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Amino acid composition of the serum proteins. III. The chromatographic isolation of human and bovine serum albumins, and the amino composition of the fractions
ARCHIVES
OF
BIOCHEMISTRY
AND
86, 366373 (19%)
BIOPHYSICS
Amino Acid Composition of the Serum Proteins. III. The Chromatographic Isolation of Human and Bovine Serum Albumins, and the Amino Composition of the Fraction@ 2 Stephen Keller and Richard J. Block From
the Boyce
Thompson
Institute
for
Plant
Research,
Inc.,
Yonkers,
New
York
Received April 4, 1959 INTRODUCTION
The complexity of serum albumins has been demonstrated by free electrophoresis (1, 2), starch electrophoresis (3), fractional salting out (4), chromatography on calcium phosphate (5), and countercurrent distribution.3 Tn this investigation, crystalline bovine mercaptalbumin was chromatographed on DEAE-SF-cellulose. Three of the resulting fractions were further characterized by electrophoresis, sedimentation in the ultracentrifuge, and by amino acid composition. Human serum albumin has been shown to be heterogeneous by Sober et al. (6). The albumin fractions obtained by Sober (6) using DEAE-SF-cellulose were contaminated with appreciable amounts of globulins. This study was, therefore, undertaken to investigate the heterogeneity, if any, of human serum albumins and of bovine mercaptalbumin. The bovine mercaptalbumin was initially free of globulins, and, therefore, was chromatographed without further purification. The human albumins obtained after DEAE-SF-chromatography were contaminated with globulins. These were removed by continuous paper electrophoresis. EXPERIMENTAL Crystalline bovine mercaptalbumin (Nutritional Biochemicals Co.), 2.5 g., was dissolved in 40 ml. of a 0.005 M phosphate buffer at pH 6.8, and dialyzed for 16 hr. at 4°C. against 11. of the same buffer. The protein solution was then applied to a 2.2 X 1 Aided by grant H-2487 (c), National Heart Institute, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland. 2 Part of a thesis submitted by Stephen Keller in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Physiology and Biochemistry, Rutgers University, New Brunswick, N. J. 3 Personal communication from Dr. Lyman C. Craig, Rockefeller Institute for Medical Research, New York, N. Y. 366
SERUM
PROTEINS.
367
III
40 cm. column, containing 30 g. of DEAE-SF-cellulose, prepared from Solka-Floe SW-40 (7). The preparation had a pK of 9.5 in the presence of 0.5M N&l, and a capacity of 0.24 meq./g. The adsorbent was introduced into the column in the form of a slurry, which was packed by the application of 10 lb./sq. in. pressure. The proteins were fractionally eluted from the column using stepwise increases in the NaCl concentration of the eluting agent as follows: Composition
Bllff‘er
of buffer
Volume
pH 6.8 “ “ “ “ “ “ “ “ “
225 150 300 150 “ “ “ “ “ “
ml.
8 9 10
0.005 M phosphate 0.013 M “ ‘L “ “ L‘ “ “ “ ,‘ ‘I <‘ “ “ “ “ “ “
buffer, “ “ “ “ “ “ “ “ “
in in in in in in in in in
0.073 0.093 0.110 0.130 0.160 0.190 0.210 0.240 0.270
M M M M M M M M M
NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl
A similar procedure was employed earlier (8) for the proteins of human serum. The buffers were washed through the column under a pressure of 4 lb. nitrogen. Fractions of 15 ml. were collected at 4-min. intervals. The amount of protein in each tube was estimated by ultraviolet absorption at 280 rnp; the protein solutions comprising each peak were pooled and dialyzed, and the resulting salt-free proteins were lyophilized. Nitrogen and paper electrophoretic analyses were carried out on all of the fractions. For the electrophoresis, a potential difference of 150 v. was applied across the papers for 16 hr. The electrolyte was a 0.075 M barbital