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A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2
ANALYTICAL
BIOCHEMISTRY
A Simple
110,
Method
431-436 (1981)
for Preparation of Valency Hybrid ((~~+p~+)~and (ar2+p3+),
AKIOTOMODAANDYOSHIMASA Department
of Biochemistry,
Kanazwa
University
School
Hemoglobins,
YONEYAMA of Medicine,
Kanazawa,
Ishikawa
920, Japan
Received April 22, 1980 The improved methods for the preparation of valency hybrid hemoglobins, (~~*/3*+)~ and (a*+@+), were presented. The (~r~+$‘)~ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (aZ+j33+)2 valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with fenicyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (cE”p*+)2 and (az+&?a+)*valency hybrids.
Since Banerjee and Cassoly reported that the valency hybrid hemoglobins such as (a3+/32+)2and ((u~+@~+)~ are preparable by combining the isolated ferrous (Y and ferric p chains or the isolated ferric (Yand ferrous /3chains (l), thesehybrid hemoglobins were subjected to the studies of allosteric properties of hemoglobin (2-5). This method is composed of three procedures; (a) isolation of (r and /II chains, (b) oxidation of isolated chains (very unstable), (c) combination of ferrous and ferric chains. After combination of the chains, the separation of the reconstituted valency hybrid hemoglobins from excess of free chains is necessary. Therefore, it takes about a week to prepare the reconstituted valency hybrid hemoglobins, when this method is used. Recently we studied the changes in the intermediate hemoglobins such as ( a3+P2f)2 and (~r”+p”+)~ valency hybrids during the redox reaction of hemoglobin by various reagents and enzymes (6-9). During the reduction of methemoglobin by ascorbic acid under anaerobic conditions, we observed that the (a3+p2’J2 valency hybrid is dominant (6). Furthermore, we found that the (01~+@+)~valency hybrid is predomi431
nantly present in hemoglobin solutions which were partially oxidized by ferricyanide under anaerobic conditions (unpublished data). On the basis of these findings, we tried to purify these intermediate hemoglobins using column chromatography. In this paper, the method for the preparation of (a3+p2+), and ((Y~+@+)~valency hybrids using CM 32 column chromatography is presented. This method has the merit of a very simple procedure, because preparation of isolated 1yand /3 chains of hemoglobin is excluded, and so the bulk preparation of valency hybrids such as ( (Y~+P~+)~ and ((Y~+P~+)~ is feasible. MATERIALS
AND METHODS
Materials. Sephadex G-25 (coarse), and CM Sephadex C-50 were purchased from Pharmacia. CM 32, and Dowex 1 x 8 were obtained from Whatman and Muromachi (Tokyo), respectively. Sodium ascorbate, potassium ferricyanide, and p-chloromercuribenzoate (PCMB)’ were purchased from Wako (Tokyo). Ampholine plate gel (pH 3.5-9.5) was obtained from LKB. Cata’ Abbreviation
used:
PCMB,p-chloromercuribenzoate.
0003-2697/81/020431-06$02.00/O Coptight 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
432
TOMODAANDYONEYAMA
lase and superoxide dismutase were purchased from Boehringer and Sigma, respectively . Preparation of hemoglobin and methemoglobin solutions. Freshly obtained hu-
man bloods were centrifuged at 3000 t-pm for 10 min, and red cells were collected after removal of the buffy coats. These red cells were washed twice with 10 vol of icecold 0.9% NaCl, and were hemolyzed with 5 vol of ice-cold distilled water. After addition of 5-ml aliquots of 1 M NaCl to 45 ml hemoglobin solutions, the mixture was centrifuged at 10000r-pm for 20 min at 4°C. Half of the supernatant was converted to methemoglobin with ferricyanide (molar ratio; heme:ferricyanide = 1:6). After standing 15 min at room temperature, the solutions were passed through a column of Dowex 1 x 8 (washed with distilled water until neutralized, 1 x 3 cm length) for removing ferricyanide from the solutions. The elution of methemoglobin was carried out by a distilled water. The effluents were used for the partial reduction of methemoglobin by ascorbic acid under anaerobic conditions as mentioned in next paragraph. The other half of the supernatant was used for the partial oxidation with ferricyanide under anaerobic conditions as described later. Purification
of (~8+/3”‘)~ valency hybrid.
effluents (610 pM in heme) was applied on a column of CM 32 (1.8 x 20 cm length) equilibrated with 10 mM potassium phosphate buffer, pH 6.8. The sample which adsorbed at the top of the cellulose, was eluted by 400 ml of 10 mM potassium phosphate buffer, pH 7.48. In each tubes of the fraction collector, 20 ~1 of catalase and superoxide dismutase solutions (13,000 units and 7500 units/ml, respectively) were preloaded in order to prevent the autoxidation of the (LY~+/~~+)~ valency hybrid. The flow rate was about 15 ml effluents per hour. Purijcation
of (~2+@+)~ valency hybrid.
