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Mouse hemoglobin. I. Chromatographic analysis
ANALYTICAL
BIOCHEMlSTKY
Mouse
8, 407-414 (1964)
Hemoglobin.
HERBERT BETTY From the Meadville, Biological
I. Chromatographic
S. RHINESMITH, LOU MILLIKIN,
AND
HERSCHEL LEONELL
Carnegie Laboratory of Chemistry, Pennsylvania and Roswell Park Station
(New
York
Sprkgville,
Department New York
Analysis HUI-CHIU LI, C. STRONG
Allegheny Memorial of Public
College, Institute, Health),
Received July 22, 1963 INTRODUCTION Huisman and Prins (1) accomplished the separation of human hemoglobins F, A, E, S, and C by varying both the flow rate and sodium ion concentration during chromatography on the cation-exchange resin IRC-50 using citrate buffers. With the same resin Morrison and Cook (2) demonstrated that oxyhemoglobin from hemolyzates of human hemoglobin was actually heterogeneous, and could be separated by chromatography into a main zone (84%) and two minor zones of 10 and 6%) respectively. By refinement of these techniques, a very sensitive chromatographic method for the separation of the major component of whole normal human hemoglobin from six minor components was first described by Allen, Schroeder, and Balog (3) and later perfected by Clegg and Schroeder (4) and Schnek and Schroeder (5). Amberlite IRC-50 was again employed for the separation and a series of six phosphate buffer developers, varying both in pH and sodium ion concentration, was employed for the elution. Hutton, Bishop, Schweet, and Russell (6) applied the above techniques to the problem of “single” and “diffuse” hemoglobins in inbred mouse strains, hemoglobins previously characterized only by a variety of electrophoretic techniques (7-11). They found that the use of Developer No. 2 of Allen et al. (3) showed for the “single” hemoglobins (C57Bl and SEC) one single major component and a fast moving minor peak which was observed but not further studied. Four “diffuse” hemoglobins (AKR, FL, C3H, and DBA), on the other hand, showed a major component in the same region as the “single” hemoglobins plus one additional minor component. 407
408
RHINESMITH,
LI,
MILLIKIN,
AND
STRONG
Allen et al. (3) noted that their Developer No. 2 did not always give reproducible results for minor components and work in our laboratory indicated that Developers 1 through 5 exhibited poor resolving power when used to chromatograph mouse hemoglobins. Thus with strain C3HB Developer No. 1 gave three broad zones at 0°C. At 6” the three peaks were somewhat sharper and a fourth zone appeared which was removed as a single zone only by raising the temperature to 28”. With Developers No. 3 and 5 the strongly adsorbed fourth zone showed no movement even at 28”, but could be removed by increasing the sodium ion concentration. Hence the present investigation was begun to determine whether additional components might be found in both “single” and “diffuse” types of inbred mouse hemoglobin by use of developers capable of sharper resolution. Our work has led to an extension of the methods developed by Schroeder et ~2. (3-5) for human hemoglobins, primarily through the combination of their Developer No. 5 with a two-chamber “autograd” to give a linear gradient of increasing sodium ion concentration. As a result, the detection of additional minor components as well as nonheme protein has been achieved for both “single” and “diffuse” inbred strains; additional major components have been found for the “diffuse” forms. With a constant flow rate and one change in temperature all components can be resolved in a single chromatogram in a reasonable period of time. EXPERIMENTAL Mouse Strains. The mice used in this investigation were obtained from inbred colonies maintained at Roswell Park Memorial Institute Biological Station. The strains used were C3H/St, C3HB/St, AKR, A/St, C57BL/St, F/St, and N/St. They are referred to in this paper as C3H, CIHB, AKR, A, C57, F, and N, respectively. Preparation of Hemoglobin. Blood was taken directly from the left ventricle by syringe and mixed