- Email: [email protected]
Quantitative determination of the minor hemoglobin component Hb-A2 by DEAE-cellulose chromatography
SHORT COMMUNICATIONS
400
Because of possible aldehyde losses as demonstrated above, it is important to determine that a stoichiometric relation exists between tyramine utilized and product formed in a given system before using it routinely. Such determinations are not difficult with purified preparations on which oxygen consumption or NH, formation may be estimated; however, with crude preparations neither of the above procedures may be practical. Under these conditions, the use of a calorimetric method and an internal standard is essential for accurate estimations of MAO activity. REFERENCES 1. GREEN, A. 2. LANaHELD, 3. AUTERHOE’F,
4. 5. 6.
L., AND HAUGHTON, T. M., &o&em. J. 78, 172 (1961). K., Ber. 42, 2360 (1909). H., AND ROTH, H. J., Arch. Pharm. 289, 470 (1956). RICHTER, D., Biochem. .I. 31, 2022 (1937). CAEASEY, N. H., Biochem. J. 64, 178 (1956). SCHNEIDER, W. C., J. Biol. Chem. 176, 259 (1948). IVAN
J. STERN
SHERWIN RICHARD
WILK
D. HOLLIFIELD
Biochemistry Section, Eaton Laboratories The Norwich Pharmacal Company Norwich, New York Received April 24, 1961
Quantitative Component
Determination Hb-A,
of
the
Minor
by DEAE-Cellulose
Hemoglobin
Chromatography’
Quantitative analyses of the minor hemoglobin fraction Hb-A, in different hemoglobin samples have been successfully carried out using starch electrophoresis (1, 2) and chromatography with the cation exchanger carboxymethylcellulose (CMC) as adsorbent (3, 4). With both methods the amount of Hb-A, in the blood of normal adults was found to range from 1.8 to 2.5% of the total hemoglobin, while the percentages of Hb-A, were’increased to 4-6s in most casesof thalassemia trait. In recent investigations* the chromatographic behavior of different ‘Supported by U. S. Public Health Service Grant No. “T. H. J. Huisman and A. M. Dozy, J. Chromatography.
H 5168. In press
human hemoglobin types on the anion exchanger diethylaminoethylcellulose (DEAE-cellulose) was studied. It was found that the elution rates of many different hemoglobin types (with the exception of fetal hemoglobin) were in accordance with differences in their isoelectric points. With the use of this anion exchanger it was possible to separate the hemoglobin of a normal adult into three components: the minor Hb-A, fraction being eluted first followed by the main hemoglobin component (A,) and then the electrophoretically fast moving Hb-A, fraction. Since the separation between the Hb-A, and the Hb-A, components after isolation, behaving electrowas complete, with both fractions, phoretically pure [tested by starch gel electrophoresis (5, S)], new possibilities were presented for the determination of Hb-A, percentages in blood samples. The following routine procedure was developed: A large quantity of DEAE-cellulose (Selecta cel-DEAE,3 type 40) is carefully equilibrated by repeated washings with 0.005 M sodium phosphate buffer pH 8.6 containing 100 mg KCN/l. A small quantity of absorbent is washed once again with the same buffer just prior to the experiment. The equilibrated DEAE-cellulose is poured into columns of 20 X 0.9 cm a,nd packed under atmospheric pressure until a column height of 15 cm is obtained. Prior to chromatography hemoglobin solutions are dialyzed for 24 hr against the buffer used for the equilibration. From 10 to 25 mg oxyhemoglobin, dissolved in 0.5-2.0 ml, is chromatographed. Elution of the Hb-A, fraction is performed by applying a 0.01 M sodium phosphate buffer pH 8.6 (with 100 mg KCN/l) to the column from a separatory funnel. The flow rate is adjusted t.o 3040 ml/hr. Fractions of 3.54.0 ml are collected in tubes graduated to 4 ml. After t.he elution of the Hb-A2 fraction (about 40 ml of the effluent is required), the column is mounted above a volumetric flask (200 ml) and the remaining hemoglobin eluted with the use of 0.01 N sodium phosphate buffer pH 6.0 to which 0.3M NaCl is added. The volumes of the fractions are finally adjusted to 4 and 200 ml, respectively. Examination of the fractions is carried out at 415 mp in the Beckman DU spectrophotometer. The percentage of Hb-A, is calculated using the formula
in which A represents the combined optical densities of the Hb-A,containing fractions and B the optical density of t.he hemoglobin solution collected in the 200-ml volumetric flask. ‘Brown
Company,
55 Main
Street,
Berlin,
N.
