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Electrophoretic properties of the globins of bovine hemoglobins A and B
ARCHIVES
OF
BIOCHEMISTRY
AND
Electrophoretic
BIOPHYSICS
84,
3%-&i
(1959)
Properties of the Globins of Bovine Hemoglobins A and B1
R. M. Grimes2 and C. W. Duncan From the Department of Agricultural Chemistry, Michigan University, East Lansing, Michigan Received
March
State
13, 1959
The occurrence of a second normal type of adult hemoglobin in some individuals of certains breeds of cattle has recently been reported (14). The more common type, hemoglobin A, has been found in all breeds so far investigated; whereas the second variant, hemoglobin B, which has the greater anodic mobility at pH 9.0, apparently does not occur in all breeds. According to Bangham (2) and Salisbury and Shreffler (3), hemoglobin types in cattle are probably controlled by an allelic pair of genes at a single locus, with neither allele being dominant. When the two hemoglobins occur together in the heterozygous animal, their ratio is always close to 1: 1 (4). If the genetic control theory of hemoglobin types in cattle is correct, the structural differences between hemoglobins A and B would be of considerable interest as the reflection, on a molecular level, of the mutations undergone by the genes HbA or HbB. The purpose of the present investigation was to compare the electrophoretic properties of the globins of bovine hemoglobins A and B, and to isolate the individual globin components by continuous flow paper electrophoresis as first steps in determining these structural differences. METHODS
Preparation of Globins Globins A and B were isolated from blood samples from cows previously shown by paper electrophoresis to possess only hemoglobin A or B (4). The globins were separated from heme and precipitated by the acid-acetone method of Anson and Mirsky (5)) modified by the use of carboxyhemoglobin and low temperatures (-5 to 1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 2356. 2 Present address: Chemistry Department, Montana State University, Missoula, Montana. 393
394
GRIMES
AND
DUNCAN
-10°C.) as recommended by Havinga (6). The dry, nearly white globin powders were stored in the dark in air-tight containers at 4°C. All subsequent operations, including dialysis and centrifugation, were carried out at temperatures not higher than 4°C. Three types of globin solutions were prepared from these powders for electrophoretic analysis. 1. Crude globin sloutions were obtained by dissolving 2 g. of globin powder in 100 ml. of cold distilled water. The pH of these solutions was about 2.5 because of the hydrochloric acid adsorbed in the acid-acetone precipitation. A small amount of cellular debris was removed by centrifugation, and the resulting clear, almost color less, solution was dialyzed against three 2-l. changes of pH 2.4, 4.25, or 5.4 buffer for a total of at least 3 days. 2. “Renatured” globin solutions were prepared either by slow titration in an ice bath of 2% aqueous solutions of crude globin with 0.2 N sodium hydroxide to pH 7.4 (5), or by dialysis at 4°C. against frequent changes of 0.01 M disodium phosphate for 3 days. The insoluble denatured globin was removed by centrifugation (refrigerated “renatured” globin, was dialyzed centrifuge), and the supernatant, containing against the buffer to be used in electrophoretic analysis. 3. Denatured globin solutions were prepared by suspending the precipitate obtained by neutralization of crude globin solutions in distilled water and adding 2 N hydrochloric acid dropwise, with stirring, until the precipitate was completely dissolved. Solution was usually complete before the pH reached 4.5. These solutions were dialyzed as before in preparation for electrophoresis.
Electrophoresis Electrophoretic analyses were carried out in a Klett-Tiselius apparatus with the standard 11-ml. analytical cell. The following buffers were used: pH 5.4 and 4.25 acetate, and pH 2.4 and 10.7 glycine. The pH 2.4 buffer contained 0.1 M glycine, 0.06 M hydrochloric acid, and 0.04 M sodium chloride, and the pH 10.7 buffer was 0.05 M with respect to glycine, sodium hydroxide, and sodium chloride. The ionic strength of all buffers was 0.1. Runs were made at 2°C. withpotential gradientsof 7.0-7.4 V./cm. with the acetate buffers, 4.0-4.3 V./cm. with the pH 2.4 glycine buffer, and 5.3-5.4 V./cm. with the pH 10.7 glycine buffer. The duration of electrophoresis was 150 min. at pH 2.4, 180 min. at pH 4.25, 240 min. at pH 5.4, and 300 min. at pH 10.7. Because resolution was generally poor in the ascending limb at low pH values, descending patterns were used throughout for both mobility and concentration measurements.