Since the (cP/~~+)~valency hybrid is mainly present in hemoglobin solutions which were partially oxidized by ferricyanide under anaerobic conditions, this valency hybrid was purified by using CM 32 column chromatography. A 5-ml hemoglobin solution (3.7 mM in heme) was placed in a Thunberg-type cell, and was deoxygenated by replacement of air with Q gas. After 30 min standing at room temperature, the oxidation of hemoglobin was initiated by the addition of 25 ~1 of 400 mM ferricyanide solution, which was previously placed in the side arm of the cell. The reaction mixture was collected from the cell after 2 min, and immediately applied on a column of Sephadex G-25 (3 x 25 cm length) previously equilibrated with 10 mM potassium phosphate buffer (pH 6.8). A 25-ml sample of the effluent (685 PM in heme) was applied on a column of CM 32 (1.8 x 24 cm) previously equilibrated with 10 mM potassium phosphate buffer, pH 6.8. The samples were eluted with 400 ml of 10 mM potassium phosphate buffer, pH 7.41. The flow rate was about 25 ml effluents per hour. Catalase and superoxide dismutase were already preloaded in the fraction tubes for the prevention of autoxidation of ( cw2+p3+)2 valency hybrid.
A lo-ml aliquot of methemoglobin solutions (1.8 mM in heme) was placed in a Thunberg-type cell, and was deoxygenated by the replacement of air with Q gas (helium: isobutane = 99.05:0.95). The reduction of methemoglobin was initiated by the addition of 0.5 ml of sodium ascorbate solution (400 m&f) which was previously placed in the side arm of the cell, and the solution was kept at 25°C for an hour. Then the reaction mixture was passed through a column of Sephadex G-25 (coarse, 3 x 25 cm length) previously equilibrated with 10 mM potasIsoelectric focusing electrophoresis for sium phosphate buffer, pH 6.8,4”C. (All the column chromatographic procedures were the vafency hybrids. The partially oxidized carried out at 4°C). A 25-ml aliquot of the hemoglobin with ferricyanide, and the puri-
VALENCY
HYBRID
HEMOGLOBIN
PREPARATION
(aS+/30’)2 AND (aZ+@‘)2
433
$28 20 4 4I f 1.0 2 * I 0
IO
A
0 0
20
30
fraction
IO
20
30 Fraction
40
50
60
60
70
Number
40
50
80
Number
FIG. 1. Purification of (a3’f12+)2 and (a2’@+)2 valency hybrids by CM 32 column chromatography. (A) Elution pattern of the partially reduced methemoglobin solutions with ascorbic acid. After methemoglobin was partially reduced by ascorbic acid under anaerobic conditions, the reaction mixture was passed through a column of Sephadex G-25 (coarse, 3 x 25 cm length), and applied on a column of CM 32 (1.8 x 20 cm length). The samples were eluted with 10 mM potassium phosphate buffer, pH 7.48, CC at 15 ml/h flow rates. (B) Elution pattern for the partially oxidized hemoglobin solutions with ferricyanide. The solutions of hemoglobin were partially oxidized by ferricyanide under anaerobic conditions, and were passed through a column of Sephadex G-25 (coarse, 3 x 25 cm length). The effluents were subsequently applied on a column of CM 32 (1.8 x 24 cm length). The samples were eluted with 10 mM potassium phosphate buffer, pH 7.41,4”C at 25 ml/h flow rates. The arrow in both figures shows the addition of 10 mM K,HPG, solution.
fied valency hybrids were applied to an Ampholine plate gel (pH 3.5-9.5) and isoelectric focusing was performed at 4°C for 1.5 h (settled at initial current 8 mA, final voltage 1200 V and constant 6 W). Then the gel plate was fixed with solutions containing 0.7 M trichloroacetic acid, 0.14 M sulphosalicylate, and 7 M methanol. The gel scanning was carried out at 630 nm.