buffer at pH 8.6. Three milliliters of a 0.8% solution of the bovine mercaptalbumin in a 0.02 ionic strength acetate buffer at pH 4.69 was used for the moving-boundary electrophoresis in a Perkin-Elmer model 38 apparatus. Time: 40 min. at 3 ma. Ten milligrams each of fractions A, B, and E were dissolved in 1 ml. of 0.15M NaC1-0.02 M sodium acetate0.03 M acetic acid, and schlieren patterns were traced after approximately 65 min. of centrifugation at 59,780 r.p.m. Amino acid analyses of fractions A, B, and E were carried out using paper chromatography (9). The proteins of human serum were fractionated on a 2.2 X 40 cm. column of DEAESF-cellulose previously equilibrated with a 0.01 M phosphate buffer at pH 8.0. Fifty milliliters of pooled human serum was dialyzed for 16 hr. against 1 1. of the same buffer at 4°C. and then placed on the column. The proteins were eluted using a continuous gradient of decreasing pH and increasing ionic strength. A cone-sphere type of apparatus was employed, similar to that of Fahey et al. (10). The mixing chamber contained 1 1. of 0.01 M phosphate buffer at pH 8.0, while the reservoir contained 500 ml. of 0.30 M NaHzPOd . The hydrostatic head was adjusted to give a flow rate of 1.0 ml./min., and IO-ml. fractions were collected. The protein concentration was estimated by absorption at 280 mp, and the appropriate effluents were combined and dialyzed free of salts. Three albumin-containing fractions, as determined by paperstrip electrophoresis, were subjected to continuous paper electrophoresis, employing the Karler-Misco apparatus. The protein solutions were applied to the paper by a motor-driven syringe over a period of 24 hr., with a 0.01 M ionic strength barbital buffer at pH 8.6 as the electrolyte, and using a potential difference of 500 v. The resulting albumin fractions were dialyzed free of salts, lyophilized, and analyzed for nitrogen and amino acids.
368
KELLER
AND
BLOCK
RESULTS
The column chromatography of crystalline bovine mercaptalbumin yielded eight fractions under the conditions of these experiments (Fig. 1). The heterogeneity of this sample of mercaptalbumin is also indicated by its behavior in free electrophoresis, in which three well-resolved peaks were revealed (Fig. 2). However, some uncertainty exists at present as to the interpretation of electrophoretic patterns obtained at low ionic strength, isoelectric conditions (11). Ultracentrifugal analysis of fractions A, B, and E showed that fractions A and B consisted entirely of monomer (XzO,,V= 4.3). Fraction E was composed of 51% monomer, 43% dimer (X,,,, = 6.7), and 6% of higher molecular weight material. Paper electrophoretic analyses of all eight fractions at pH 8.6 showed only one peak, albumin, as did the analysis of the starting material. Despite the differences in their physical properties, the nitrogen and amino acid contents of fractions A, B, and E were essentially the same (Table I). The standard deviation and coefficient of variation for each of the amino acid values were calculated. The greatest variation was 4.8 % in the case of arginine. The separation of the human serum proteins on DEAE-SF-cellulose yielded a number of protein fractions (Fig. 3) of which the three designated A, B, and C contained albumins. These three fractions, after removal of BUFFER
NUMBER
t
40
I
60 TUBE
I
60
100
I
1
120
NUMBER
FIG. 1. Fractionation of bovine mercaptalbumin cellulose, employing stepwise elution of the protein.
on a column of DEAE-SF-
SERUM
PROTEINS.
369
III
D
X-
:’
FIG. 2. Electrophoretic patterns of bovine mercaptalbumin in moving-boundary apparatus, at pH 4.69. Figure 2A is the ascending boundary at the start of the run; Fig. 2B after 40 min.; Fig. 2C represents the descending boundary at the start; Fig. 20 after 40 min.