Continuous Paper Electrophoresis Crude globins were fractionated in a Spinco (Beckman) model CP continuous flow paper electrophoresis apparatus. All fractionations were carried out in a refrigerator at an ambient temperature of 5-8”C., and an acetate buffer of pH 4.25, ionic strength 0.025, was used for all runs. The other conditions given below could be varied slightly without noticeable effect on the success of the operation. The overflow tube in the electrolyte reservoir was adjusted to 6.0 cm., and a current of 35 ma. was applied to the curtain. The wicks were irrigated at the maximum rate. After equilibrium was established, the voltage was 530 f 10 v. A 2% globin solution in pH 4.25 acetate buffer was applied to the tab 4 in. from the anode side of the lower curtain at a rate of 1.5 ml./hr. Thirty minutes after protein was first detected at the drip points, collection was started. The contents of tubes 1-12 were analyzed for protein by the Folin-Ciocalteu method as modified by Lowry et al. (7), and the results were used to determine the location of components. Because the location of
ELECTROPHORETIC
PROPERTIES
OF
GLOBINS
395
the pattern shifted slowly toward the right during the 94.hr. run, reliance could not be placed on the pattern revealed by staining the curtain with bromophenol blue after completion of the run; and it was necessary to analyze each rack of collection tubes. The tubes suspected to contain the pure fast component, both components, and the pure slow component were pooled. The three fractions were then concentrated to about one-third of the original volume by pervaporation, dialyzed against distilled water for 12 hr., and again concentrated by pervaporation to a protein concentration of 0.6-0.870. Portions of the three fractions were dialyzed against the appropriate buffers and analyzed electrophoretically. RESULTS
Electrophoretic Comparison of Globins A and B Crude and renatured globin preparations from two animals having only hemoglobin A and from two with only hemoglobin B were examined at pH’s 5.4,4.25, and 2.4. No qualitative differences between the patterns of globins A and B could be detected. The patterns presented in Fig. 1 are typical of both globins. Both types separated into two components at pH’s 5.4 and 4.25, and a third small component appeared in patterns obtained at pH 2.4. The data recorded in Table I, however, reveal a quantitative difference in the distribution of the components of the two globins at pH 5.4. At this pH, component 1 (slow) madeup one-tenth of thetotal of globin A; whereas, component 1 of globin B accounted for almost one-fifth of the total. Moreover, globin B appeared to be less stable than A at this pH level. A constantly increasing opacity which could not be permanently removed by centrifugation developed during dialysis prior to electrophoresis in both the crude and renatured globin solutions from animal B 1. The reliability of the results obtained with globin B at pH 5.4 is therefore questionable. At pH 4.25, the relative concentration of the slow component was found to have increased to about 30% for both globins A and B. The slightly higher average proportion of component 1 in globin B cannot be considered significant in view of the greater difference between the globins from animals B 1 and B 2. A further increase in acidity to pH 2.4 caused the appearance of a third component in the patterns of both globins. The small differences between the compositions of the two globins may be insignificant when the differences between animals having the same type of hemoglobin are considered. The electrophoretic mobilities recorded in Table II are essentially the same for corresponding components of globins A and B at all three pH levels. Similar to the data for relative concentrations, the variation between animals was greater than the variation between the two globins. It is probably significant, and should be noted, that the globins from animals A 1 and B :l were isolated and processed at the same time, as were the globins from A 2 and B 2.
396
GRIMES
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DUNChN
c
FIG. 1. Descending electrophoretic A, pH 5.4; B, pH 4.25; C, pH 2.4.
patterns of crude globin A at three pH levels.
To determine whether the pH-induced dissociation effect is reversible, a sample of whole globin B was dialyzed first against pH 2.4 buffer for 3 days. The electrophoretic pattern of a portion of this sample was identical to the ones previously obtained at pH 2.4. The remainder, after dialysis for 3 days against pH 4.25 buffer, was then subjected to electrophoretic analysis at the higher pH. The pattern could not be distinguished from that shown in Fig. 13. The mobilities and relative concentrations of the two components were within the range recorded in Tables I and II. Within the pH range 2.44.25, the dissociation appears to be completely reversible. No consistent differences were found between the crude and renatured globin preparations. The results of an experiment in which the denatured and renatured fractions of globins A and B were compared at pH 10.7, as well as 2.4 and 4.25, are presented in Table III. The denatured fraction of globin B at pH 10.7
ELECTROPHORETIC
Relative
cz
OF
GLOBINS
397
TABLE I Concentrations of Components of Crude and Renatured Bovine Globins A and B in Acid Bu$ers (Protein
*2’
PROPERTIES
Globin preparationb
concentrations:
l&2.0%)
pH 5.4 Component
pH 4.25 Component
1
2
1
pH 2.4 Component 2
1
2
3
30 31 28 30
70 69 72 70
37 38 34 36
44 44 45 40
19 18 21 24
35 33 29 31
65 67 71 69
36 37 35 36
47 48 46 41
17 15 19 23
% of total
Al Al A2 A2
C R C R
11 9 10 10
Bl Bl B2 .B 2
C R C R
c c 18 --
89 91 90 90
82
(1The letters A and B indicate the type of hemoglobin. b C denotes crude globin; R, renatured globin. c These solutions became slightly cloudy during dialysis against buffer and could not be permanently cleared by centrifugation.
TABLE Electrophoretic
N0.L”
II
Mobilities of Components of Crude and Renatured Bovine Globins A and B in Acid Buffers (Protein
Animal
pH 5.4 acetate
Globin preparationb
concentrations pH 5.4 Compment 1
: 1 S-2.0%) pH 4.25 Component
2
1 sq. cm./o./sec.
Al Al A2 A2
C R C R
Bl Bl B2 B2
C R C R
3.4 3.5 3.3 3.3
4.7 4.7 4.6 4.5
c c 3.5 --
4.5
pH 2.4 Component 2
1
2
3
x lo-~
4.2 4.3 4.2 4.1
6.3 6.4 6.2 6.2
5.8 5.7 5.4 5.4
7.8 7.7 7.4 7.4
12.3 12.2 11.8 11.8
4.3 4.4 4.1 4.1
6.5 6.5 6.1 6.1
5.5 5.7 5.5 5.4
7.5 7.7 7.4 7.3
12.1 12.3 11.8 11.8
0 The letters A and B indicate the type of hemoglobin. b C denotes crude globin; R, renatured globin. c These solutions became slightly cloudy during dialysis against buffer and could not be permanently cleared by centrifugation.
pH 5.4 acetate
398
GRIMES
AND
DUNCAN
TABLE
III
Electrophoretic Analysis of Denatured and Renatured Globins A and B in Acid and Alkaline Bujers (Protein concentrations : 0.7-0.9%) Relative concentration
Globin
Mobility component
component
PR Type Fraction
1
2
3
1
% of lotal
2.4 2.4 2.4 2.4
A A B B
D R D R
36 39 37 39
44 40 43 42
4.25 4.25
A B
D D
34 30
66 70
A A B B
D R D R
59 72 56 59
41 28 44 41
10.7 10.7 10.7 10.7
Bovine
2 sq. cm./n./sec.