H.
402
SHORT
COMMUNICATIONS
Examples of elution diagrams for the hemoglobins of a normal adult and a patient with thalassemia trait are presented in Fig. 1. The Hb-A, was completely separated from the main component. In starch gel elect.rophoresis the isolated Hb-A, was found to be free of any other Hb fraction (Fig. 1). Quantitative data are presented in Table 1. The
FIG. 1. Left: Elution trait and of a normal Hb-AZ fraction (2 and by CMC chromatography
diagrams of the control. Right: 3) and of normal (3).
hemoglobins Starch-gel Hb-A. The
of a patient electrophoresis eluted Hb-A,
with thalassemia of the isolated was concentrated
mean Hb-A, level of 2.1% found in blood samples of 34 normal individuals is in close agreement with the percentages determined either by starch-block electrophoresis or by CMC chromatography. In the blood of 15 patients wit,h thalassemia minor the Hb-A, level was increased t,o 4.4% with a range of 3.8-5.20/o. The method offers reproducible data since repeated Hb-A, analyses in one hemoglobin sample carried out at the same time revealed similar results (Table 1). The new chromatographic technique can also be applied to hemoglobin samples from cases with various hemoglobinopathies. Electrophoretically slow-moving hemoglobins, however, will interfere with the elution of the Hb-A, component. Application is therefore restricted to hemoglobin samples of normal individuals, patients with thalassemia minor, and carriers of hemoglobin anomalies possessing higher electrophoretic mobil-
40s
COMMI~NIC.\TIONS
SHORT
ities than Hb-A. Since fetal hemoglobin does not interfere with the elution of the Hb-A, component,* the method can be used in the study of samples from cord blood and from patients with the persistent high TABLE Hb-An
PERCENTAGES
WITH
THAL~SSEMIA
C&e
Normal Thal. minor C&e
Normal Normal Thai. Thal. Thal. Thai. Thal.
trait, trait trait trait trait
IN MINOR
BLOOD
1
SAMPLES
OF NORA~AL
DETERMINED
ADULTS
.~ND
BY DEAE-CELLULOSE
No.
Hb-A?
34 15
2.1 4.4
OF PATIENTS
CHROMATOGRAPHY Ran&F!
f 0.2 * 0.3
I .6-2.5 3.8-5.2
Cd. 1
Cd. 2
Cd. 3
1.8 2.0 5.25 4.5 4.6 4.8 4.3
1.95 2 2 5.25 4.3 4.8 4.7 4.3
1.85 2. 2 5.05 4.1 4.5 4.3 4.1
Cd. 4
Mm1
1.95 5.25 4.1 4.4 4.0
1 .9 3. 1 5.2 4.25 4.6 4.55 4.2
fetal hemoglobin abnormality or thalassemia major. The use of DEAEcellulose chromatography for the preparation of large quantities of different hemoglobin fract.ions will be report.ed elsewhere.? REFERENCES H. G., AND WALLENIUS, G., Science 122, 920 (1955). P. S., AND DIAMOND, L. K., Blood 12, 61 (1957). T. H. J., .~ND MEYERING, C. A., C&n. C&z. Acta 5, 103 ( 1960). 1. MEYERING, C. A., ISRAELS, A. L. M., SERENS, T., AND HUIRMAN, T. H. J., Chim. Acta 5, 208 (1960). 5. SMITHIES, O., Biochem. J. 71, 585 (1959). 6. HUISMAN, T. H. J., Clin. Chim. Actn 5, 709 (1960). 1. K~NKEL,
2. GERALD, 3. HUISMAN,
TITUS ANDREE Departments of Biochemistry Medical College o,f Georgia Augusta, Georgia Received May 8, 1961
and
Pathologll
H. J. I-IUISM.~N M. DOZY
Clir~.