the globulins by continuous paper electrophoresis, were analyzed for their constituent amino acids. The amino acid compositions of the three albumins were very similar, with the exception of cystine (Table II, Fig. 4). DISCUSSION
The albumins of mammalian sera have been shown to consist of a number of components by various fractionation procedures. However, this heterogeneity appears to be primarily physical in nature. The fractionation of mercaptalbumin resembles the findings of Tiselius et al. (5) who reported that various serum albumins were eluted from Ca3(P0Jz columns by stepwise increases in the ionic strength of the eluant. The separation of proteins on DEAE-SF-cellulose is believed t,o be based primarily on their electric charges. The results with mercaptalbumin, how-
Amino
TABLE I Composition of the Bovine Serum Mercaptalbumin (Amino acids as mmoles/l.6 g. N)
Acid
Fraction
Amino acid
Arginine Histidine Lysine Tyrosine Phenylalanine Cystine Methionine Serine Threonine Leucine + isoleucine Valine Glutamic acid Aspartic acid Glycine Alanine Nitrogen, ‘%
A
B
E
2.8 2.2 5.8 2.0 3.6 2.9 0.50 2.5 4.0 5.5
3.2 2.2 5.6 2.0 3.5 2.9 0.47 2.5 3.5 5.5
3.3 2.4 6.3 2.2 3.4 3.0 0.52 2.5 3.7 5.8
4.3 15.9 6.8 1.9 4.9 13.7 I
4.4 16.6 6.4 1.8 4.9 13.7 I
4.3 17.5 7.0 1.9 5.2 13.3
1
TUBE
3. Fractionation employing a cone-sphere FIG.
1
S.D.
Co&. of var.
3.1 2.3 5.9 2.1 3.5 2.9 0.50 2.5 3.7 5.6
0.15 0.07 0.21 0.07 0.06 0.04 0.015 0 0.15 0.10
4.8 3.1 3.6 3.4 1.6 1.4 3.0 0 4.0 1.8
0.04 0.47 0.18 0.04 0.10
1.0 2.8 2.7 2.2 2.0
1
I
1
NUMBER
of human serum protein gradient for elution. 370
Meall
4.3 16.7 6.7 1.9 5.0
I
Fractions
on a column of DEAE-SF-cellulose,
SERUM
PROTEINS.
TABLE Amino
Acid
Composition (Amino
Arginine Histidine Lysine Tyrosine Phenylalanine Cystine Serine Threonine Leucine + isoleutine Valine Glutamic acid Aspartic acid Glycine Alanine Nitrogen, y0
’
GLY
II
of the Human
TYR.
S.D.
Coeff. of var.
3.0 2.1 6.0 1.9 2.8 4.4 2.5 3.0 5.4
3.4 2.2 6.2 2.0 3.2 4.0 2.3 2.9 5.2
0.20 0.04 0.25 0.07 0.22 0.61 0.10 0.12 0.23
6.0 1.8 4.0 3.5 6.9 15.0 4.4 4.1 4.4
3.1 15.0 5.3 2.0 7.1 12.5
3.6 14.8 5.2 1.9 7.0
0.26 0.56 0.12 0.06 0.09
7.2 3.8 2.3 3.1 1.3
B
C
3.4 2.2 5.9 2.1 3.5 4.8 2.2 2.7 4.7
3.7 2.2 6.7 2.1 3.4 2.8 2.2 3.1 5.4
3.6 13.8 5.0 1.8 6.8 14.4
4.0 15.7 5.4 1.9 7.0 13.6
SER.
CYS.
Fractions
g. N) Mean
A
HIST,
Serum Albumin
acids as mmoles/l.6
Fraction
Amino acid
371
III
ARG.
PHE.
THR
VAL.
ALA.
LEU.
LYS.
ASI?
GLU.
IS’O.
FIG. 4. Amino acid compositions of three bovine three human serum albumin fractions.
mercaptalbumin
fractions
and of
ever, indicate that molecular size is also of experimental significance (Fig. 1). In this case, changes in size may have affected the distribution of charges on the protein. The finding that the three fractions of mercaptalbumin analyzed possess the same amino acid composition suggests that the peptide
372
KELLER AND BLOCK
chains in the various fractions are similar but that these peptides can exist in a number of steric modifications. Craig3 indicated that human plasma albumins, separated by countercurrent distribution, consist of at least two fractions having the same amino acid composition. He also showed that human and bovine mercaptalbumins were heterogeneous. The type of heterogeneity found in the albumin fractions differs from that of the globulins of mammalian sera reported previously (9). In the earlier experiments, the various human y- and /?-globulins, obtained by cellulose ion-exchange chromatography, were found to differ markedly in their amino acid composition (9). The explanation of the finding of a number of albumin fractions is not clear at present. The different physical properties may be the result of the binding of various nonprotein components to a single type of albumin, or to differential denaturation, with the resultant production of a number of physically different albumins. On the other hand, a number of albumins may actually be produced which differ from each other by only a few amino acid residues or in other respects which would not be detected by the analytical procedures used here. The amino acid patterns of the three human serum albumins were very similar except that the cystine content of fraction B was markedly lower than that of fractions A and C (Table II, Fig. 4). Thisindicates the presence of at least two polypeptide chains in human serum albumins. It is interesting that the amino acid composition of human fraction B is practically the same as that of the bovine mercaptalbumin fractions, except that the amounts of alanine and aspartic acid are interchanged. The threonine content of the human albumins was also found to be somewhat lower than that of the bovine mercaptalbumins. ACKNOWLEDGMENT We are indebted the ultracentrifugal
to Dr. Frederick analyses.