immediately with cold 3.2% sodium citrate solution. The procedure for purification of the hemoglobin was essentially that of Clegg and Schroeder (4). After final centrifugation for 1 hr at 23,500 X g the pooled, uncrystallized samples of oxyhemoglobin were dialyzed for a minimum of 24 hr at &6’ against the appropriate developer. Since all of the developers used were 0.01 M in KCN, the problem discussedby Allen et al. (3) of extraneous zones due to traces of methemoglobin was avoided. During purification and dialysis of the hemoglobins, great care must be exercised to see that no crystallization occurs. Mouse hemoglobin is much less soluble in phosphate buffers than human hemoglobin, especially at low temperatures. Popp and Cosgrove (10) have characterized the amorphous mouse hemoglobin precipitates
MOUSE
HEMOGLOBIN
ANALYSIS
409
and hexagonal crystals salted out of homozygous-“diffuse” and Xngle” types, respectively, as well as the mixed precipitates obt’ained from heterozygous-“diffuse” types. Our work with these heterogeneous solid mouse hemoglobins indicates that preferential solution of individual components may occur and hence the possibility of selective precipit,ation should be avoided. Chromatographic Procedures. Columns (1 X 35 cm) of Amberlite IRC-50 resin were equilibrated with Developer No. 5 of Allen et al. (3) and maintained at 6”. The top 3 cm of the column was stirred and allowed to settle. The dialyzed oxyhemoglobin was diluted to a concentration of approximately 60 mg in 3 ml and was added to the column with a bent-tip dropper without disturbing the surface of the resin. Instead of rinsing the sample onto the column, the new technique described by Schnek and Schroeder (5) of carefully overlaying the sample with 1 or 2 ml of developer achieved quantitative transfer and produced welldefined chromatographic zones. In order to obtain a gradient of [Na+] a two-chamber “autograd” was used for the elution. This apparatus was first suggestedby Bock and Ling (12) and further developed by Peterson and Sober (13), who used the term “varigrad” to describe the apparatus. The flow rate was set at 10 ml/hr and 4-ml fractions were collected. The optical densities were read at 280 rnp on the DU spectrophotometer and at 415 mp on the Bausch & Lomb Spectronic 20. Chamber I of the “autograd” contained 400 ml of Developer No. 5, phosphate buffer pH 6.85 and [Na+] 0.055 M. This solution was stirred magnetically. Chamber 2 contained 400 ml of phosphate buffer pH 6.85 and [Na;] 0.20M. The eluent from Chamber I of the “autograd” was delivered directly to the capillary ball joint at the top of the chromatographic column. By varying both the initial [Na’] and the limiting [Na+], a flexibility in the resolving power of the eluent was achieved which made the procedure applicable to hemoglobins differing widely in solubility, as well as in the number of major and minor components. RESULTS
The results of the chromatography are summarized in Fig. 1 and in Table 1, which compares the relative percentages of both major and minor components. As a system of nomenclature we propose an extension of the original designation of Allen et al. (3) used for human hemoglobins: the letter M to indicate mouse hemoglobin, with Roman numerals and small letters as subscripts to represent the major and minor components, respectively, the genetic strain to be included as a prefix and the hemin coordinating complex to he added as a suffix. Thus in Fig. 11)
410
RHINESMITH,
LI,
:
__x::;-, 100
200
300
STRONG
: ‘MX
0 40 80
0 0
AND
M/; :: I[.,.. : ,’ : +a
-
0 40 80
MILLIKIN,
400
500 Effluent
. . . . _..
600
700
000
900
IO00
1100
(ml 1
FIG.
la
25 t
0
100
200
300
400
500 Effluent
FIG.
600
700
800
900
1000
1100
(ml)
lb
the second minor component obtained from the C3H strain of mouse oxyhemoglobin would be completely described by the expression C3HMIb-HbO,; the major component would be written as C3H-MII-HbO,.
MOUSE
30-
HEMOGLOBIN
ANALYSIS
-111
0.0 al 415 M/L F---
25-
c57. N-
-
OD.at 415 Mp0.13 at 280 Mp ., ....’
Effluent FIG.