20 21 20 19
6.0 6.0 6.1 6.1
8.2 8.5 8.3 8.5
4.5 4.6
6.7 6.7
-4.6 -4.7 -5.5 -5.6
3 x 10-6
11.4 11.5 11.5 11.3
-3.6 -3.4 -3.5 -3.5
amounted to 45 % of the total as compared to 33 % in the case of A. The distribution of components at pH’s 2.4 and 4.25 was essentially the same as before; there were no gross differences either between globins A and B or between the renatured and denatured fractions. Although the mobilities of corresponding globin A and B components in acidic buffers were again practically identical in this experiment (Table III), components 1 and 2 had higher mobilities and component 3 had a lower mobility at pH’s 2.4 and 4.25 than in the previous experiment (Table II). The difference in protein concentration used for electrophoresis may possibly be the reason for these changes. At pH 10.7, the slow components (labeled component 2 on the assumption that the component with the greater cathodic mobility at pH 4.25 is the slower component at pH values above the isoelectric point) of globins A and B had the same mobility and may be identical, but component 1 of globin A had a mobility of -4.7 compared to -5.6 sq. cm./v./sec. for component 1 of globin B. The descending patterns are shown in Fig. 2. This result is in agreement with the previous finding that intact hemoglobin B has a greater anodic mobility than does hemoglobin A at pH 9.0 on paper-strip electrophoresis (4). The lack of agreement between the distribution of the components at pH 10.7 may be due, in part, to the precipitation of the components at different rates during dialysis, but the amount of precipitate was not great enough to account for all the difference. It will
ELECTROPHORETIC
FIG.
renatured
PROPERTIES
2. Descending electrophoretic patterns globin A; B, renatured globin B.
OF
GLOBINS
of globins
399
A and B at pH 10.7. A,
also be noted (Table III) that at pH 10.7 component 1 was present in greater concentration than component 2. This is the opposite to the relative proportions at the lower pH values. Isolation of Globin Components The technique of continuous paper electrophoresis was found to be suitable for the simultaneous isolation of globin components. In typical runs, separation of crude globin into two distinct bands with some mixed material between was observed. The bromophenol blue-stained curtain in Fig. 3 shows that the protein migrated toward the cathode at a rate which brought the bands to drip points l-11. The faster-migrating band on the left was found by electrophoretic analysis to be component 2. Most of the protein in each band was found in two or three collection tubes, and the mixed fraction between the bands was confined to two tubes. Over-all recovery, after concentration of the three fractions by pervaporation was 84-91%. Protein in the mixed fraction amounted to 19-28 % of the total recovered. No differences in the behavior of globins A and B were observed. Surprisingly, the amount of component 1 isolated was approximately equal to that of component 2 for both globins; the ratio was not 30:70 as found in the electrophoretic patterns of unfractionated globins in pH 4.25 buffer. This cannot be explained by the composition of the mixed fraction which in one case was found by electrophoretic analysis at pH 4.25 to contain 41% slow component and 59 % fast.
GKIMES
AKD
DUSCAS
FIG. 3. Bromophenol blue-stained continuous paper electrophoresis curtain used for fractionation of crude globin A. The cathode side is at the left. Only the lower left portion of the curtain is shown. The band on the right is component 1.
The isolated fractions were examined electrophoretically at pH’s 4.25 and 2.4, and the results are given in Fig. 4 and Table IV. Single components were obtained from both bands of both globins at pH 4.25, and the mobility of corresponding components from globins A and B was practically identical. At pH 2.4, both components of both globins dissociated, and the new component, 3, had the same mobility whatever its origin. The amount found depended on the fraction being analyzed, but was independent of the type of globin. For component 1, the figure was 1516%; for component 2, 32 %. The mobilities recorded in Table IV are not in complete agreement with those in Table III. Component 1 had a higher mobility at pH 4.25, but the same mobility as before at pH 2.4. On the other hand, the mobility of component 2 at pH 4.25 is the same in Tables III and IV, but is lower in Table IV at pH 2.4. The mobility of component 3 is lower in Table IV. No explanation of these discrepancies can be offered, except that the lack of interference from other components may have influenced the migration.
ELECTROPHORETIC
PROPERTIES
OF
401
GLOBINS
FIG. 4. Descending elcctrophoretic patterns of glohin components isolated 1)) continuous paper electrophoresis. A: Component 1 of globin A, pH 4.25. H: Component 2 of globin A, pH 4.25. C: Component 1 of globin A, pII 2.4. 11: Component 2 of globin -4, pH 2.4.
Electrophoretic
of Isolated
Analysis
(Protein PH
Globin type
Comwnent”
Componenls
concentrations:
Relative concentration Component .__ ! 2 3
% o/ 2.4 2.4 2.4 2.4
.4 B A B
1 1 2 2
4.25 4.25 4.25 4.25
A B A B
1 1 2 2
of Bovine
85 84 _106 I()()
Globins
A and B
O.M.8~)
robd
Motility ComponEnt 1
-
2
3
sp. cm./v./scc.
x IcrJ
68 68
15 16 32 32
6.0 6.0
-8.1 8.1
100 100
-._ -
4.9 4.9
6.7 6.8
11.1 11.0 11.2 11.1
* Component in this column refers to the slow (1) and fast (2) bands on the cont.inuous flow paper elcctrophoresis apparatus.