Gutter
of the National
Institutes
of Health
for
SUMMARY
Crystalline bovine mercaptalbumin and human serum albumins were fractionated by chromatography on DEAE-SF-cellulose. The human albumins were further fractionated by continuous paper electrophoresis. It was found that the nitrogen content of all the albumins were similar. Amino acid analyses revealed, however, that while the three bovine mercaptalbumin fractions had the identical compositions, one of the three human albumins differed markedly in its cystine content from the other two. Furthermore, the albumins from human and bovine sera differed primarily in the amounts of alanine and of aspartic acid.
SERUM
PROTEINS.
III
373
REFERENCES 1. LONGSWORTH, L. G., AND JACOBSEN, C. F., J. Phys. & Colloid Chem. 63, 126 (1949). 2. SAIFER, A., AND COREY, H., 1. Biol. Chem. 217, 23 (1955). 3. RAACKE, I. D., Arch. Biochem. Biophys. 62, 184 (1956). 4. DERRIEN, Y., Biochim. et Biophys. Acta 8, 631 (1952). 5. TISELIUS, A., HJERTEN, S., AND LEVIN, a., Arch. Biochem. Biophys. 66,132 (1956). 6. SOBER, H. A., GUTTER, F. J., WYCKOFF, M. M., AND PETERSON, E. A., J. Am. Chews. Sot. 78, 756 (1956). 7. BLOCK, R. J., KELLER, S., AND MILLER, D. W., Arch. Biochem. Biophys. 83, 426 (1959). 8. BLOCK, R. J., MANDL, R. H., KELLER, S., AND WERNER, S. C., Arch. Biochem. Biophys. 76, 608 (1958). 9. KELLER, S., AND BLOCK, R. J., Contribs. Boyce Thompson Inst. 19, 451 (1958). 10. FAHEY, J. L., MCCOY, P. F., AND GOULIAN, M., J. Clin. Invest. 37, 272 (1958). 11. COREY, H., SAIFER, A., AND STEIGMAN, J., Abstr. Am. Chem. Sot., p. 79C. 132nd Meeting, New York City, September 8-13,1957; CANN, J. R., J. Am. Chem. Sot. SO, 4263 (1958). 12. SCHULTZ, J., GRANNIS, G., KIMMEL, H., AND SHAY, H., Arch. Biochem. Biophys. 67, 174 (1955).
OF
BIOCHEMISTRY
AND
86, 366373 (19%)
BIOPHYSICS
Amino Acid Composition of the Serum Proteins. III. The Chromatographic Isolation of Human and Bovine Serum Albumins, and the Amino Composition of the Fraction@ 2 Stephen Keller and Richard J. Block From
the Boyce
Thompson
Institute
for
Plant
Research,
Inc.,
Yonkers,
New
York
Received April 4, 1959 INTRODUCTION
The complexity of serum albumins has been demonstrated by free electrophoresis (1, 2), starch electrophoresis (3), fractional salting out (4), chromatography on calcium phosphate (5), and countercurrent distribution.3 Tn this investigation, crystalline bovine mercaptalbumin was chromatographed on DEAE-SF-cellulose. Three of the resulting fractions were further characterized by electrophoresis, sedimentation in the ultracentrifuge, and by amino acid composition. Human serum albumin has been shown to be heterogeneous by Sober et al. (6). The albumin fractions obtained by Sober (6) using DEAE-SF-cellulose were contaminated with appreciable amounts of globulins. This study was, therefore, undertaken to investigate the heterogeneity, if any, of human serum albumins and of bovine mercaptalbumin. The bovine mercaptalbumin was initially free of globulins, and, therefore, was chromatographed without further purification. The human albumins obtained after DEAE-SF-chromatography were contaminated with globulins. These were removed by continuous paper electrophoresis. EXPERIMENTAL Crystalline bovine mercaptalbumin (Nutritional Biochemicals Co.), 2.5 g., was dissolved in 40 ml. of a 0.005 M phosphate buffer at pH 6.8, and dialyzed for 16 hr. at 4°C. against 11. of the same buffer. The protein solution was then applied to a 2.2 X 1 Aided by grant H-2487 (c), National Heart Institute, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland. 2 Part of a thesis submitted by Stephen Keller in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Physiology and Biochemistry, Rutgers University, New Brunswick, N. J. 3 Personal communication from Dr. Lyman C. Craig, Rockefeller Institute for Medical Research, New York, N. Y. 366
SERUM
PROTEINS.
