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Amino acid composition of monomeric and polymeric human serum albumin
ANALYTICAL
BIOCHEMISTRY
Amino
27, 1-14 (1969)
Acid Composition of Monomeric Polymeric Human Serum Albumin’ ABRAHAM
Biochemistry of the Kingsbrook
SAIFER
AND
JORMA
and
PALO”
Department, Isaac Albert Research Znstitute Jew&h Medical Center, Brooklyn, New York 11203 Received December
18, 1967
Although crystalline human serum albumin (HSA) is among the best studied of all “purified” proteins (1, 2), few accurate analyses of its amino acid composition have been performed. The most widely quoted values in the literature are those of Brand and his co-workers (3, 4). Since these investigators found no alanine, which is present in HSA to at least 6 amino acid residues (gm/lOO gm protein), many of their other amino values are of questionable validity. Replacement of the relatively inaccurate microbiological and paper chromatographic techniques (5) with automated ion-exchange column chromatographic methods (6, 7) has made possible accurate amino acid analysis of protein hydrolyzates. Only two significant reports of the amino acid composition of HSA have a,ppeared in the lit,erature (8, 9) and there are appreciable differences between them for several amino acids. There is definitive experimental evidence for at least two kinds of heterogeneity in crystalline HSA preparations as obtained by the Cohn low-temperature alcohol fractionation method (10). The first is due to the presence of a fraction reacting with mercuric ions, i.e., mercaptalbumin (11)) and an unreactive fraction, i.e., nonmercaptalbumin. The second is the presence of polymeric forms in both the Yotal” crystallized albumin preparation and in the mercaptalbumin fraction, as was first demonstrated by means of starch gel electrophoresis (12). The work of Spahr and Edsall (8) has shown that there are no significant differences in the amino acid composition of human serum mercaptalbumin as compared to the “total” crystallized albumin. The present study was undertaken to determine whether the amino acid composition of “monomeric” ‘This investigation was supported by grants from the John A. Hartford Foundation, New York, N. Y., and by Grant NB-285C14 of the U. S. Public Health Service. *On leave from Department of Neurology, University of Helsinki, Helsinki, Finland. Recipient of a Fulbright travel grant. @ 1968 by Academic
Press, Inc.
2
SAIFER
AND
PALO
HSA is the same or different from that of the “polymeric” form and to furnish additional analytical data for this important protein that might clarify existing discrepancies among published values. Such analyses are important in the interpretation of the physical chemical properties of HSA (such as ion binding, acid-base equilibria, molecular weight determinations, etc.) and for the study of the inherited variants of albumin which differ in their electrophoretic mobility in starch gel media (13). EXPERIMENTAL
Gel electrophoresis: Commercially available crystalline human serum albumin is usually prepared from outdated blood plasma by some modification of the Cohn cold-ethanol fractionation procedure (10). Five albumin preparations, obtained from different manufacturers, were subjected to acrylamide gel disc electrophoresis with a modification of the technique described by Ornstein and Davis (14). The modification consisted of changing the composition of solutions C and D so as to give the small-pore gel an acrylamide concentration of lo%, as follows: Solution C: Acrylamide 40.0 gm Bisacrylamide 0.12 gm Hz0 to 100 ml
Solution D: Acrylamide 14.0 gm Bkacrylamide 0.25 gm Hz0 to 100 ml
The sample gel was replaced by a mixture of the sample and a buffer (pH 8.3) plus 40% sucrose solution (7: 1 v/v). For a 1% protein solution, 0.1 volume of protein to 0.4 volume of buffer/sucrose was found to be suitable. After filling the buffer reservoirs with buffer, this mixture was introduced into the electrophoresis tubes by means of a capillary pipet, through the buffer solution. The sample was gently expelled and permitted to layer between the top of the surface of the spacer gel and the less dense buffer solution above. Electrophoresis was carried out for 1.5 hr at about 2.5 mA per column. Staining with amido black and electrophoretic destaining were carried out as described by Ornstein and Davis (14). Disc electrophoresis showed that all five samples of human albumin analyzed (Fig. 1) consisted of one major monomeric component together with 2 to 7 polymeric fractions. Sample No. 1 (Kabi albumin) contained the highest percentage of the monomeric form and was employed in all subsequent experiments. In order to separate the albumin preparation into its monomeric and polymeric forms, exclusion chromatography on Sephadex G-150 columns was utilized with 0.1 M Tris/O.2 M NaCl buffer (pH 8.1) as the eluting fluid, as proposed by Pedersen (15). The optical density of the outflow from the column was continuously monitored at 280 rnp and recorded by
AMINO
ACIDS
OF
MONOMERIC
ALBUMIN
3
means of a Gilford model 2000 spectrophotometer. The elution curve obtained is shown in Figure 2. The fractions containing the monomer and the combined dimer plus trimer were collected, dialyzed against water at 4O, and freeze dried. Disc gel electrophoretic patterns of the separated fractions as compared to the original albumin preparation are shown in Figure 3 and substantiate the adequacy of the separation into relatively pure monomer and dimer plus trimer fractions. Blue dextran 2000 wac run on the column prior to each sample in order to determine void volume and as a check on the operation of the column. The homogeneity of each
FIG. 1. Disc gel electrophoresis available human serum albumin. monomeric form. Kabi albumin sample A. Albumin samples (B to
patterns obtained for 5 samples of commercially The fastest moving component (bottom) is the (Tu‘o. RD030, AB Kabi, Stockholm, Sweden) is E) are those of other manufacturers.