(ml)
lc
FIG. 1. (a) Chromatogram of two homozygous-“diffuse” mouse oxyhemoglobins on a 1 x 35 cm column of IRC-50 with Developer No. 5 and a gradient of sodium ion concentration. The inserts show on a reduced vertical scale the nonheme protein with zone MI, at the beginning of the chromatogram and zone Mv. at the end. The temperature was changed from 6 to 28°C after 336 ml of effluent for AKR and after 1162 ml for A; the temperature change automatically increased the flow rate from 10 to 15 ml/hr. (b) Chromatogram of two “diffuse” mouse oxyhemoglobins, C3H and C3HB, under conditions similar to (a) except that the temperature was changed after 716 ml of effluent for C3H and after 724 ml for C3HB. (c) Chromatogram of three “single” mouse oxyhemoglobins under conditions similar to (a) except that the entire chromatogram was run at 6”.
DISCUSSION
It should first be pointed out that the Roman numerals and small letters used to designate the major and minor components of the hemoglobins are assigned to indicate only the order of emergence of the zones; the fact that several zones have the same designation does not mean that they are identical. Thus in Fig. 1 the seven zones variously labeled Mrr show effluent volumes at which the peaks emerge varying from 480 to 580 ml. Indeed, the point of emergence of the two peaks MI1 in Fig. la is very close to that of MI, of Fig. lb. Since the values calculated in Table 1 represent fractions of the total optical density, we might expect any variations in the point of emergence of the peaks to depend sharply on the degree of resolution of the zones and the reproducibility of the
412
RHINESMITH,
LI,
MILLIKIN,
TABLE
AND
STRONG
1
:\MOUNT OF EACH OF CHROMATOGRAPHICALLY SEPARATED COMPONENTS OF HEMOGLOBIN FROM SEVEN INBRED STRAINS OF MICE EXPRESSED AS PERCENTAGE OF TOTAL OPTICAL DENSITY AT 4%MM Total Hb type
“Diffuse”
‘ ‘Single”
Strain
AKR run run A run run C3H C3HB c57 F N
Ml.
1 2 1 2
2 4 3 5 2 2 8 2 4
MB
MIO
MI1
MI11
MN
-
16 19 2 10
23 22 26 24 38 27 70 90 82
22 20 20 21 36 46 -
48 48 43 42 -
8 6 21 5 4
Mv.
5 6 8 8 -
H%
100 100 100 100 100 100 99 99 100
% nonheme proteina
2 -) 3 2 3 3 5 2 2
Q Values for nonheme protein were obtained by dividing optical density of nonheme protein at 280 mp by total optical density at 415 mp; they represent a percentage of the total protein of the sample. b Not determined for this run.
results. For chromatograms of AKR and A we have included duplicate runs to show that the major peaks are reproducible within limits of error of the order of lt5 to 10%. In general we find this to be true for all the chromatograms, including replicate chromatograms on the same sample as well as hemoglobin samples obtained from various mice of the same strain, provided they are chromatographed under similar conditions. We are less certain about the minor components, however, especially when they amount to less than 10% of the sample. None of these zones returns to baseline; they occasionally overlap with a second major zone and the resolution seems to depend to some extent on the size of the sample, improving somewhat with increasing amounts of hemoglobin. For more consistent results the minor zones should be rechromatographed and the effect of a reduction in sodium ion also studied. This work is now in progress. Recently we accomplished the removal of zone MIII (Fig. lb) from the column by raising [Na+] to 0.3M and holding the temperature at 6”; thus it too may be further resolvable, especially in view of the fact that AKR and A possess a fourth major zone and an additional minor zone Mva. Hemoglobins of the A and AKR strains are shown to be the “diffuse” type. Both contain three major zones totaling in each case about 90% of the protein, as well as a small, fast moving zone MI, and a somewhat larger slow moving zone M,,. They are thus remarkably similar chromatographically considering that their biological characteristics and their genetic origins differ widely. Strain A was obtained from
MOUSE
HEMOGLOBIN
AKALTSIS
413