402
GRIMES
9ND
DUNCAN
DISCUSSION
The results support the conclusion that bovine globins of both types consist of two primary components of different electrokinetic properties. Both components dissociate into subunits at low pH values, releasing a third component which is possibly the same whether it comes from the slow or fast component of globin A or B. At low pH values, no differences between the electrophoretic behavior of globins A and B were found, but at pH 10.7 one component of globin B was found to have a greater anodic mobility than the corresponding globin A component. Two bovine globin components were previously reported by Reiner et al. (8)) Munro and Munro (9)) and Moore and Reiner (10). Munro and Munro (9), however, only found a single component at pH 5.2-7.9, and two at pH 2.6. Moore and Reiner (lo), on the other hand, found two components at all pH values from 2.5 to 11.3, and noted, as we did, that the relative proportions of the fast and slow fractions changed as pH varied. No reference has been found in the literature concerning the presence of a third component in bovine globins noted in our patterns, but, qualitatively similar results were obtained with horse globin by Reichmann and Colvin (11). The similarity between denatured and renatured globin preparations found in this investigation has also been reported previously (9, 10). The globins of other species are also probably made up of at least two proteins. Electrophoretic evidence of this has been obtained for the globin of the rabbit (9), horse (10, ll), and man (9, 10). Rhinesmith et al. (12) found two different N-terminal amino acid sequences in normal adult human hemoglobin. The nearly equal yields of components 1 and 2 obtained by continuous paper electrophoresis suggest that they may actually occur in a 1: 1 ratio in spite of the fact that this ratio was not encountered in moving-boundary electrophoresis. One of the two peaks observed in patterns obtained at pH’s 5.4 and 4.25 may represent a loose association in which the relative proportions of the two chains depends partly on the pH. In moving-boundary electrophoresis the components would be free to establish equilibrium, but, on the paper curtain, where movement of solutes once separated is more restricted, the equilibrium could be upset and result in complete dissociation of the complex. A second factor which might be involved, but has not been investigated, is ionic strength. Reichmann and Colvin (11) found that in buffers of high ionic strength, the slow component of horse globin could not be resolved in some experiments at pH 4.0. In our experiments, the ionic strength of the buffer used for continuous paper electrophoresis was 0.025, and that for moving-boundary electrophoresis 0.10. This question is being studied further.
ELECTROPHORETIC PROPERTIES OF GLOBINS
403
The electrophoretic patterns obtained at pH values on the acid side of the isoelectric point provided no conclusive evidence that globins A and B are different. Because of the apparent association of components, the difference in relative proportions of components in globins A and B at pH 5.4 is not convincing, especially since the mobilities of corresponding components were the same. At pH 10.7, however, the difference between the globins was definite, and it is of some interest that it is confined to a single component. Ingram (13) and Hunt and Ingram (14) have recently presented evidence that human hemoglobins A, S, and C differ only by a single amino acid residue out of nearly 300 in the half molecule. A glutamic acid residue in hemoglobin A is replaced by valine in hemoglobin S and by lysine in hemoglobin C. Since apparently only one of the components of bovine globins A and B differs, such a relatively small structural difference as that found for the human hemoglobins could conceivably account for the difference in electrophoretic behavior of bovine hemoglobins A and B. SUMMARY
The globins isolated from bovine hemoglobins A and B were investigated by moving-boundary electrophoresis at pH’s 2.4, 4.25, 5.4, and 10.7. At the intermediate pH levels, both globins had two components, the relative proportions of which changed in favor of the slow component as the pH was decreased. At pH 2.4, both globins had three components in the same relative proportions. The mobilities of corresponding components were essentially the same for the two globins at the three lower pH levels. At pH 10.7, both globins had two components. The slower component had the same mobility in the two globins, but the mobility of the faster components differed significantly. No differences between crude and renatured, or between denatured and renatured globin fractions were observed. The two primary components of globins A and B were isolated by continuous flow paper electrophoresis. At pH 4.25, both components of both globins appeared to be electrophoretically homogeneous, but at pH 2.4 both fractions of both globins dissociated into subunits. The newly dissociated component had the same mobility whatever its source, but the amount depended on whether it came from the fast or slow primary component . REFERENCES 1. CABANNES, R., AND SERAIN, CH., Compt. rend. sot. biol. 149, 7 (1955). 2. BAKGHAM, A. D., Nature 179, 467 (1957). 3. SALMBURY, G. W., AND SHREFFLER, D. C., J. Dairy Sci. 40, 1198 (1957). 4. GRIMES, R. M., DUNCAN, C. W., AND LASSITER, C. A., J. Dairy Sci. 40, (1957).
1338
404
GRIMES
AND
DUNCAB
5. ANSON, M. L., AXD MIRSKY, A. E., J. Gen. Ph~+siol. 13, 469 (1930). 6. HAVINGA, E., Proc. N&l. Acad. Sci. U. S. 39, 59 (1953). 7. LOWRY, 0. H., RO~EBROUGH, N. J., FARR, A. L., AND RAXDALL, R. J., J. Bid. Chem. 193, 265 (1951). 8. REINER, L., MOORE, D. H., LANG, E. H., AND GREEN, M., J. Biol. Chem. 146, 583 (1942) 9. MUNRO, M. P., AND MUNRO, F. L., J. Biol. Chem. 160, 427 (1943). 10. MOORE, D. H., AND REIXER, L., J. Bid. Chem. 166, 411 (1944). 11. REICHMANN, M. E., AND COLVIN, J. R., Can. J. Chem. 34, 411 (1956). 12. RHINESMITH, H. S., SHROEDER, W. A., AND PAULING, L., J. Am. Chem. Sot. 79, 4682 (1957). 13. INGRAM, V. M., Biochim. et Biophys. Acta 28, 539 (1958). 14. HUNT, J. A., AND INGRAM, V. M., Nature 181, 1062 (1958).