Isoelectric focusing of PCMB-treated valency hybrid hemoglobins on Ampholine plate gel. The fractions containing (013+@2+)z,
which were eluted through CM 32 column (fraction Nos. 28-36), were pooled (4%ml samples, 31.8 PM in heme), and were applied on a column of CM Sephadex C-50 (1.5; x 2 cm length) previously equilibrated with 10 mM potassium phosphate buffer,
434
TOMODA
AND YONEYAMA
anaerobic conditions (7). This valency hybrid was separated from the partially reduced methemoglobin solutions with ascorbic acid through the CM 32 column as shown in Fig. 1A. The elution was carried out by 10 mM potassium phosphate buffer, pH 7.48. The intermediate hemoglobin was eluted subsequently after the fractions of **2 oxygenated hemoglobin. From the spectrophotometrical measurements, the interme+ Of 0 diate hemoglobin was found to be ( LY~+/~~+)~ FIG. 2. Isoelectric focusing profiles of the partially valency hybrid (not shown here). oxidized deoxyhemoglobin solutions with ferricyanide Figure 2 shows the isoelectric focusing on Ampholine plate gel. pattern of the hemoglobin solutions, which were partially oxidized by fenicyanide, on pH 6.8. The samples were eluted by the the Ampholine plate gel. As a result, there addition of 10 mM K,HPO,. By this pro- were four main bands including oxygenated cedure, the (L~+P~+)~was concentrated hemoglobin, intermediate hemoglobins, and to 278 PM in heme (5 ml). The (LY~+S~“)~ methemoglobin. The amounts of (LU~+/~~‘)~ valency hybrid, which was also concen- were, however, as much as six times trated by the same procedure, (from 34.5 greater than those of (cw~+~“+)~. We tried PM in heme, 45 ml to 290 PM in heme, 5 ml). to separate the (a2+p3+)* valency hybrid A solution containing these valency hy- from oxygenated hemoglobin, (cu3’j3”‘h, brid hemoglobins or oxyhemoglobin was and methemoglobin by using CM 32 column bubbled with CO gas and treated with chromatography. As shown in Fig. lB, PCMB (15-fold concentrations against oxygenated hemoglobins, (cr2fp”‘)2 and heme) according to the method of Mansouri (cx~+@+)~ clearly separatedfrom one another. and Winterhalter (10) for 4 h at 4°C. Since The identification of (ar2+p3+)2is mentioned ferric chains are insoluble and readily pre- in the next section. cipitate in the presence of PCMB, they These intermediate hemoglobins (fraction were removed from carboxy ferrous chains by centrifugation at 10,000 rpm for 15 min at 4°C. Then the solution was passed through Sephadex G-25 column equilibrated cx3+r32+) 2 with 10 mM potassium phosphate buffer (pH 7.0) and applied on the Ampholine plate gel. The isoelectric focusing was performed within 1.5 h at 4°C. The plate gel was fixed as mentioned above.
cc12+B3+? 2
RESULTS AND DISCUSSION
Purification of the (a”+$+) and (Q”$+)~ Valency Hybrids by CM 32 Column Chromatography
@I--+
0-
FIG. 3. Isoelectric focusing profiles of the purified intermediate hemoglobins. The samples including (aS’pO+)2 or (a”$+),, which were obtained by the We previously showed that only ( (Y~+/Y+)~ CM 32 column chromatography, were applied on valency hybrid is produced during methe- Ampholine plate gel, and the isoelectric focusing moglobin reduction by ascorbic acid under was performed at 4°C.
VALENCY
HYBRID
HEMOGLOBIN
PREPARATION
(a3+j?+), AND (oz+p3+)z
435
k!2+83*) 2
FIG. 4. Isoelectric focusing profiles of the PCMB-treated purified intermediate hemoglobins on Ampholine plate gel. PCMB-treated purified intermediate hemoglobins [( c&+B*+)~ and ( a2’/3”+)J were applied on Ampholine plate gel along with PCMB-treated control carboxyhemoglobin, after removal of precipitates by centrifugation. The isoelectric focusing was carried out at 4°C. The upper sample shows the isoelectric focusing pattern of the PCMB-treated (a3+pZ+)2. The middle and lower samples show the pattern of the PCMB-treated (oP+@+), and carboxyhemoglobin.
