolar extinction of 93 at 417 nm was used for the partially purified, but unresolved, mixtures of forms I and II. After resolution, a millimolar extinction of 93 at 416 nm was used for form I and 87 at 418 nm for form II (2’2).

Fraction vol- Yield Purification ume Amount

Overall 8teP Overall 8mP ml pLol %

127.0 3.48 5.4 x 104 18.7

-fold -

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2950 Isolation and Study of Bovine Erythrocyte Green Hemoproteins

have identical electrophoretic mobilities on cellulose acetate at pH 8.6 and on polyacrylamide gel at pH 8.9, 7.6, and 6.8 and show identical behavior upon isoelectric focusing, suggest- ing the possibility that the resolution on DEAE-Sephadex was not effected on the basis of net charge differences. Separation might be accomplished on the basis of differing abilities of the hemoproteins to be liganded by functional groups of the DEAE-cellulose and DEAE-Sephadex. In this sense the resin would be effecting separation more on the basis of affinity than ion exchange. Precedence for a separation of this type has been seen with the separation of free hemin dicarboxylic acids on poly-f-caprolactam (polyamide) resin (19). Alterna- tively, the separation may be due to dissimilar charge distri- bution on the protein surfaces of the two forms.

Elwtrophoretic Behavior- Forms I and II exhibited identi- cal mobilities upon electrophoresis in the presence of SDS by the methods of Laemmli (pH 8.8 in Tris buffer; Ref 6) or Weber and Osborn (pH 7.0 in phosphate buffer; Ref. 5). The two forms co-migrated when placed on a single gel. Each form gave a single protein band in each electrophoretic system. However, in some cases an opaque white band of unknown origin was detected approximately 1.5 cm down the gel.

Forms I and II also exhibited homogeneity upon electropho- resis on cellulose acetate strips at pH 8.6. The two forms exhibited identical mobilities in this system.

The two hemoproteins likewise co-migrated upon polyacryl- amide gel electrophoresis at pH 7.6 in potassium/Tricine buffer and at pH 6.9 in potassium phosphate buffer. In these systems both forms I and II migrated as a single holoprotein band with only a small amount of tailing.

In the pH 8.9 Tris/glycine system the migration of the two holoproteins was also indistinguishable. However, form II ran as a single band with a small amount of tailing, whereas form I exhibited multiple peaks after electrophoresis at pH 8.9 in the absence of stacking gel. Scanning at 280 and 416 nm

demonstrates that in addition to the holoprotein band (at 3.2 cm), a second hemoprotein (at 2.8 cm), a protein without heme (at 2.5 cm), and free hemin (at 5.2 cm) were also present (see Fig. 4, Scans A of Plates a and b). No protein migrated with the free heme as evidenced by a complete lack of Coomassie blue staining. When a l-cm stacking gel was used on top of the separating gel, the amount of heme-free protein and the amount of free heme increased while the amount of holopro- tein decreased; with a 2.5cm stacking gel this apparent dissociation of holoprotein into apoprotein and free hemin proceeded to such an extent that the heme-free protein became the major protein (data not shown). A large degree of dissocia- tion of the hemoprotein was also accomplished by performing the electrophoresis in the presence of cyanide (Fig. 4, Scan B), as reported previously (20). A higher cyanide concentration resulted in more dissociation (Scan C). Scan D shows that dissociation was nearly complete when electrophoresis was performed in the presence of cyanide and with a stacking gel. Proteins migrated somewhat faster in the presence of cyanide, apparently as a result of cyanide increasing the pH of the upper electrophoresis buffer.

Purified holoprotein was also shown to dissociate to apopro- tein and free hemin. Purified protein was obtained by conserv- atively excising the holoprotein band from a standard pH 8.9 gel and eluting it with water. When this fraction was rerun on a similar gel, the apoprotein band (at 2.5 cm), the holopro- tein band (at 3.2 cm), and the free hemin band (at 5.2 cm) were again present in their usual proportion.

The origin of the minor hemoprotein band (at 2.8 cm) was not established. It possesses approximately 3% of the Soret absorption of the main hemoprotein band. It was not gener- ated when the holoprotein was rerun on a gel and the amount of this species did not appear to be enhanced when stacking gels were employed. Like forms I and II, this hemoprotein showed the unusual property of losing its hemin during

a

FIG. 4. Absorbance scans of polyacrylamide gels (pH 8.9) of form I directly after electrophoresis in the presence and absence of cyanide. The separating gels were prepared from 10% acrylamide and 0.2% bisacrylamide. Tracings labeled A show absorbance of sample electrophoresed in absence of cyanide. Tracings labeled B and C show absorbance of samples electrophoresed in the presence of 20 and 30 rnM cyanide, respectively. The conditions of electrophoresis

I I I ,

b

ofD were the same as C except that a l-cm stacking gel was layered on top of the gel column. Approximately 3 nmol of the hemoprotein had been layered on top of each gel column. PZate a, absorbance at 416 (tracing A) and 430 nm (tracings B to D) detects holoprotein, free hemin, and cyanide complexes of holoprotein and hemin. Plate b, absorbance at 280 nm, of same gels as above, detects both protein- containing and hemin-containing species.