367
III
40 cm. column, containing 30 g. of DEAE-SF-cellulose, prepared from Solka-Floe SW-40 (7). The preparation had a pK of 9.5 in the presence of 0.5M N&l, and a capacity of 0.24 meq./g. The adsorbent was introduced into the column in the form of a slurry, which was packed by the application of 10 lb./sq. in. pressure. The proteins were fractionally eluted from the column using stepwise increases in the NaCl concentration of the eluting agent as follows: Composition
Bllff‘er
of buffer
Volume
pH 6.8 “ “ “ “ “ “ “ “ “
225 150 300 150 “ “ “ “ “ “
ml.
8 9 10
0.005 M phosphate 0.013 M “ ‘L “ “ L‘ “ “ “ ,‘ ‘I <‘ “ “ “ “ “ “
buffer, “ “ “ “ “ “ “ “ “
in in in in in in in in in
0.073 0.093 0.110 0.130 0.160 0.190 0.210 0.240 0.270
M M M M M M M M M
NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl
A similar procedure was employed earlier (8) for the proteins of human serum. The buffers were washed through the column under a pressure of 4 lb. nitrogen. Fractions of 15 ml. were collected at 4-min. intervals. The amount of protein in each tube was estimated by ultraviolet absorption at 280 rnp; the protein solutions comprising each peak were pooled and dialyzed, and the resulting salt-free proteins were lyophilized. Nitrogen and paper electrophoretic analyses were carried out on all of the fractions. For the electrophoresis, a potential difference of 150 v. was applied across the papers for 16 hr. The electrolyte was a 0.075 M barbital buffer at pH 8.6. Three milliliters of a 0.8% solution of the bovine mercaptalbumin in a 0.02 ionic strength acetate buffer at pH 4.69 was used for the moving-boundary electrophoresis in a Perkin-Elmer model 38 apparatus. Time: 40 min. at 3 ma. Ten milligrams each of fractions A, B, and E were dissolved in 1 ml. of 0.15M NaC1-0.02 M sodium acetate0.03 M acetic acid, and schlieren patterns were traced after approximately 65 min. of centrifugation at 59,780 r.p.m. Amino acid analyses of fractions A, B, and E were carried out using paper chromatography (9). The proteins of human serum were fractionated on a 2.2 X 40 cm. column of DEAESF-cellulose previously equilibrated with a 0.01 M phosphate buffer at pH 8.0. Fifty milliliters of pooled human serum was dialyzed for 16 hr. against 1 1. of the same buffer at 4°C. and then placed on the column. The proteins were eluted using a continuous gradient of decreasing pH and increasing ionic strength. A cone-sphere type of apparatus was employed, similar to that of Fahey et al. (10). The mixing chamber contained 1 1. of 0.01 M phosphate buffer at pH 8.0, while the reservoir contained 500 ml. of 0.30 M NaHzPOd . The hydrostatic head was adjusted to give a flow rate of 1.0 ml./min., and IO-ml. fractions were collected. The protein concentration was estimated by absorption at 280 mp, and the appropriate effluents were combined and dialyzed free of salts. Three albumin-containing fractions, as determined by paperstrip electrophoresis, were subjected to continuous paper electrophoresis, employing the Karler-Misco apparatus. The protein solutions were applied to the paper by a motor-driven syringe over a period of 24 hr., with a 0.01 M ionic strength barbital buffer at pH 8.6 as the electrolyte, and using a potential difference of 500 v. The resulting albumin fractions were dialyzed free of salts, lyophilized, and analyzed for nitrogen and amino acids.