a cross made in 1921 of albino from Cold Spring Harbor and Bagg albino. Inbreeding was carried out for 138 generations. The breeders show a high incidence of mammary gland tumors. The AKR was obtained from Carworth Farms in 1955 and since that time has undergone 23 additional generations of inbreeding at Springville; 70% of all these breeders become leukemic with age and show some mammary gland tumors. The hemoglobins of the C3H and C3HB strains are also shown to be of the “diffuse” type. They differ from AKR and A in that zones MIv and Mva are completely lacking, although we do not preclude the possibility that zones M III of C3H and C3HB may be further resolved. These hemoglobins differ also in that C3H has about 10% more of the MI1 component but 10% less of M,, ; in each case, however, the sum of MI1 + MIXI may be associated with the mutation at the microphthalmicwhite anemia locus. Mice of both strains show a very high incidence of breast tumors, but it would be unwise to draw any conclusions now between any hemoglobin type and the origin of spontaneous tumors. Mice of the three strains that show the “single” type of hemoglobin, C57, F, and N, differ in many biological characteristics. They are all relatively resistent to spontaneous breast tumors; F mice, however, develop a high incidence of both myelogenous and lymphatic leukemia. Here again it is premature to draw correlations. Chromatographically, all three hemoglobins of the “single” type (C57, F and N) lack completely the M III zone which is a major component in the “diffuse” types (C3H, CSHB, AKR, and A). The LLsingle” types also lack the M,, zone which appears in AKR and A, as well as the strongly adsorbed Mv,, which can be removed only by raising the temperature to 28”. The missing zones for the “single” types are shown clearly in Table 1. It is also striking that even though F and N mice have no common ancestry these mice possess very similar hemoglobins. In fact, all strains used in this investigation except C3H and C3HB have had independent origins for at least forty years of inbreeding. All of the hemoglobins examined contain nonheme protein in amounts varying from 2 to 5%. Since the nonheme protein appears in the first 50 ml of effluent and is of interest mainly in terms of its relative amount, it seemed unnecessary to read all of the fractions at 280 mp in order to get the total optical density at 280 m ,LL.Consequently the values for the nonheme protein were calculated as a percentage of the total protein, as indicated in footnote a of Table 1. SUMMARY 1. The %ingle” type mouse hemoglobin has one major, three minor, and one nonheme component. Strain C57 appears to be exceptional (zone
414
RHINESMITH,
LI,
MILLIKIN,
AND
STRONG
MI, is missing) but there is some existing evidence that the minor component MI,, may be further resolved. 2. Two types of “diffuse” hemoglobins were found. Strains C3H and CSHB contain two major, three minor, and one nonheme component; AKR and A contain three major, two minor, and one nonheme component. 3. While the “single” type mouse hemoglobins seem to be characterized by one large major component, very real differences exist in the over-all composition of all the hemoglobins studied, both in the major and minor components. 4. It is proposed that a modification of the nomenclature of Allen et a,l. (3) for the components of human hemoglobin be developed for other species. ACKNOWLEDGMENTS This investigation has been supported in part by grants from the National Science Foundation (G-7970) and the National Institutes of Health (GM-11838-01). We acknowledge with thanks the technical assistance of Wilson W. Strong, Jr., and Mrs. Leona Reichert. REFERENCES 1. HUISMAN,
T. H. J., AND PRINS, K. K., J. Lab. Clin. Med. 46, 255 (1956). AND COOK, J. L., Science 122, 920 (1955). SCHROEDER, W. A., AND BALOG, J., J. Am. Chem. Sot. SO, 1628
2. MORRISON, M., 3. ALLEN, D. W.,
(1958). CLEQQ, M. D., AND SCHROEDER, W. A., J. Am. Chem. Sot. 81, 6065(1959). 5. SCENEK, A. G., AND SCHROEDER, W. A,, J. Am. Chem. Sot. 83, 1472 (1961). 6. HUT~VN, J. J., BISHOP, J., SCHWEET, R., AND RUSSELL, E. S., Proc. Natl. Acad. sci. u. s. 48, 1505 (1962). 7, RANNEY, H. M., AND GLUECKSOHN-WAELSCH, S., Ann. Human Genetics 19, 269 (1955). 8. RUSSELL, E. S., AND GERALD, P. S., Science 128, 1569 (1958). 9. WELLINQ, W., AND VAN BEKKUM, D. W., Nature 182, 946 (1953). 10. POPP, R. A., AND COSGROVE, G. E., Proc. Sot. Ezptl. Biol. Med. 101, 754 (1959). 11. GLUECKSOHN-WAELSCH, S., J. Cell. Comp. Physiol. 56, Suppl. 1, 89 (1960). 12. BOCK, R. M., AND LING, N. S., Anal. Chem. 26, 1543 (1954). 13. PETERSON, E. A., AND SOBER, H. A., Anal. Chem. 31, 857 (1959). 4.