OF
BIOCHEMISTRY
AND
Electrophoretic
BIOPHYSICS
84,
3%-&i
(1959)
Properties of the Globins of Bovine Hemoglobins A and B1
R. M. Grimes2 and C. W. Duncan From the Department of Agricultural Chemistry, Michigan University, East Lansing, Michigan Received
March
State
13, 1959
The occurrence of a second normal type of adult hemoglobin in some individuals of certains breeds of cattle has recently been reported (14). The more common type, hemoglobin A, has been found in all breeds so far investigated; whereas the second variant, hemoglobin B, which has the greater anodic mobility at pH 9.0, apparently does not occur in all breeds. According to Bangham (2) and Salisbury and Shreffler (3), hemoglobin types in cattle are probably controlled by an allelic pair of genes at a single locus, with neither allele being dominant. When the two hemoglobins occur together in the heterozygous animal, their ratio is always close to 1: 1 (4). If the genetic control theory of hemoglobin types in cattle is correct, the structural differences between hemoglobins A and B would be of considerable interest as the reflection, on a molecular level, of the mutations undergone by the genes HbA or HbB. The purpose of the present investigation was to compare the electrophoretic properties of the globins of bovine hemoglobins A and B, and to isolate the individual globin components by continuous flow paper electrophoresis as first steps in determining these structural differences. METHODS
Preparation of Globins Globins A and B were isolated from blood samples from cows previously shown by paper electrophoresis to possess only hemoglobin A or B (4). The globins were separated from heme and precipitated by the acid-acetone method of Anson and Mirsky (5)) modified by the use of carboxyhemoglobin and low temperatures (-5 to 1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 2356. 2 Present address: Chemistry Department, Montana State University, Missoula, Montana. 393
394
GRIMES
AND
DUNCAN
-10°C.) as recommended by Havinga (6). The dry, nearly white globin powders were stored in the dark in air-tight containers at 4°C. All subsequent operations, including dialysis and centrifugation, were carried out at temperatures not higher than 4°C. Three types of globin solutions were prepared from these powders for electrophoretic analysis. 1. Crude globin sloutions were obtained by dissolving 2 g. of globin powder in 100 ml. of cold distilled water. The pH of these solutions was about 2.5 because of the hydrochloric acid adsorbed in the acid-acetone precipitation. A small amount of cellular debris was removed by centrifugation, and the resulting clear, almost color less, solution was dialyzed against three 2-l. changes of pH 2.4, 4.25, or 5.4 buffer for a total of at least 3 days. 2. “Renatured” globin solutions were prepared either by slow titration in an ice bath of 2% aqueous solutions of crude globin with 0.2 N sodium hydroxide to pH 7.4 (5), or by dialysis at 4°C. against frequent changes of 0.01 M disodium phosphate for 3 days. The insoluble denatured globin was removed by centrifugation (refrigerated “renatured” globin, was dialyzed centrifuge), and the supernatant, containing against the buffer to be used in electrophoretic analysis. 3. Denatured globin solutions were prepared by suspending the precipitate obtained by neutralization of crude globin solutions in distilled water and adding 2 N hydrochloric acid dropwise, with stirring, until the precipitate was completely dissolved. Solution was usually complete before the pH reached 4.5. These solutions were dialyzed as before in preparation for electrophoresis.
Electrophoresis Electrophoretic analyses were carried out in a Klett-Tiselius apparatus with the standard 11-ml. analytical cell. The following buffers were used: pH 5.4 and 4.25 acetate, and pH 2.4 and 10.7 glycine. The pH 2.4 buffer contained 0.1 M glycine, 0.06 M hydrochloric acid, and 0.04 M sodium chloride, and the pH 10.7 buffer was 0.05 M with respect to glycine, sodium hydroxide, and sodium chloride. The ionic strength of all buffers was 0.1. Runs were made at 2°C. withpotential gradientsof 7.0-7.4 V./cm. with the acetate buffers, 4.0-4.3 V./cm. with the pH 2.4 glycine buffer, and 5.3-5.4 V./cm. with the pH 10.7 glycine buffer. The duration of electrophoresis was 150 min. at pH 2.4, 180 min. at pH 4.25, 240 min. at pH 5.4, and 300 min. at pH 10.7. Because resolution was generally poor in the ascending limb at low pH values, descending patterns were used throughout for both mobility and concentration measurements.