368
KELLER
AND
BLOCK
RESULTS
The column chromatography of crystalline bovine mercaptalbumin yielded eight fractions under the conditions of these experiments (Fig. 1). The heterogeneity of this sample of mercaptalbumin is also indicated by its behavior in free electrophoresis, in which three well-resolved peaks were revealed (Fig. 2). However, some uncertainty exists at present as to the interpretation of electrophoretic patterns obtained at low ionic strength, isoelectric conditions (11). Ultracentrifugal analysis of fractions A, B, and E showed that fractions A and B consisted entirely of monomer (XzO,,V= 4.3). Fraction E was composed of 51% monomer, 43% dimer (X,,,, = 6.7), and 6% of higher molecular weight material. Paper electrophoretic analyses of all eight fractions at pH 8.6 showed only one peak, albumin, as did the analysis of the starting material. Despite the differences in their physical properties, the nitrogen and amino acid contents of fractions A, B, and E were essentially the same (Table I). The standard deviation and coefficient of variation for each of the amino acid values were calculated. The greatest variation was 4.8 % in the case of arginine. The separation of the human serum proteins on DEAE-SF-cellulose yielded a number of protein fractions (Fig. 3) of which the three designated A, B, and C contained albumins. These three fractions, after removal of BUFFER
NUMBER
t
40
I
60 TUBE
I
60
100
I
1
120
NUMBER
FIG. 1. Fractionation of bovine mercaptalbumin cellulose, employing stepwise elution of the protein.
on a column of DEAE-SF-
SERUM
PROTEINS.
369
III
D
X-
:’
FIG. 2. Electrophoretic patterns of bovine mercaptalbumin in moving-boundary apparatus, at pH 4.69. Figure 2A is the ascending boundary at the start of the run; Fig. 2B after 40 min.; Fig. 2C represents the descending boundary at the start; Fig. 20 after 40 min.
the globulins by continuous paper electrophoresis, were analyzed for their constituent amino acids. The amino acid compositions of the three albumins were very similar, with the exception of cystine (Table II, Fig. 4). DISCUSSION
The albumins of mammalian sera have been shown to consist of a number of components by various fractionation procedures. However, this heterogeneity appears to be primarily physical in nature. The fractionation of mercaptalbumin resembles the findings of Tiselius et al. (5) who reported that various serum albumins were eluted from Ca3(P0Jz columns by stepwise increases in the ionic strength of the eluant. The separation of proteins on DEAE-SF-cellulose is believed t,o be based primarily on their electric charges. The results with mercaptalbumin, how-
Amino
TABLE I Composition of the Bovine Serum Mercaptalbumin (Amino acids as mmoles/l.6 g. N)
Acid
Fraction
Amino acid
Arginine Histidine Lysine Tyrosine Phenylalanine Cystine Methionine Serine Threonine Leucine + isoleucine Valine Glutamic acid Aspartic acid Glycine Alanine Nitrogen, ‘%
A
B
E
2.8 2.2 5.8 2.0 3.6 2.9 0.50 2.5 4.0 5.5
3.2 2.2 5.6 2.0 3.5 2.9 0.47 2.5 3.5 5.5
3.3 2.4 6.3 2.2 3.4 3.0 0.52 2.5 3.7 5.8
4.3 15.9 6.8 1.9 4.9 13.7 I
4.4 16.6 6.4 1.8 4.9 13.7 I
4.3 17.5 7.0 1.9 5.2 13.3
1
TUBE
3. Fractionation employing a cone-sphere FIG.
1
S.D.
Co&. of var.
3.1 2.3 5.9 2.1 3.5 2.9 0.50 2.5 3.7 5.6
0.15 0.07 0.21 0.07 0.06 0.04 0.015 0 0.15 0.10
4.8 3.1 3.6 3.4 1.6 1.4 3.0 0 4.0 1.8
0.04 0.47 0.18 0.04 0.10
1.0 2.8 2.7 2.2 2.0
1
I
1
NUMBER
of human serum protein gradient for elution. 370
Meall
4.3 16.7 6.7 1.9 5.0
I
Fractions
on a column of DEAE-SF-cellulose,
SERUM
PROTEINS.