BIOCHEMlSTKY
Mouse
8, 407-414 (1964)
Hemoglobin.
HERBERT BETTY From the Meadville, Biological
I. Chromatographic
S. RHINESMITH, LOU MILLIKIN,
AND
HERSCHEL LEONELL
Carnegie Laboratory of Chemistry, Pennsylvania and Roswell Park Station
(New
York
Sprkgville,
Department New York
Analysis HUI-CHIU LI, C. STRONG
Allegheny Memorial of Public
College, Institute, Health),
Received July 22, 1963 INTRODUCTION Huisman and Prins (1) accomplished the separation of human hemoglobins F, A, E, S, and C by varying both the flow rate and sodium ion concentration during chromatography on the cation-exchange resin IRC-50 using citrate buffers. With the same resin Morrison and Cook (2) demonstrated that oxyhemoglobin from hemolyzates of human hemoglobin was actually heterogeneous, and could be separated by chromatography into a main zone (84%) and two minor zones of 10 and 6%) respectively. By refinement of these techniques, a very sensitive chromatographic method for the separation of the major component of whole normal human hemoglobin from six minor components was first described by Allen, Schroeder, and Balog (3) and later perfected by Clegg and Schroeder (4) and Schnek and Schroeder (5). Amberlite IRC-50 was again employed for the separation and a series of six phosphate buffer developers, varying both in pH and sodium ion concentration, was employed for the elution. Hutton, Bishop, Schweet, and Russell (6) applied the above techniques to the problem of “single” and “diffuse” hemoglobins in inbred mouse strains, hemoglobins previously characterized only by a variety of electrophoretic techniques (7-11). They found that the use of Developer No. 2 of Allen et al. (3) showed for the “single” hemoglobins (C57Bl and SEC) one single major component and a fast moving minor peak which was observed but not further studied. Four “diffuse” hemoglobins (AKR, FL, C3H, and DBA), on the other hand, showed a major component in the same region as the “single” hemoglobins plus one additional minor component. 407
408
RHINESMITH,
LI,
MILLIKIN,
AND
STRONG
Allen et al. (3) noted that their Developer No. 2 did not always give reproducible results for minor components and work in our laboratory indicated that Developers 1 through 5 exhibited poor resolving power when used to chromatograph mouse hemoglobins. Thus with strain C3HB Developer No. 1 gave three broad zones at 0°C. At 6” the three peaks were somewhat sharper and a fourth zone appeared which was removed as a single zone only by raising the temperature to 28”. With Developers No. 3 and 5 the strongly adsorbed fourth zone showed no movement even at 28”, but could be removed by increasing the sodium ion concentration. Hence the present investigation was begun to determine whether additional components might be found in both “single” and “diffuse” types of inbred mouse hemoglobin by use of developers capable of sharper resolution. Our work has led to an extension of the methods developed by Schroeder et ~2. (3-5) for human hemoglobins, primarily through the combination of their Developer No. 5 with a two-chamber “autograd” to give a linear gradient of increasing sodium ion concentration. As a result, the detection of additional minor components as well as nonheme protein has been achieved for both “single” and “diffuse” inbred strains; additional major components have been found for the “diffuse” forms. With a constant flow rate and one change in temperature all components can be resolved in a single chromatogram in a reasonable period of time. EXPERIMENTAL Mouse Strains. The mice used in this investigation were obtained from inbred colonies maintained at Roswell Park Memorial Institute Biological Station. The strains used were C3H/St, C3HB/St, AKR, A/St, C57BL/St, F/St, and N/St. They are referred to in this paper as C3H, CIHB, AKR, A, C57, F, and N, respectively. Preparation of Hemoglobin. Blood was taken directly from the left ventricle by syringe and mixed immediately with cold 3.2% sodium citrate solution. The