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Isolation and Study of Bovine Erythrocyte Green Hemoproteins 2951

electrophoresis in the presence of cyanide. It is possible that this species is an artifact derived from the green hemopro- teins.

The addition of reducing agents increased the heterogeneity of form I. Incubation of form I with 25 mM reduced glutathione at 37°C for 5 h resulted in its almost complete destruction as evidenced by the absence of any discrete protein bands on the gel. Such destruction of the hemoprotein by reduced glutathi- one and other reducing agents is associated with the formation of peroxides in the system (21).2 Preincubation of the sample with low concentrations of 2-mercaptoethanol, 2-thioglycol- late, or reduced glutathione at 4°C for a few minutes produced additional heterogeneity of the preparation at the expense of the holoprotein. These findings make it unlikely that the bands of form I are due to heterogeneity of oxidation state of the protein-sulfhydryl groups.

Forms I and II of the hemoprotein showed identical behavior upon electrofocusing in polyacrylamide gels (Fig. 5). For each, two major bands were detected with isoelectric points of 5.95 and 5.83. A lesser band with an isoelectric point of 5.74 could also be observed. The proteins precipitated in the gel upon reaching their isoelectric points.

Molecular Weight and Heme to Protein Ratio - Forms I and II showed the same molecular weight as determined by SDS gel electrophoresis in the Tris and phosphate buffer systems. The average of two molecular weight determinations in the potassium phosphate system and one in the Tris system was 23,000 for each protein. By gel exclusion chromatography on Bio-Gel P-60 the two native green hemoproteins gave a molecular weight of 27,000 and co-eluted when run simulta- neously. The correspondence of this value with that deter- mined by SDS-gel electrophoresis shows the native protein to be present in solution as a single subunit.

The number of heme groups per monomer was calculated for forms I and II. Heme content was based on absorbance of the ferric forms at the Soret peak. A millimolar extinction of 93 at 416 nm was used for form I and millimolar extinction of 87 at 418 nm was used for form II (22). Protein was calculated from amino acid analysis. The value of 1 heme133,OOO daltons of protein was obtained for form I and 1 heme/30,000 daltons for form II. These values are consistent with the presence of one heme per monomer. The somewhat high values for protein in these determinations suggest the presence of apoprotein in these preparations.

Immunological Properties - Immunoglobulins prepared against a crude preparation of the green hemoprotein (anti- crude) showed seven discernible bands of varying intensities when diffused against a serial dilution of a crude preparation of the protein. The combined concentration of the two forms was approximately 8 nmol/ml (0.18 mg/ml) in the first well and was diluted by one-half in each successive well. The cross- reactivity of the anticrude with the purified hemoproteins was quite weak, as was the cross-reactivity of the antibody elicited by injection of a mixture of the hemoproteins that were highly purified by DEAE-Sephadex. Antibody elicited against the green hemoproteins purified by preparative polyacrylamide gel electrophoresis, however, gave a strong response when diffused against purified protein. This preparation was used in the experiments reported here. Possibly, the presence of small amounts of polyacrylamide improved the antigenic response of the rabbit against the green hemoproteins. Poly- acrylamide gel has been used as an adjuvant by others (23).

* L. J. DeFilippi, D. P. Ballou, and D. E. Hultquist, manuscript in preparation.

4 12 20 20 36 44 52 60 FRACTION NUEHBER

FIG. 5. The determination of the isoelectric points of forms I and II of the hemoprotein upon gel isoelectric focusing. Samples were applied to each gel under a protective layer of sucrose and electro- phoresis was carried out as described under “Methods.” Photographs of the stained gels, containing form I (top) or form II (bottom), are inserted beneath the pH profile of a third, blank gel. The arrow heads show the positions of the three protein bands of forms I and II. The numbers are the pH values corresponding to each band. The anode is to the left and the cathode to the right of the figure. The electrofocusing and the pH determinations were performed at 0-4°C.

With anti-green hemoprotein in the center well and serial dilution of form I of the hemoprotein in the peripheral wells, a single sharp precipitin band was formed when diffusion was allowed to occur for 12 to 24 h. When allowed to diffuse for many days the single band separated into multiple bands. This is interpreted as being due to loss of heme, a phenomenon seen with agar diffusion of hemoglobin against antihemoglo- bin (24). In support of this interpretation, diffusion of apo- form I against anti-green hemoprotein (20) gave a single sharp band with no spurs, even after a few days.