Continuous Paper Electrophoresis Crude globins were fractionated in a Spinco (Beckman) model CP continuous flow paper electrophoresis apparatus. All fractionations were carried out in a refrigerator at an ambient temperature of 5-8”C., and an acetate buffer of pH 4.25, ionic strength 0.025, was used for all runs. The other conditions given below could be varied slightly without noticeable effect on the success of the operation. The overflow tube in the electrolyte reservoir was adjusted to 6.0 cm., and a current of 35 ma. was applied to the curtain. The wicks were irrigated at the maximum rate. After equilibrium was established, the voltage was 530 f 10 v. A 2% globin solution in pH 4.25 acetate buffer was applied to the tab 4 in. from the anode side of the lower curtain at a rate of 1.5 ml./hr. Thirty minutes after protein was first detected at the drip points, collection was started. The contents of tubes 1-12 were analyzed for protein by the Folin-Ciocalteu method as modified by Lowry et al. (7), and the results were used to determine the location of components. Because the location of
ELECTROPHORETIC
PROPERTIES
OF
GLOBINS
395
the pattern shifted slowly toward the right during the 94.hr. run, reliance could not be placed on the pattern revealed by staining the curtain with bromophenol blue after completion of the run; and it was necessary to analyze each rack of collection tubes. The tubes suspected to contain the pure fast component, both components, and the pure slow component were pooled. The three fractions were then concentrated to about one-third of the original volume by pervaporation, dialyzed against distilled water for 12 hr., and again concentrated by pervaporation to a protein concentration of 0.6-0.870. Portions of the three fractions were dialyzed against the appropriate buffers and analyzed electrophoretically. RESULTS
Electrophoretic Comparison of Globins A and B Crude and renatured globin preparations from two animals having only hemoglobin A and from two with only hemoglobin B were examined at pH’s 5.4,4.25, and 2.4. No qualitative differences between the patterns of globins A and B could be detected. The patterns presented in Fig. 1 are typical of both globins. Both types separated into two components at pH’s 5.4 and 4.25, and a third small component appeared in patterns obtained at pH 2.4. The data recorded in Table I, however, reveal a quantitative difference in the distribution of the components of the two globins at pH 5.4. At this pH, component 1 (slow) madeup one-tenth of thetotal of globin A; whereas, component 1 of globin B accounted for almost one-fifth of the total. Moreover, globin B appeared to be less stable than A at this pH level. A constantly increasing opacity which could not be permanently removed by centrifugation developed during dialysis prior to electrophoresis in both the crude and renatured globin solutions from animal B 1. The reliability of the results obtained with globin B at pH 5.4 is therefore questionable. At pH 4.25, the relative concentration of the slow component was found to have increased to about 30% for both globins A and B. The slightly higher average proportion of component 1 in globin B cannot be considered significant in view of the greater difference between the globins from animals B 1 and B 2. A further increase in acidity to pH 2.4 caused the appearance of a third component in the patterns of both globins. The small differences between the compositions of the two globins may be insignificant when the differences between animals having the same type of hemoglobin are considered. The electrophoretic mobilities recorded in Table II are essentially the same for corresponding components of globins A and B at all three pH levels. Similar to the data for relative concentrations, the variation between animals was greater than the variation between the two globins. It is probably significant, and should be noted, that the globins from animals A 1 and B :l were isolated and processed at the same time, as were the globins from A 2 and B 2.
396
GRIMES
AND
DUNChN
c
FIG. 1. Descending electrophoretic A, pH 5.4; B, pH 4.25; C, pH 2.4.
patterns of crude globin A at three pH levels.
To determine whether the pH-induced dissociation effect is reversible, a sample of whole globin B was dialyzed first against pH 2.4 buffer for 3 days. The electrophoretic pattern of a portion of this sample was identical to the ones previously obtained at pH 2.4. The remainder, after dialysis for 3 days against pH 4.25 buffer, was then subjected to electrophoretic analysis at the higher pH. The pattern could not be distinguished from that shown in Fig. 13. The mobilities and relative concentrations of the two components were within the range recorded in Tables I and II. Within the pH range 2.44.25, the dissociation appears to be completely reversible. No consistent differences were found between the crude and renatured globin preparations. The results of an experiment in which the denatured and renatured fractions of globins A and B were compared at pH 10.7, as well as 2.4 and 4.25, are presented in Table III. The denatured fraction of globin B at pH 10.7
ELECTROPHORETIC
Relative
cz
OF
GLOBINS
397
TABLE I Concentrations of Components of Crude and Renatured Bovine Globins A and B in Acid Bu$ers (Protein
*2’
PROPERTIES
Globin preparationb
concentrations:
l&2.0%)
pH 5.4 Component
pH 4.25 Component
1
2
1
pH 2.4 Component 2
1
2
3
30 31 28 30
70 69 72 70
37 38 34 36
44 44 45 40
19 18 21 24
35 33 29 31
65 67 71 69
36 37 35 36
47 48 46 41
17 15 19 23
% of total
Al Al A2 A2
C R C R
11 9 10 10
Bl Bl B2 .B 2
C R C R
c c 18 --
89 91 90 90
82
(1The letters A and B indicate the type of hemoglobin. b C denotes crude globin; R, renatured globin. c These solutions became slightly cloudy during dialysis against buffer and could not be permanently cleared by centrifugation.
TABLE Electrophoretic
N0.L”
II
Mobilities of Components of Crude and Renatured Bovine Globins A and B in Acid Buffers (Protein
Animal
pH 5.4 acetate
Globin preparationb
concentrations pH 5.4 Compment 1
: 1 S-2.0%) pH 4.25 Component
2
1 sq. cm./o./sec.
Al Al A2 A2
C R C R
Bl Bl B2 B2
C R C R
3.4 3.5 3.3 3.3
4.7 4.7 4.6 4.5
c c 3.5 --
4.5
pH 2.4 Component 2
1
2
3
x lo-~
4.2 4.3 4.2 4.1
6.3 6.4 6.2 6.2
5.8 5.7 5.4 5.4
7.8 7.7 7.4 7.4
12.3 12.2 11.8 11.8
4.3 4.4 4.1 4.1
6.5 6.5 6.1 6.1
5.5 5.7 5.5 5.4
7.5 7.7 7.4 7.3
12.1 12.3 11.8 11.8
0 The letters A and B indicate the type of hemoglobin. b C denotes crude globin; R, renatured globin. c These solutions became slightly cloudy during dialysis against buffer and could not be permanently cleared by centrifugation.
pH 5.4 acetate
398
GRIMES
AND
DUNCAN
TABLE
III
Electrophoretic Analysis of Denatured and Renatured Globins A and B in Acid and Alkaline Bujers (Protein concentrations : 0.7-0.9%) Relative concentration
Globin
Mobility component
component
PR Type Fraction
1
2
3
1
% of lotal
2.4 2.4 2.4 2.4
A A B B
D R D R
36 39 37 39
44 40 43 42
4.25 4.25
A B
D D
34 30
66 70
A A B B
D R D R
59 72 56 59
41 28 44 41
10.7 10.7 10.7 10.7
Bovine
2 sq. cm./n./sec.