TABLE Amino
Acid
Composition (Amino
Arginine Histidine Lysine Tyrosine Phenylalanine Cystine Serine Threonine Leucine + isoleutine Valine Glutamic acid Aspartic acid Glycine Alanine Nitrogen, y0
’
GLY
II
of the Human
TYR.
S.D.
Coeff. of var.
3.0 2.1 6.0 1.9 2.8 4.4 2.5 3.0 5.4
3.4 2.2 6.2 2.0 3.2 4.0 2.3 2.9 5.2
0.20 0.04 0.25 0.07 0.22 0.61 0.10 0.12 0.23
6.0 1.8 4.0 3.5 6.9 15.0 4.4 4.1 4.4
3.1 15.0 5.3 2.0 7.1 12.5
3.6 14.8 5.2 1.9 7.0
0.26 0.56 0.12 0.06 0.09
7.2 3.8 2.3 3.1 1.3
B
C
3.4 2.2 5.9 2.1 3.5 4.8 2.2 2.7 4.7
3.7 2.2 6.7 2.1 3.4 2.8 2.2 3.1 5.4
3.6 13.8 5.0 1.8 6.8 14.4
4.0 15.7 5.4 1.9 7.0 13.6
SER.
CYS.
Fractions
g. N) Mean
A
HIST,
Serum Albumin
acids as mmoles/l.6
Fraction
Amino acid
371
III
ARG.
PHE.
THR
VAL.
ALA.
LEU.
LYS.
ASI?
GLU.
IS’O.
FIG. 4. Amino acid compositions of three bovine three human serum albumin fractions.
mercaptalbumin
fractions
and of
ever, indicate that molecular size is also of experimental significance (Fig. 1). In this case, changes in size may have affected the distribution of charges on the protein. The finding that the three fractions of mercaptalbumin analyzed possess the same amino acid composition suggests that the peptide
372
KELLER AND BLOCK
chains in the various fractions are similar but that these peptides can exist in a number of steric modifications. Craig3 indicated that human plasma albumins, separated by countercurrent distribution, consist of at least two fractions having the same amino acid composition. He also showed that human and bovine mercaptalbumins were heterogeneous. The type of heterogeneity found in the albumin fractions differs from that of the globulins of mammalian sera reported previously (9). In the earlier experiments, the various human y- and /?-globulins, obtained by cellulose ion-exchange chromatography, were found to differ markedly in their amino acid composition (9). The explanation of the finding of a number of albumin fractions is not clear at present. The different physical properties may be the result of the binding of various nonprotein components to a single type of albumin, or to differential denaturation, with the resultant production of a number of physically different albumins. On the other hand, a number of albumins may actually be produced which differ from each other by only a few amino acid residues or in other respects which would not be detected by the analytical procedures used here. The amino acid patterns of the three human serum albumins were very similar except that the cystine content of fraction B was markedly lower than that of fractions A and C (Table II, Fig. 4). Thisindicates the presence of at least two polypeptide chains in human serum albumins. It is interesting that the amino acid composition of human fraction B is practically the same as that of the bovine mercaptalbumin fractions, except that the amounts of alanine and aspartic acid are interchanged. The threonine content of the human albumins was also found to be somewhat lower than that of the bovine mercaptalbumins. ACKNOWLEDGMENT We are indebted the ultracentrifugal
to Dr. Frederick analyses.
Gutter
of the National
Institutes
of Health
for
SUMMARY
Crystalline bovine mercaptalbumin and human serum albumins were fractionated by chromatography on DEAE-SF-cellulose. The human albumins were further fractionated by continuous paper electrophoresis. It was found that the nitrogen content of all the albumins were similar. Amino acid analyses revealed, however, that while the three bovine mercaptalbumin fractions had the identical compositions, one of the three human albumins differed markedly in its cystine content from the other two. Furthermore, the albumins from human and bovine sera differed primarily in the amounts of alanine and of aspartic acid.
SERUM
PROTEINS.
III
373
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