As shown in Fig. 6 diffusion of this antibody against highly purified forms I and II gives a reaction of complete identity (between the center well and Wells 1 and 2), demonstrating that these two antigens are either completely identical or they have a number of antigenic groups in common and the antibodies present are against these common antigens only. The antibodies have no cross-reactivity against bovine hemo- globin (Well 3) or the other proteins present in the crude antigenic mixture (Wells 4, 5, and 6). Likewise, antibodies prepared against bovine hemoglobin do not cross-react with either form I or II (data not shown).

A single sharp precipitin band was formed when the anti- crude preparation (whose titre against green hemoprotein had been boosted by the addition of antibody against purified protein) was placed in the center well and allowed to diffuse against the two forms of the hemoprotein (adjacent to Wells 1 and 2 in Fig. 7). There was no cross-reactivity against hemo- globin (Well 3) while multiple bands were detected upon

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Isolation and Study of Bovine Erythrocyte Green Hemoproteins

.

FIG. 6 (left). Agar-gel diffusion of anti-green hemoprotein against form I, form II, hemoglobin, and crude green hemoprotein by the Ouchterlony method. The central well contained an (NH&SO,-purified antiserum from a rabbit that had been injected with the two green hemoproteins, purified by preparative polyacryl- amide gel electrophoresis. Well 1 contained 18 pg of form I; Well 2, 18 Fg of form II; Well 3, 30 pg of bovine hemoglobin; Well 4, 80 yg of protein of a sample of crude green hemoproteins eluted from the Amberlite CC-50 column; Well 5, same as Well 4, but 40 /*g; Well 6, same as Well 4, but 20 pg. Diffusion was allowed to proceed at room temperature for approximately 36 h.

FIG. 7 (right). Agar-gel diffusion of anticrude green hemoprotein against form I, form II, hemoglobin, and crude green hemoprotein by the Ouchterlony method. The central well contained (NH&SO,- purified antiserum from a rabbit that had been injected with crude green hemoprotein obtained from the Amberlite CG-50 column. Diffusion was allowed to proceed at room temperature for approxi- mately 36 h. See Fig. 6 for composition and concentration of moieties in peripheral wells.

diffusion against the crude protein preparation (Wells 4, 5, and 6). Since we visualized multiple antigens as precipitin bands in the crude preparation but only one antigen in the purified preparation, this is further evidence for homogeneity of the purified hemoproteins.

After immunoelectrophoresis in an agar gel, form I ex- hibited a single precipitin band when diffused against either anticrude or antipurified green hemoprotein. An identical pattern was exhibited by form II. Forms I and II had identical mobilities and immunological behavior when electrophoresed together or side by side.

These results clearly demonstrate that, within the limita- tions of immunological and electrophoretic techniques, the two forms are extremely similar if not identical, that the purified preparation used to elicit an antigenic response was essentially homogeneous, and that the hemoproteins bear no immunologically detectable structural relationship to hemo- globin.

Spectral Comparison of Two Hemes - The reduced pyridine hemochromes of the proteins show Soret, p, and (Y absorption maxima for form I at 434, 544, and 580.5 nm, respectively, and those for form II at 431, 538, and 573 nm, respectively. The hemochrome spectrum of form I of the bovine protein corre- sponds closely to that of the green hemoprotein isolated in this laboratory from human erythrocytes (3). It is interesting that the reduced pyridine hemochrome of the heme of form II of the bovine protein is spectrally similar to that first reported for the human protein by Morrison (1). Thus, it is likely that

two forms of the green hemoprotein are also present in human erythrocytes.

Since the hemochrome is formed under conditions which denature proteins and completely replace the natural ligands to the heme by pyridine, the differences in the spectra of the hemochromes of forms I and II reflect a structural difference between the prosthetic groups of the two forms. Evidence that the pyridine hemochrome formation procedure indeed results in formation of the dipyridine heme complexes is provided by the finding that these spectra are identical to the spectra of the pyridine hemochromes of the isolated free hemes.3 The Soret, /3, and (Y absorbance maxima of the hemochrome of form I are reproducibly found 3, 6, and 7.5 nm to the red of the corresponding maxima of form II. It has been demonstrated by others (25) that shifts of hemochrome spectra to longer wavelengths result from increases in the electron-attracting power of side chain substituents. Thus, the hemin of form I possesses substituents that have a greater electron withdraw- ing ability than the substituents of form II. To our knowledge, this is the first time a hemoprotein has been shown to exist as two different forms differing in prosthetic group.

Acknowledgments -We would like to thank Richard Dean and Richard Douglas for their help and ideas during the initial phases of this research, Linda Toler for her help with electrophoresis experiments, Minor J. Coon and Eugene E. Dekker for use of various facilities in their laboratories, and the laboratory of Halvor Christensen for performing the amino acid analyses.

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3 Unpublished observations.

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L J DeFilippi and D E Hultquistcharacterization.

The green hemoproteins of bovine erythrocytes. I purification and

1978, 253:2946-2953.J. Biol. Chem. 

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