Nos. 28-36 in Fig. IA and Nos. 28-36 in Fig. 1B) were subjected to the isoelectric focusing on Ampholine plate gel, and were found to be a single entity electrophoretitally (Fig. 3). Identification of the ( CX~+P”+)~ and ( a2+@+j2 Vaiency Hybrids
It is well known that ferrous a and p chains are obtained when either oxyhemoglobin or carboxyhemoglobin was treated by PCMB (10). We observed that the a and /3 chains precipitate easily for their instability, when unliganded methemoglobin was split into their subunits by treatment with PCMB. It is therefore expected that only ferrous p chains or a chains will be obtained if the (a3+p2+)2 or the (a2+p3+)2 is separated into its subunits in the presence of PCMB. Consequently, only ferrous p chains were obtained when the (a3+fi2+)2 (fraction NOS. 28- 36 in Fig. 1A) was treated with PCMB (Fig. 4). Only ferrous OLchains were obtained in the case for the (a2+p3+)2 (fraction Nos. 28-36 in Fig. 1B). However, carboxyhemoglobin was separated into two main bands including ferrous a and /3 chains when treated with PCMB. These re-
sults show that the intermediate hemoglobins obtained by the present procedure are the ( (Y~+@~+)~and ( (Y~+P~+)~valency hybrids. The absorption spectra of these intermediate hemoglobins were consistent with those of the reconstituted (a3+fi2’)2 and (cu~+~~+)~ valency hybrids as shown prevously (6) (not shown here). Advantage of the Present Procedure for the Preparation of the ( CY~+$+)~ and ((Y~+P~+)~Valency Hybrids
Though we previously showed that the (cx~+~~+)~ valency hybrid produced during methemoglobin reduction by ascorbic acid is separable by CM Sephadex C-50 column chromatography (6), the rechromatography of the effluents was necessary for the purification of the (cx~+~~+)~because of the small amount of contamination of methemoglobin. According to the present method, not only the ((~~+p~+)~ but also the ( a2+p3+h valency hybrid is readily prepared without contamination of methemoglobin by oneprocess chromatography. By the present procedure, 45ml aliquots of the (a3’@2’)2 valency hybrid (31.8 PM in heme) were obtained from 2.5 ml aliquots of the partially
436
TOMODA
AND YONEYAMA TABLE
PURIFICATION
1
OF (~#+p*')~
AND (@p+)*
@‘P’),
(a”‘P’h
Step
Protein (rmol in heme)
Yield m
Protein (pm01 in heme)
Yield @)
Gel filtration on Sephadex Chromatography on CM cellulose
15.25 1.43
100 8.5
17.1 1.55
100 9.1
reduced methemoglobin solutions (610 PM in heme). The final yield of the valency hybrid was 8.5% (Table 1). With regard to the ( CX~+$+)~ valency hybrid, 45ml aliquots of the sample (34.5 PM in heme) were obtamed from 25-ml aliquots of the partially oxidized hemoglobin solutions with fen-icyanide (685 PM in heme). The final yield of the viency hybrid was 9.1%. This present method is very time saving compared with the method of Banetjee and Cassoly (1). They prepared the (c?+$+)~ and (cY~+~~+)~ valency hybrids by combining the isolated a and p chains, which were obtained by PCMB treatment of oxyhemoglobin tetramer. However, the isolated (Y and /3 chains are relatively unstable, and it takes about a week to prepare these valency hybrids. By the present method, the fractionation of (02+@$ and ((u3+/32+)2 can be carried out within several hours. Therefore, the valency hybrids are obtainable the next morning when the application and elution of the sample were initiated in the evening. Furthermore, these valency hybrids
can be prepared in quantity when higher concentrations of hemoglobin are applied to the cellulose. ACKNOWLEDGMENT We wish to discussion
thank Dr. M. Nagai for her useful
REFERENCES 1. Banejee, R., and Cassoly, R. (1%9) J. Mol. Biol. 42, 351-361. 2. Cassoly, R., and Gibson, Q. H. (1972) .I. Biol. Chem. 247,7332-7341. 3. Cassoly, R. (1975) J. Mol. Biol. 98, 581-595. 4. Cassoly, R. (1976) Eur. J. Biochem. 65,461-464. 5. Nagai, K. (1977) J. Mol. Biol. 111, 41-53. 6. Tomoda, A., Takeshita, M., and Yoneyama, Y. (1978) J. Biol. Chem. 253,7415-7419. 7. Tomoda, A., Tsuji, A., Matsukawa, S., Takeshita, M., and Yoneyama, Y. (1978) J. Biol. Chem. 253, 7420-7423. 8. Tomoda, A., Yubishui, T., Tsuji, A., and Yoneyama, Y. (1979) J. Biol. Chem. 254, 3119-3123. 9. Tomoda, A., and Yoneyama, Y. (1979) Biochim. Biophys. Acta 581, 128- 135. 10. Mansouri, A., and Winterhalter, K. H. (1973) Biochemistry 12,4946-4949.