20 21 20 19
6.0 6.0 6.1 6.1
8.2 8.5 8.3 8.5
4.5 4.6
6.7 6.7
-4.6 -4.7 -5.5 -5.6
3 x 10-6
11.4 11.5 11.5 11.3
-3.6 -3.4 -3.5 -3.5
amounted to 45 % of the total as compared to 33 % in the case of A. The distribution of components at pH’s 2.4 and 4.25 was essentially the same as before; there were no gross differences either between globins A and B or between the renatured and denatured fractions. Although the mobilities of corresponding globin A and B components in acidic buffers were again practically identical in this experiment (Table III), components 1 and 2 had higher mobilities and component 3 had a lower mobility at pH’s 2.4 and 4.25 than in the previous experiment (Table II). The difference in protein concentration used for electrophoresis may possibly be the reason for these changes. At pH 10.7, the slow components (labeled component 2 on the assumption that the component with the greater cathodic mobility at pH 4.25 is the slower component at pH values above the isoelectric point) of globins A and B had the same mobility and may be identical, but component 1 of globin A had a mobility of -4.7 compared to -5.6 sq. cm./v./sec. for component 1 of globin B. The descending patterns are shown in Fig. 2. This result is in agreement with the previous finding that intact hemoglobin B has a greater anodic mobility than does hemoglobin A at pH 9.0 on paper-strip electrophoresis (4). The lack of agreement between the distribution of the components at pH 10.7 may be due, in part, to the precipitation of the components at different rates during dialysis, but the amount of precipitate was not great enough to account for all the difference. It will
ELECTROPHORETIC
FIG.
renatured
PROPERTIES
2. Descending electrophoretic patterns globin A; B, renatured globin B.
OF
GLOBINS
of globins
399
A and B at pH 10.7. A,
also be noted (Table III) that at pH 10.7 component 1 was present in greater concentration than component 2. This is the opposite to the relative proportions at the lower pH values. Isolation of Globin Components The technique of continuous paper electrophoresis was found to be suitable for the simultaneous isolation of globin components. In typical runs, separation of crude globin into two distinct bands with some mixed material between was observed. The bromophenol blue-stained curtain in Fig. 3 shows that the protein migrated toward the cathode at a rate which brought the bands to drip points l-11. The faster-migrating band on the left was found by electrophoretic analysis to be component 2. Most of the protein in each band was found in two or three collection tubes, and the mixed fraction between the bands was confined to two tubes. Over-all recovery, after concentration of the three fractions by pervaporation was 84-91%. Protein in the mixed fraction amounted to 19-28 % of the total recovered. No differences in the behavior of globins A and B were observed. Surprisingly, the amount of component 1 isolated was approximately equal to that of component 2 for both globins; the ratio was not 30:70 as found in the electrophoretic patterns of unfractionated globins in pH 4.25 buffer. This cannot be explained by the composition of the mixed fraction which in one case was found by electrophoretic analysis at pH 4.25 to contain 41% slow component and 59 % fast.
GKIMES
AKD
DUSCAS
FIG. 3. Bromophenol blue-stained continuous paper electrophoresis curtain used for fractionation of crude globin A. The cathode side is at the left. Only the lower left portion of the curtain is shown. The band on the right is component 1.
The isolated fractions were examined electrophoretically at pH’s 4.25 and 2.4, and the results are given in Fig. 4 and Table IV. Single components were obtained from both bands of both globins at pH 4.25, and the mobility of corresponding components from globins A and B was practically identical. At pH 2.4, both components of both globins dissociated, and the new component, 3, had the same mobility whatever its origin. The amount found depended on the fraction being analyzed, but was independent of the type of globin. For component 1, the figure was 1516%; for component 2, 32 %. The mobilities recorded in Table IV are not in complete agreement with those in Table III. Component 1 had a higher mobility at pH 4.25, but the same mobility as before at pH 2.4. On the other hand, the mobility of component 2 at pH 4.25 is the same in Tables III and IV, but is lower in Table IV at pH 2.4. The mobility of component 3 is lower in Table IV. No explanation of these discrepancies can be offered, except that the lack of interference from other components may have influenced the migration.
ELECTROPHORETIC
PROPERTIES
OF
401
GLOBINS
FIG. 4. Descending elcctrophoretic patterns of glohin components isolated 1)) continuous paper electrophoresis. A: Component 1 of globin A, pH 4.25. H: Component 2 of globin A, pH 4.25. C: Component 1 of globin A, pII 2.4. 11: Component 2 of globin -4, pH 2.4.
Electrophoretic
of Isolated
Analysis
(Protein PH
Globin type
Comwnent”
Componenls
concentrations:
Relative concentration Component .__ ! 2 3
% o/ 2.4 2.4 2.4 2.4
.4 B A B
1 1 2 2
4.25 4.25 4.25 4.25
A B A B
1 1 2 2
of Bovine
85 84 _106 I()()
Globins
A and B
O.M.8~)
robd
Motility ComponEnt 1
-
2
3
sp. cm./v./scc.
x IcrJ
68 68
15 16 32 32
6.0 6.0
-8.1 8.1
100 100
-._ -
4.9 4.9
6.7 6.8
11.1 11.0 11.2 11.1
* Component in this column refers to the slow (1) and fast (2) bands on the cont.inuous flow paper elcctrophoresis apparatus.