procedure for purification of the hemoglobin was essentially that of Clegg and Schroeder (4). After final centrifugation for 1 hr at 23,500 X g the pooled, uncrystallized samples of oxyhemoglobin were dialyzed for a minimum of 24 hr at &6’ against the appropriate developer. Since all of the developers used were 0.01 M in KCN, the problem discussedby Allen et al. (3) of extraneous zones due to traces of methemoglobin was avoided. During purification and dialysis of the hemoglobins, great care must be exercised to see that no crystallization occurs. Mouse hemoglobin is much less soluble in phosphate buffers than human hemoglobin, especially at low temperatures. Popp and Cosgrove (10) have characterized the amorphous mouse hemoglobin precipitates
MOUSE
HEMOGLOBIN
ANALYSIS
409
and hexagonal crystals salted out of homozygous-“diffuse” and Xngle” types, respectively, as well as the mixed precipitates obt’ained from heterozygous-“diffuse” types. Our work with these heterogeneous solid mouse hemoglobins indicates that preferential solution of individual components may occur and hence the possibility of selective precipit,ation should be avoided. Chromatographic Procedures. Columns (1 X 35 cm) of Amberlite IRC-50 resin were equilibrated with Developer No. 5 of Allen et al. (3) and maintained at 6”. The top 3 cm of the column was stirred and allowed to settle. The dialyzed oxyhemoglobin was diluted to a concentration of approximately 60 mg in 3 ml and was added to the column with a bent-tip dropper without disturbing the surface of the resin. Instead of rinsing the sample onto the column, the new technique described by Schnek and Schroeder (5) of carefully overlaying the sample with 1 or 2 ml of developer achieved quantitative transfer and produced welldefined chromatographic zones. In order to obtain a gradient of [Na+] a two-chamber “autograd” was used for the elution. This apparatus was first suggestedby Bock and Ling (12) and further developed by Peterson and Sober (13), who used the term “varigrad” to describe the apparatus. The flow rate was set at 10 ml/hr and 4-ml fractions were collected. The optical densities were read at 280 rnp on the DU spectrophotometer and at 415 mp on the Bausch & Lomb Spectronic 20. Chamber I of the “autograd” contained 400 ml of Developer No. 5, phosphate buffer pH 6.85 and [Na+] 0.055 M. This solution was stirred magnetically. Chamber 2 contained 400 ml of phosphate buffer pH 6.85 and [Na;] 0.20M. The eluent from Chamber I of the “autograd” was delivered directly to the capillary ball joint at the top of the chromatographic column. By varying both the initial [Na’] and the limiting [Na+], a flexibility in the resolving power of the eluent was achieved which made the procedure applicable to hemoglobins differing widely in solubility, as well as in the number of major and minor components. RESULTS
The results of the chromatography are summarized in Fig. 1 and in Table 1, which compares the relative percentages of both major and minor components. As a system of nomenclature we propose an extension of the original designation of Allen et al. (3) used for human hemoglobins: the letter M to indicate mouse hemoglobin, with Roman numerals and small letters as subscripts to represent the major and minor components, respectively, the genetic strain to be included as a prefix and the hemin coordinating complex to he added as a suffix. Thus in Fig. 11)
410
RHINESMITH,
LI,
:
__x::;-, 100
200
300
STRONG
: ‘MX
0 40 80
0 0
AND
M/; :: I[.,.. : ,’ : +a
-
0 40 80
MILLIKIN,
400
500 Effluent
. . . . _..
600
700
000
900
IO00
1100
(ml 1
FIG.
la
25 t
0
100
200
300
400
500 Effluent
FIG.
600
700
800
900
1000
1100
(ml)
lb
the second minor component obtained from the C3H strain of mouse oxyhemoglobin would be completely described by the expression C3HMIb-HbO,; the major component would be written as C3H-MII-HbO,.
MOUSE
30-
HEMOGLOBIN
ANALYSIS
-111
0.0 al 415 M/L F---
25-
c57. N-
-
OD.at 415 Mp0.13 at 280 Mp ., ....’
Effluent FIG.