BIOCHEMISTRY
A Simple
110,
Method
431-436 (1981)
for Preparation of Valency Hybrid ((~~+p~+)~and (ar2+p3+),
AKIOTOMODAANDYOSHIMASA Department
of Biochemistry,
Kanazwa
University
School
Hemoglobins,
YONEYAMA of Medicine,
Kanazawa,
Ishikawa
920, Japan
Received April 22, 1980 The improved methods for the preparation of valency hybrid hemoglobins, (~~*/3*+)~ and (a*+@+), were presented. The (~r~+$‘)~ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (aZ+j33+)2 valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with fenicyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (cE”p*+)2 and (az+&?a+)*valency hybrids.
Since Banerjee and Cassoly reported that the valency hybrid hemoglobins such as (a3+/32+)2and ((u~+@~+)~ are preparable by combining the isolated ferrous (Y and ferric p chains or the isolated ferric (Yand ferrous /3chains (l), thesehybrid hemoglobins were subjected to the studies of allosteric properties of hemoglobin (2-5). This method is composed of three procedures; (a) isolation of (r and /II chains, (b) oxidation of isolated chains (very unstable), (c) combination of ferrous and ferric chains. After combination of the chains, the separation of the reconstituted valency hybrid hemoglobins from excess of free chains is necessary. Therefore, it takes about a week to prepare the reconstituted valency hybrid hemoglobins, when this method is used. Recently we studied the changes in the intermediate hemoglobins such as ( a3+P2f)2 and (~r”+p”+)~ valency hybrids during the redox reaction of hemoglobin by various reagents and enzymes (6-9). During the reduction of methemoglobin by ascorbic acid under anaerobic conditions, we observed that the (a3+p2’J2 valency hybrid is dominant (6). Furthermore, we found that the (01~+@+)~valency hybrid is predomi431
nantly present in hemoglobin solutions which were partially oxidized by ferricyanide under anaerobic conditions (unpublished data). On the basis of these findings, we tried to purify these intermediate hemoglobins using column chromatography. In this paper, the method for the preparation of (a3+p2+), and ((Y~+@+)~valency hybrids using CM 32 column chromatography is presented. This method has the merit of a very simple procedure, because preparation of isolated 1yand /3 chains of hemoglobin is excluded, and so the bulk preparation of valency hybrids such as ( (Y~+P~+)~ and ((Y~+P~+)~ is feasible. MATERIALS
AND METHODS
Materials. Sephadex G-25 (coarse), and CM Sephadex C-50 were purchased from Pharmacia. CM 32, and Dowex 1 x 8 were obtained from Whatman and Muromachi (Tokyo), respectively. Sodium ascorbate, potassium ferricyanide, and p-chloromercuribenzoate (PCMB)’ were purchased from Wako (Tokyo). Ampholine plate gel (pH 3.5-9.5) was obtained from LKB. Cata’ Abbreviation
used:
PCMB,p-chloromercuribenzoate.
0003-2697/81/020431-06$02.00/O Coptight 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
432
TOMODAANDYONEYAMA
lase and superoxide dismutase were purchased from Boehringer and Sigma, respectively . Preparation of hemoglobin and methemoglobin solutions. Freshly obtained hu-
man bloods were centrifuged at 3000 t-pm for 10 min, and red cells were collected after removal of the buffy coats. These red cells were washed twice with 10 vol of icecold 0.9% NaCl, and were hemolyzed with 5 vol of ice-cold distilled water. After addition of 5-ml aliquots of 1 M NaCl to 45 ml hemoglobin solutions, the mixture was centrifuged at 10000r-pm for 20 min at 4°C. Half of the supernatant was converted to methemoglobin with ferricyanide (molar ratio; heme:ferricyanide = 1:6). After standing 15 min at room temperature, the solutions were passed through a column of Dowex 1 x 8 (washed with distilled water until neutralized, 1 x 3 cm length) for removing ferricyanide from the solutions. The elution of methemoglobin was carried out by a distilled water. The effluents were used for the partial reduction of methemoglobin by ascorbic acid under anaerobic conditions as mentioned in next paragraph. The other half of the supernatant was used for the partial oxidation with ferricyanide under anaerobic conditions as described later. Purification
of (~8+/3”‘)~ valency hybrid.
effluents (610 pM in heme) was applied on a column of CM 32 (1.8 x 20 cm length) equilibrated with 10 mM potassium phosphate buffer, pH 6.8. The sample which adsorbed at the top of the cellulose, was eluted by 400 ml of 10 mM potassium phosphate buffer, pH 7.48. In each tubes of the fraction collector, 20 ~1 of catalase and superoxide dismutase solutions (13,000 units and 7500 units/ml, respectively) were preloaded in order to prevent the autoxidation of the (LY~+/~~+)~ valency hybrid. The flow rate was about 15 ml effluents per hour. Purijcation
of (~2+@+)~ valency hybrid.