400
Because of possible aldehyde losses as demonstrated above, it is important to determine that a stoichiometric relation exists between tyramine utilized and product formed in a given system before using it routinely. Such determinations are not difficult with purified preparations on which oxygen consumption or NH, formation may be estimated; however, with crude preparations neither of the above procedures may be practical. Under these conditions, the use of a calorimetric method and an internal standard is essential for accurate estimations of MAO activity. REFERENCES 1. GREEN, A. 2. LANaHELD, 3. AUTERHOE’F,
4. 5. 6.
L., AND HAUGHTON, T. M., &o&em. J. 78, 172 (1961). K., Ber. 42, 2360 (1909). H., AND ROTH, H. J., Arch. Pharm. 289, 470 (1956). RICHTER, D., Biochem. .I. 31, 2022 (1937). CAEASEY, N. H., Biochem. J. 64, 178 (1956). SCHNEIDER, W. C., J. Biol. Chem. 176, 259 (1948). IVAN
J. STERN
SHERWIN RICHARD
WILK
D. HOLLIFIELD
Biochemistry Section, Eaton Laboratories The Norwich Pharmacal Company Norwich, New York Received April 24, 1961
Quantitative Component
Determination Hb-A,
of
the
Minor
by DEAE-Cellulose
Hemoglobin
Chromatography’
Quantitative analyses of the minor hemoglobin fraction Hb-A, in different hemoglobin samples have been successfully carried out using starch electrophoresis (1, 2) and chromatography with the cation exchanger carboxymethylcellulose (CMC) as adsorbent (3, 4). With both methods the amount of Hb-A, in the blood of normal adults was found to range from 1.8 to 2.5% of the total hemoglobin, while the percentages of Hb-A, were’increased to 4-6s in most casesof thalassemia trait. In recent investigations* the chromatographic behavior of different ‘Supported by U. S. Public Health Service Grant No. “T. H. J. Huisman and A. M. Dozy, J. Chromatography.
H 5168. In press
human hemoglobin types on the anion exchanger diethylaminoethylcellulose (DEAE-cellulose) was studied. It was found that the elution rates of many different hemoglobin types (with the exception of fetal hemoglobin) were in accordance with differences in their isoelectric points. With the use of this anion exchanger it was possible to separate the hemoglobin of a normal adult into three components: the minor Hb-A, fraction being eluted first followed by the main hemoglobin component (A,) and then the electrophoretically fast moving Hb-A, fraction. Since the separation between the Hb-A, and the Hb-A, components after isolation, behaving electrowas complete, with both fractions, phoretically pure [tested by starch gel electrophoresis (5, S)], new possibilities were presented for the determination of Hb-A, percentages in blood samples. The following routine procedure was developed: A large quantity of DEAE-cellulose (Selecta cel-DEAE,3 type 40) is carefully equilibrated by repeated washings with 0.005 M sodium phosphate buffer pH 8.6 containing 100 mg KCN/l. A small quantity of absorbent is washed once again with the same buffer just prior to the experiment. The equilibrated DEAE-cellulose is poured into columns of 20 X 0.9 cm a,nd packed under atmospheric pressure until a column height of 15 cm is obtained. Prior to chromatography hemoglobin solutions are dialyzed for 24 hr against the buffer used for the equilibration. From 10 to 25 mg oxyhemoglobin, dissolved in 0.5-2.0 ml, is chromatographed. Elution of the Hb-A, fraction is performed by applying a 0.01 M sodium phosphate buffer pH 8.6 (with 100 mg KCN/l) to the column from a separatory funnel. The flow rate is adjusted t.o 3040 ml/hr. Fractions of 3.54.0 ml are collected in tubes graduated to 4 ml. After t.he elution of the Hb-A2 fraction (about 40 ml of the effluent is required), the column is mounted above a volumetric flask (200 ml) and the remaining hemoglobin eluted with the use of 0.01 N sodium phosphate buffer pH 6.0 to which 0.3M NaCl is added. The volumes of the fractions are finally adjusted to 4 and 200 ml, respectively. Examination of the fractions is carried out at 415 mp in the Beckman DU spectrophotometer. The percentage of Hb-A, is calculated using the formula
in which A represents the combined optical densities of the Hb-A,containing fractions and B the optical density of t.he hemoglobin solution collected in the 200-ml volumetric flask. ‘Brown
Company,
55 Main
Street,
Berlin,
N.