(ml)
lc
FIG. 1. (a) Chromatogram of two homozygous-“diffuse” mouse oxyhemoglobins on a 1 x 35 cm column of IRC-50 with Developer No. 5 and a gradient of sodium ion concentration. The inserts show on a reduced vertical scale the nonheme protein with zone MI, at the beginning of the chromatogram and zone Mv. at the end. The temperature was changed from 6 to 28°C after 336 ml of effluent for AKR and after 1162 ml for A; the temperature change automatically increased the flow rate from 10 to 15 ml/hr. (b) Chromatogram of two “diffuse” mouse oxyhemoglobins, C3H and C3HB, under conditions similar to (a) except that the temperature was changed after 716 ml of effluent for C3H and after 724 ml for C3HB. (c) Chromatogram of three “single” mouse oxyhemoglobins under conditions similar to (a) except that the entire chromatogram was run at 6”.
DISCUSSION
It should first be pointed out that the Roman numerals and small letters used to designate the major and minor components of the hemoglobins are assigned to indicate only the order of emergence of the zones; the fact that several zones have the same designation does not mean that they are identical. Thus in Fig. 1 the seven zones variously labeled Mrr show effluent volumes at which the peaks emerge varying from 480 to 580 ml. Indeed, the point of emergence of the two peaks MI1 in Fig. la is very close to that of MI, of Fig. lb. Since the values calculated in Table 1 represent fractions of the total optical density, we might expect any variations in the point of emergence of the peaks to depend sharply on the degree of resolution of the zones and the reproducibility of the
412
RHINESMITH,
LI,
MILLIKIN,
TABLE
AND
STRONG
1
:\MOUNT OF EACH OF CHROMATOGRAPHICALLY SEPARATED COMPONENTS OF HEMOGLOBIN FROM SEVEN INBRED STRAINS OF MICE EXPRESSED AS PERCENTAGE OF TOTAL OPTICAL DENSITY AT 4%MM Total Hb type
“Diffuse”
‘ ‘Single”
Strain
AKR run run A run run C3H C3HB c57 F N
Ml.
1 2 1 2
2 4 3 5 2 2 8 2 4
MB
MIO
MI1
MI11
MN
-
16 19 2 10
23 22 26 24 38 27 70 90 82
22 20 20 21 36 46 -
48 48 43 42 -
8 6 21 5 4
Mv.
5 6 8 8 -
H%
100 100 100 100 100 100 99 99 100
% nonheme proteina
2 -) 3 2 3 3 5 2 2
Q Values for nonheme protein were obtained by dividing optical density of nonheme protein at 280 mp by total optical density at 415 mp; they represent a percentage of the total protein of the sample. b Not determined for this run.
results. For chromatograms of AKR and A we have included duplicate runs to show that the major peaks are reproducible within limits of error of the order of lt5 to 10%. In general we find this to be true for all the chromatograms, including replicate chromatograms on the same sample as well as hemoglobin samples obtained from various mice of the same strain, provided they are chromatographed under similar conditions. We are less certain about the minor components, however, especially when they amount to less than 10% of the sample. None of these zones returns to baseline; they occasionally overlap with a second major zone and the resolution seems to depend to some extent on the size of the sample, improving somewhat with increasing amounts of hemoglobin. For more consistent results the minor zones should be rechromatographed and the effect of a reduction in sodium ion also studied. This work is now in progress. Recently we accomplished the removal of zone MIII (Fig. lb) from the column by raising [Na+] to 0.3M and holding the temperature at 6”; thus it too may be further resolvable, especially in view of the fact that AKR and A possess a fourth major zone and an additional minor zone Mva. Hemoglobins of the A and AKR strains are shown to be the “diffuse” type. Both contain three major zones totaling in each case about 90% of the protein, as well as a small, fast moving zone MI, and a somewhat larger slow moving zone M,,. They are thus remarkably similar chromatographically considering that their biological characteristics and their genetic origins differ widely. Strain A was obtained from
MOUSE
HEMOGLOBIN
AKALTSIS
413