Since the (cP/~~+)~valency hybrid is mainly present in hemoglobin solutions which were partially oxidized by ferricyanide under anaerobic conditions, this valency hybrid was purified by using CM 32 column chromatography. A 5-ml hemoglobin solution (3.7 mM in heme) was placed in a Thunberg-type cell, and was deoxygenated by replacement of air with Q gas. After 30 min standing at room temperature, the oxidation of hemoglobin was initiated by the addition of 25 ~1 of 400 mM ferricyanide solution, which was previously placed in the side arm of the cell. The reaction mixture was collected from the cell after 2 min, and immediately applied on a column of Sephadex G-25 (3 x 25 cm length) previously equilibrated with 10 mM potassium phosphate buffer (pH 6.8). A 25-ml sample of the effluent (685 PM in heme) was applied on a column of CM 32 (1.8 x 24 cm) previously equilibrated with 10 mM potassium phosphate buffer, pH 6.8. The samples were eluted with 400 ml of 10 mM potassium phosphate buffer, pH 7.41. The flow rate was about 25 ml effluents per hour. Catalase and superoxide dismutase were already preloaded in the fraction tubes for the prevention of autoxidation of ( cw2+p3+)2 valency hybrid.
A lo-ml aliquot of methemoglobin solutions (1.8 mM in heme) was placed in a Thunberg-type cell, and was deoxygenated by the replacement of air with Q gas (helium: isobutane = 99.05:0.95). The reduction of methemoglobin was initiated by the addition of 0.5 ml of sodium ascorbate solution (400 m&f) which was previously placed in the side arm of the cell, and the solution was kept at 25°C for an hour. Then the reaction mixture was passed through a column of Sephadex G-25 (coarse, 3 x 25 cm length) previously equilibrated with 10 mM potasIsoelectric focusing electrophoresis for sium phosphate buffer, pH 6.8,4”C. (All the column chromatographic procedures were the vafency hybrids. The partially oxidized carried out at 4°C). A 25-ml aliquot of the hemoglobin with ferricyanide, and the puri-
VALENCY
HYBRID
HEMOGLOBIN
PREPARATION
(aS+/30’)2 AND (aZ+@‘)2
433
$28 20 4 4I f 1.0 2 * I 0
IO
A
0 0
20
30
fraction
IO
20
30 Fraction
40
50
60
60
70
Number
40
50
80
Number
FIG. 1. Purification of (a3’f12+)2 and (a2’@+)2 valency hybrids by CM 32 column chromatography. (A) Elution pattern of the partially reduced methemoglobin solutions with ascorbic acid. After methemoglobin was partially reduced by ascorbic acid under anaerobic conditions, the reaction mixture was passed through a column of Sephadex G-25 (coarse, 3 x 25 cm length), and applied on a column of CM 32 (1.8 x 20 cm length). The samples were eluted with 10 mM potassium phosphate buffer, pH 7.48, CC at 15 ml/h flow rates. (B) Elution pattern for the partially oxidized hemoglobin solutions with ferricyanide. The solutions of hemoglobin were partially oxidized by ferricyanide under anaerobic conditions, and were passed through a column of Sephadex G-25 (coarse, 3 x 25 cm length). The effluents were subsequently applied on a column of CM 32 (1.8 x 24 cm length). The samples were eluted with 10 mM potassium phosphate buffer, pH 7.41,4”C at 25 ml/h flow rates. The arrow in both figures shows the addition of 10 mM K,HPG, solution.
fied valency hybrids were applied to an Ampholine plate gel (pH 3.5-9.5) and isoelectric focusing was performed at 4°C for 1.5 h (settled at initial current 8 mA, final voltage 1200 V and constant 6 W). Then the gel plate was fixed with solutions containing 0.7 M trichloroacetic acid, 0.14 M sulphosalicylate, and 7 M methanol. The gel scanning was carried out at 630 nm.