H.
402
SHORT
COMMUNICATIONS
Examples of elution diagrams for the hemoglobins of a normal adult and a patient with thalassemia trait are presented in Fig. 1. The Hb-A, was completely separated from the main component. In starch gel elect.rophoresis the isolated Hb-A, was found to be free of any other Hb fraction (Fig. 1). Quantitative data are presented in Table 1. The
FIG. 1. Left: Elution trait and of a normal Hb-AZ fraction (2 and by CMC chromatography
diagrams of the control. Right: 3) and of normal (3).
hemoglobins Starch-gel Hb-A. The
of a patient electrophoresis eluted Hb-A,
with thalassemia of the isolated was concentrated
mean Hb-A, level of 2.1% found in blood samples of 34 normal individuals is in close agreement with the percentages determined either by starch-block electrophoresis or by CMC chromatography. In the blood of 15 patients wit,h thalassemia minor the Hb-A, level was increased t,o 4.4% with a range of 3.8-5.20/o. The method offers reproducible data since repeated Hb-A, analyses in one hemoglobin sample carried out at the same time revealed similar results (Table 1). The new chromatographic technique can also be applied to hemoglobin samples from cases with various hemoglobinopathies. Electrophoretically slow-moving hemoglobins, however, will interfere with the elution of the Hb-A, component. Application is therefore restricted to hemoglobin samples of normal individuals, patients with thalassemia minor, and carriers of hemoglobin anomalies possessing higher electrophoretic mobil-
40s
COMMI~NIC.\TIONS
SHORT
ities than Hb-A. Since fetal hemoglobin does not interfere with the elution of the Hb-A, component,* the method can be used in the study of samples from cord blood and from patients with the persistent high TABLE Hb-An
PERCENTAGES
WITH
THAL~SSEMIA
C&e
Normal Thal. minor C&e
Normal Normal Thai. Thal. Thal. Thai. Thal.
trait, trait trait trait trait
IN MINOR
BLOOD
1
SAMPLES
OF NORA~AL
DETERMINED
ADULTS
.~ND
BY DEAE-CELLULOSE
No.
Hb-A?
34 15
2.1 4.4
OF PATIENTS
CHROMATOGRAPHY Ran&F!
f 0.2 * 0.3
I .6-2.5 3.8-5.2
Cd. 1
Cd. 2
Cd. 3
1.8 2.0 5.25 4.5 4.6 4.8 4.3
1.95 2 2 5.25 4.3 4.8 4.7 4.3
1.85 2. 2 5.05 4.1 4.5 4.3 4.1
Cd. 4
Mm1
1.95 5.25 4.1 4.4 4.0
1 .9 3. 1 5.2 4.25 4.6 4.55 4.2
fetal hemoglobin abnormality or thalassemia major. The use of DEAEcellulose chromatography for the preparation of large quantities of different hemoglobin fract.ions will be report.ed elsewhere.? REFERENCES H. G., AND WALLENIUS, G., Science 122, 920 (1955). P. S., AND DIAMOND, L. K., Blood 12, 61 (1957). T. H. J., .~ND MEYERING, C. A., C&n. C&z. Acta 5, 103 ( 1960). 1. MEYERING, C. A., ISRAELS, A. L. M., SERENS, T., AND HUIRMAN, T. H. J., Chim. Acta 5, 208 (1960). 5. SMITHIES, O., Biochem. J. 71, 585 (1959). 6. HUISMAN, T. H. J., Clin. Chim. Actn 5, 709 (1960). 1. K~NKEL,
2. GERALD, 3. HUISMAN,
TITUS ANDREE Departments of Biochemistry Medical College o,f Georgia Augusta, Georgia Received May 8, 1961
and
Pathologll
H. J. I-IUISM.~N M. DOZY
Clir~.