402
GRIMES
9ND
DUNCAN
DISCUSSION
The results support the conclusion that bovine globins of both types consist of two primary components of different electrokinetic properties. Both components dissociate into subunits at low pH values, releasing a third component which is possibly the same whether it comes from the slow or fast component of globin A or B. At low pH values, no differences between the electrophoretic behavior of globins A and B were found, but at pH 10.7 one component of globin B was found to have a greater anodic mobility than the corresponding globin A component. Two bovine globin components were previously reported by Reiner et al. (8)) Munro and Munro (9)) and Moore and Reiner (10). Munro and Munro (9), however, only found a single component at pH 5.2-7.9, and two at pH 2.6. Moore and Reiner (lo), on the other hand, found two components at all pH values from 2.5 to 11.3, and noted, as we did, that the relative proportions of the fast and slow fractions changed as pH varied. No reference has been found in the literature concerning the presence of a third component in bovine globins noted in our patterns, but, qualitatively similar results were obtained with horse globin by Reichmann and Colvin (11). The similarity between denatured and renatured globin preparations found in this investigation has also been reported previously (9, 10). The globins of other species are also probably made up of at least two proteins. Electrophoretic evidence of this has been obtained for the globin of the rabbit (9), horse (10, ll), and man (9, 10). Rhinesmith et al. (12) found two different N-terminal amino acid sequences in normal adult human hemoglobin. The nearly equal yields of components 1 and 2 obtained by continuous paper electrophoresis suggest that they may actually occur in a 1: 1 ratio in spite of the fact that this ratio was not encountered in moving-boundary electrophoresis. One of the two peaks observed in patterns obtained at pH’s 5.4 and 4.25 may represent a loose association in which the relative proportions of the two chains depends partly on the pH. In moving-boundary electrophoresis the components would be free to establish equilibrium, but, on the paper curtain, where movement of solutes once separated is more restricted, the equilibrium could be upset and result in complete dissociation of the complex. A second factor which might be involved, but has not been investigated, is ionic strength. Reichmann and Colvin (11) found that in buffers of high ionic strength, the slow component of horse globin could not be resolved in some experiments at pH 4.0. In our experiments, the ionic strength of the buffer used for continuous paper electrophoresis was 0.025, and that for moving-boundary electrophoresis 0.10. This question is being studied further.
ELECTROPHORETIC PROPERTIES OF GLOBINS
403
The electrophoretic patterns obtained at pH values on the acid side of the isoelectric point provided no conclusive evidence that globins A and B are different. Because of the apparent association of components, the difference in relative proportions of components in globins A and B at pH 5.4 is not convincing, especially since the mobilities of corresponding components were the same. At pH 10.7, however, the difference between the globins was definite, and it is of some interest that it is confined to a single component. Ingram (13) and Hunt and Ingram (14) have recently presented evidence that human hemoglobins A, S, and C differ only by a single amino acid residue out of nearly 300 in the half molecule. A glutamic acid residue in hemoglobin A is replaced by valine in hemoglobin S and by lysine in hemoglobin C. Since apparently only one of the components of bovine globins A and B differs, such a relatively small structural difference as that found for the human hemoglobins could conceivably account for the difference in electrophoretic behavior of bovine hemoglobins A and B. SUMMARY
The globins isolated from bovine hemoglobins A and B were investigated by moving-boundary electrophoresis at pH’s 2.4, 4.25, 5.4, and 10.7. At the intermediate pH levels, both globins had two components, the relative proportions of which changed in favor of the slow component as the pH was decreased. At pH 2.4, both globins had three components in the same relative proportions. The mobilities of corresponding components were essentially the same for the two globins at the three lower pH levels. At pH 10.7, both globins had two components. The slower component had the same mobility in the two globins, but the mobility of the faster components differed significantly. No differences between crude and renatured, or between denatured and renatured globin fractions were observed. The two primary components of globins A and B were isolated by continuous flow paper electrophoresis. At pH 4.25, both components of both globins appeared to be electrophoretically homogeneous, but at pH 2.4 both fractions of both globins dissociated into subunits. The newly dissociated component had the same mobility whatever its source, but the amount depended on whether it came from the fast or slow primary component . REFERENCES 1. CABANNES, R., AND SERAIN, CH., Compt. rend. sot. biol. 149, 7 (1955). 2. BAKGHAM, A. D., Nature 179, 467 (1957). 3. SALMBURY, G. W., AND SHREFFLER, D. C., J. Dairy Sci. 40, 1198 (1957). 4. GRIMES, R. M., DUNCAN, C. W., AND LASSITER, C. A., J. Dairy Sci. 40, (1957).
1338
404
GRIMES
AND
DUNCAB
5. ANSON, M. L., AXD MIRSKY, A. E., J. Gen. Ph~+siol. 13, 469 (1930). 6. HAVINGA, E., Proc. N&l. Acad. Sci. U. S. 39, 59 (1953). 7. LOWRY, 0. H., RO~EBROUGH, N. J., FARR, A. L., AND RAXDALL, R. J., J. Bid. Chem. 193, 265 (1951). 8. REINER, L., MOORE, D. H., LANG, E. H., AND GREEN, M., J. Biol. Chem. 146, 583 (1942) 9. MUNRO, M. P., AND MUNRO, F. L., J. Biol. Chem. 160, 427 (1943). 10. MOORE, D. H., AND REIXER, L., J. Bid. Chem. 166, 411 (1944). 11. REICHMANN, M. E., AND COLVIN, J. R., Can. J. Chem. 34, 411 (1956). 12. RHINESMITH, H. S., SHROEDER, W. A., AND PAULING, L., J. Am. Chem. Sot. 79, 4682 (1957). 13. INGRAM, V. M., Biochim. et Biophys. Acta 28, 539 (1958). 14. HUNT, J. A., AND INGRAM, V. M., Nature 181, 1062 (1958).