Isoelectric focusing of PCMB-treated valency hybrid hemoglobins on Ampholine plate gel. The fractions containing (013+@2+)z,
which were eluted through CM 32 column (fraction Nos. 28-36), were pooled (4%ml samples, 31.8 PM in heme), and were applied on a column of CM Sephadex C-50 (1.5; x 2 cm length) previously equilibrated with 10 mM potassium phosphate buffer,
434
TOMODA
AND YONEYAMA
anaerobic conditions (7). This valency hybrid was separated from the partially reduced methemoglobin solutions with ascorbic acid through the CM 32 column as shown in Fig. 1A. The elution was carried out by 10 mM potassium phosphate buffer, pH 7.48. The intermediate hemoglobin was eluted subsequently after the fractions of **2 oxygenated hemoglobin. From the spectrophotometrical measurements, the interme+ Of 0 diate hemoglobin was found to be ( LY~+/~~+)~ FIG. 2. Isoelectric focusing profiles of the partially valency hybrid (not shown here). oxidized deoxyhemoglobin solutions with ferricyanide Figure 2 shows the isoelectric focusing on Ampholine plate gel. pattern of the hemoglobin solutions, which were partially oxidized by fenicyanide, on pH 6.8. The samples were eluted by the the Ampholine plate gel. As a result, there addition of 10 mM K,HPO,. By this pro- were four main bands including oxygenated cedure, the (L~+P~+)~was concentrated hemoglobin, intermediate hemoglobins, and to 278 PM in heme (5 ml). The (LY~+S~“)~ methemoglobin. The amounts of (LU~+/~~‘)~ valency hybrid, which was also concen- were, however, as much as six times trated by the same procedure, (from 34.5 greater than those of (cw~+~“+)~. We tried PM in heme, 45 ml to 290 PM in heme, 5 ml). to separate the (a2+p3+)* valency hybrid A solution containing these valency hy- from oxygenated hemoglobin, (cu3’j3”‘h, brid hemoglobins or oxyhemoglobin was and methemoglobin by using CM 32 column bubbled with CO gas and treated with chromatography. As shown in Fig. lB, PCMB (15-fold concentrations against oxygenated hemoglobins, (cr2fp”‘)2 and heme) according to the method of Mansouri (cx~+@+)~ clearly separatedfrom one another. and Winterhalter (10) for 4 h at 4°C. Since The identification of (ar2+p3+)2is mentioned ferric chains are insoluble and readily pre- in the next section. cipitate in the presence of PCMB, they These intermediate hemoglobins (fraction were removed from carboxy ferrous chains by centrifugation at 10,000 rpm for 15 min at 4°C. Then the solution was passed through Sephadex G-25 column equilibrated cx3+r32+) 2 with 10 mM potassium phosphate buffer (pH 7.0) and applied on the Ampholine plate gel. The isoelectric focusing was performed within 1.5 h at 4°C. The plate gel was fixed as mentioned above.
cc12+B3+? 2
RESULTS AND DISCUSSION
Purification of the (a”+$+) and (Q”$+)~ Valency Hybrids by CM 32 Column Chromatography
@I--+
0-
FIG. 3. Isoelectric focusing profiles of the purified intermediate hemoglobins. The samples including (aS’pO+)2 or (a”$+),, which were obtained by the We previously showed that only ( (Y~+/Y+)~ CM 32 column chromatography, were applied on valency hybrid is produced during methe- Ampholine plate gel, and the isoelectric focusing moglobin reduction by ascorbic acid under was performed at 4°C.
VALENCY
HYBRID
HEMOGLOBIN
PREPARATION
(a3+j?+), AND (oz+p3+)z
435
k!2+83*) 2
FIG. 4. Isoelectric focusing profiles of the PCMB-treated purified intermediate hemoglobins on Ampholine plate gel. PCMB-treated purified intermediate hemoglobins [( c&+B*+)~ and ( a2’/3”+)J were applied on Ampholine plate gel along with PCMB-treated control carboxyhemoglobin, after removal of precipitates by centrifugation. The isoelectric focusing was carried out at 4°C. The upper sample shows the isoelectric focusing pattern of the PCMB-treated (a3+pZ+)2. The middle and lower samples show the pattern of the PCMB-treated (oP+@+), and carboxyhemoglobin.