a cross made in 1921 of albino from Cold Spring Harbor and Bagg albino. Inbreeding was carried out for 138 generations. The breeders show a high incidence of mammary gland tumors. The AKR was obtained from Carworth Farms in 1955 and since that time has undergone 23 additional generations of inbreeding at Springville; 70% of all these breeders become leukemic with age and show some mammary gland tumors. The hemoglobins of the C3H and C3HB strains are also shown to be of the “diffuse” type. They differ from AKR and A in that zones MIv and Mva are completely lacking, although we do not preclude the possibility that zones M III of C3H and C3HB may be further resolved. These hemoglobins differ also in that C3H has about 10% more of the MI1 component but 10% less of M,, ; in each case, however, the sum of MI1 + MIXI may be associated with the mutation at the microphthalmicwhite anemia locus. Mice of both strains show a very high incidence of breast tumors, but it would be unwise to draw any conclusions now between any hemoglobin type and the origin of spontaneous tumors. Mice of the three strains that show the “single” type of hemoglobin, C57, F, and N, differ in many biological characteristics. They are all relatively resistent to spontaneous breast tumors; F mice, however, develop a high incidence of both myelogenous and lymphatic leukemia. Here again it is premature to draw correlations. Chromatographically, all three hemoglobins of the “single” type (C57, F and N) lack completely the M III zone which is a major component in the “diffuse” types (C3H, CSHB, AKR, and A). The LLsingle” types also lack the M,, zone which appears in AKR and A, as well as the strongly adsorbed Mv,, which can be removed only by raising the temperature to 28”. The missing zones for the “single” types are shown clearly in Table 1. It is also striking that even though F and N mice have no common ancestry these mice possess very similar hemoglobins. In fact, all strains used in this investigation except C3H and C3HB have had independent origins for at least forty years of inbreeding. All of the hemoglobins examined contain nonheme protein in amounts varying from 2 to 5%. Since the nonheme protein appears in the first 50 ml of effluent and is of interest mainly in terms of its relative amount, it seemed unnecessary to read all of the fractions at 280 mp in order to get the total optical density at 280 m ,LL.Consequently the values for the nonheme protein were calculated as a percentage of the total protein, as indicated in footnote a of Table 1. SUMMARY 1. The %ingle” type mouse hemoglobin has one major, three minor, and one nonheme component. Strain C57 appears to be exceptional (zone
414
RHINESMITH,
LI,
MILLIKIN,
AND
STRONG
MI, is missing) but there is some existing evidence that the minor component MI,, may be further resolved. 2. Two types of “diffuse” hemoglobins were found. Strains C3H and CSHB contain two major, three minor, and one nonheme component; AKR and A contain three major, two minor, and one nonheme component. 3. While the “single” type mouse hemoglobins seem to be characterized by one large major component, very real differences exist in the over-all composition of all the hemoglobins studied, both in the major and minor components. 4. It is proposed that a modification of the nomenclature of Allen et a,l. (3) for the components of human hemoglobin be developed for other species. ACKNOWLEDGMENTS This investigation has been supported in part by grants from the National Science Foundation (G-7970) and the National Institutes of Health (GM-11838-01). We acknowledge with thanks the technical assistance of Wilson W. Strong, Jr., and Mrs. Leona Reichert. REFERENCES 1. HUISMAN,
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(1958). CLEQQ, M. D., AND SCHROEDER, W. A., J. Am. Chem. Sot. 81, 6065(1959). 5. SCENEK, A. G., AND SCHROEDER, W. A,, J. Am. Chem. Sot. 83, 1472 (1961). 6. HUT~VN, J. J., BISHOP, J., SCHWEET, R., AND RUSSELL, E. S., Proc. Natl. Acad. sci. u. s. 48, 1505 (1962). 7, RANNEY, H. M., AND GLUECKSOHN-WAELSCH, S., Ann. Human Genetics 19, 269 (1955). 8. RUSSELL, E. S., AND GERALD, P. S., Science 128, 1569 (1958). 9. WELLINQ, W., AND VAN BEKKUM, D. W., Nature 182, 946 (1953). 10. POPP, R. A., AND COSGROVE, G. E., Proc. Sot. Ezptl. Biol. Med. 101, 754 (1959). 11. GLUECKSOHN-WAELSCH, S., J. Cell. Comp. Physiol. 56, Suppl. 1, 89 (1960). 12. BOCK, R. M., AND LING, N. S., Anal. Chem. 26, 1543 (1954). 13. PETERSON, E. A., AND SOBER, H. A., Anal. Chem. 31, 857 (1959). 4.