Nos. 28-36 in Fig. IA and Nos. 28-36 in Fig. 1B) were subjected to the isoelectric focusing on Ampholine plate gel, and were found to be a single entity electrophoretitally (Fig. 3). Identification of the ( CX~+P”+)~ and ( a2+@+j2 Vaiency Hybrids
It is well known that ferrous a and p chains are obtained when either oxyhemoglobin or carboxyhemoglobin was treated by PCMB (10). We observed that the a and /3 chains precipitate easily for their instability, when unliganded methemoglobin was split into their subunits by treatment with PCMB. It is therefore expected that only ferrous p chains or a chains will be obtained if the (a3+p2+)2 or the (a2+p3+)2 is separated into its subunits in the presence of PCMB. Consequently, only ferrous p chains were obtained when the (a3+fi2+)2 (fraction NOS. 28- 36 in Fig. 1A) was treated with PCMB (Fig. 4). Only ferrous OLchains were obtained in the case for the (a2+p3+)2 (fraction Nos. 28-36 in Fig. 1B). However, carboxyhemoglobin was separated into two main bands including ferrous a and /3 chains when treated with PCMB. These re-
sults show that the intermediate hemoglobins obtained by the present procedure are the ( (Y~+@~+)~and ( (Y~+P~+)~valency hybrids. The absorption spectra of these intermediate hemoglobins were consistent with those of the reconstituted (a3+fi2’)2 and (cu~+~~+)~ valency hybrids as shown prevously (6) (not shown here). Advantage of the Present Procedure for the Preparation of the ( CY~+$+)~ and ((Y~+P~+)~Valency Hybrids
Though we previously showed that the (cx~+~~+)~ valency hybrid produced during methemoglobin reduction by ascorbic acid is separable by CM Sephadex C-50 column chromatography (6), the rechromatography of the effluents was necessary for the purification of the (cx~+~~+)~because of the small amount of contamination of methemoglobin. According to the present method, not only the ((~~+p~+)~ but also the ( a2+p3+h valency hybrid is readily prepared without contamination of methemoglobin by oneprocess chromatography. By the present procedure, 45ml aliquots of the (a3’@2’)2 valency hybrid (31.8 PM in heme) were obtained from 2.5 ml aliquots of the partially
436
TOMODA
AND YONEYAMA TABLE
PURIFICATION
1
OF (~#+p*')~
AND (@p+)*
@‘P’),
(a”‘P’h
Step
Protein (rmol in heme)
Yield m
Protein (pm01 in heme)
Yield @)
Gel filtration on Sephadex Chromatography on CM cellulose
15.25 1.43
100 8.5
17.1 1.55
100 9.1
reduced methemoglobin solutions (610 PM in heme). The final yield of the valency hybrid was 8.5% (Table 1). With regard to the ( CX~+$+)~ valency hybrid, 45ml aliquots of the sample (34.5 PM in heme) were obtamed from 25-ml aliquots of the partially oxidized hemoglobin solutions with fen-icyanide (685 PM in heme). The final yield of the viency hybrid was 9.1%. This present method is very time saving compared with the method of Banetjee and Cassoly (1). They prepared the (c?+$+)~ and (cY~+~~+)~ valency hybrids by combining the isolated a and p chains, which were obtained by PCMB treatment of oxyhemoglobin tetramer. However, the isolated (Y and /3 chains are relatively unstable, and it takes about a week to prepare these valency hybrids. By the present method, the fractionation of (02+@$ and ((u3+/32+)2 can be carried out within several hours. Therefore, the valency hybrids are obtainable the next morning when the application and elution of the sample were initiated in the evening. Furthermore, these valency hybrids
can be prepared in quantity when higher concentrations of hemoglobin are applied to the cellulose. ACKNOWLEDGMENT We wish to discussion
thank Dr. M. Nagai for her useful
REFERENCES 1. Banejee, R., and Cassoly, R. (1%9) J. Mol. Biol. 42, 351-361. 2. Cassoly, R., and Gibson, Q. H. (1972) .I. Biol. Chem. 247,7332-7341. 3. Cassoly, R. (1975) J. Mol. Biol. 98, 581-595. 4. Cassoly, R. (1976) Eur. J. Biochem. 65,461-464. 5. Nagai, K. (1977) J. Mol. Biol. 111, 41-53. 6. Tomoda, A., Takeshita, M., and Yoneyama, Y. (1978) J. Biol. Chem. 253,7415-7419. 7. Tomoda, A., Tsuji, A., Matsukawa, S., Takeshita, M., and Yoneyama, Y. (1978) J. Biol. Chem. 253, 7420-7423. 8. Tomoda, A., Yubishui, T., Tsuji, A., and Yoneyama, Y. (1979) J. Biol. Chem. 254, 3119-3123. 9. Tomoda, A., and Yoneyama, Y. (1979) Biochim. Biophys. Acta 581, 128- 135. 10. Mansouri, A., and Winterhalter, K. H. (1973) Biochemistry 12,4946-4949.