albumin fraction, as determined with the sensitive acrylamide gel electrophoretic procedure, also indicates the absence of appreciable amounts of serum components other than albumin. Immuno~electrophoresis: The samples were run with the micromethod of Scheidegger (16). The antigen wells were 3 mm in diameter, the serum trenches were 5.5 cm in length, and the distance between their edges was 3 mm. A potential of approximately 5 V/cm was applied for 2 hr. Antiserum used was rabbit anti-human or rabbit anti-albumin. The results obtained are shown in Figure 4. Hydrolysis: For every 3 mg of protein, 0.5 ml of constant-boiling HCI
4
SAIFER
AND
PALO
Albumin ( KABI) Sephodex G-150 TRIS 0.1 M NoCl 0.2M pH *” FRACTION Monomeric Dlmerlc Trimerac Tubes areas
TUBE
NO
34-43 25-30 IS-22
under Ihe hatched were discarded
c TUBE
NUMBER
FM. 2. Elution curves of human serum albumin monomeric,
dimeric, and trimeric
-
(Kabi)
showing
separation
into
fractions.
was used. Usually, 6 mg of protein was weighed out for one analysis, transferred into a Pyrex glass tube together with the acid solution, flushed three times with nitrogen, and sealed under vacuum. The samples were hydrolyzed in an oven maintained at 110 * 1°C for 24,48, 72, and 96 hr. After cooling, the tubes were opened and the samples were dried in a vacuum desiccator over NaOH at room temperature. The remaining HCl was removed by dissolving the dry residue in distilled H,O and this procedure was repeated three times. The final residue was dissolved in 1.0 ml of 0.1 N HCl and stored at -20°C. Usually an aliquot of 100 J.J of this solution (corresponding to about 600 pg of the original sample) was anaiyzed with the Technicon amino acid analyzer. Chromatography: The amino acid analysis was performed with the method described by Piez and Morris (7) using the Technicon automated amino acid system. However, two modifications were made. First, methanol was incorporated into the buffer system in order to improve the separation of the acidic amino acids. Second, the ninhydrin solution was prepared without using 4 N acetate buffer and ammonia was removed from the solution by shaking with acid-washed Permutit. The stock solution was found to be stable for at least two weeks. The color produced
AMINO
ACIDS
OF
MONOMERIC
ALBUMIN
FIG. 3. Disc gel electrophoretic patterns of (A) human serum albumin (Kabi), (B) monomeric albumin component, and (C) dimer plus trimer fraction. These fractions were separated by Sephadex G-150 column chromatography.
in this reaction was measured at two wavelengths, 440 rnp for proline and 570 rnp for other amino acids. Calibration: The chromatographic system was calibrated by means of repeated runs with amino acid standard solutions. The reliability of the hydrolytic procedure was checked by heating the amino acid standard solutions with 6 N HCl and hydrolyzing the protein samples for 24, 48, 72, and 96 hr. The final check on both of these procedures was made by analyzing the composition of crystallized beef insulin (Burroughs Wellcome & Co., Inc., Tuckahoe, N. Y.) and comparing the results obtained with some earlier data (17, 18). Calculations: Norleucine served as an internal standard in each run and the amount most commonly used was 0.25 pmole. For human serum albumin, the molecular weight of 66,000 was used in order to make the final data comparable to those of other investigators (8, 9, 19). Special methods: Tryptophan was determined by the micromethod published earlier from this laboratory (20). For amide NH,, a modified procedure of Stegemann (21) was employed in which the liberated ammonia was measured with the sensitive Berthelot indophenol reaction. The half-cystine content was determined as cysteic acid (22) on some
6
SAIFER
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PALO
Fm. 4. Immunoelectrophoretic patterns of the original (Kabi) albumin preparation and the isolated monomer and polymer fractions as separated electrophoretically in buffered agar gel and the precipitin bands visualized with rabbit anti-human whole serum (A, B, and C) and with rabbit anti-human albumin (D). (A) Original 1% albumin (top) and 1% monomer (bottom). (B) 1% dimer plus trimer (top and bottom). (C) Original 6% albumin (top) and 6% monomer (bottom). (D) Original 6% albumin (top) and 6% monomer (bottom).
oxidized protein samples; but on other samples, because of the small amounts available, it was estimated on the same chromatograms as the other amino acids. The water content of the Kabi albumin sample was 5.0% by gravimetric analysis. Results of the chromatographic analyses: Five analyses were carried out on each protein sample at different hydrolysis times. Because of the good reproducibility of the method, usually only one amino acid run was performed for each hydrolyzate. Table 1 lists the percentage composition of the amino acids of the original Kabi human serum albumin
Amino
acid
1.00 6.72 6.23 10.30 1.34 3.60 6.70 4.06 0.40 3.04 3.96 5.30 1.10 5.54 3.14 10.91 9.08 15.50 0.89 98.81
This analysisO
amino
crystalline
1.05 6.67 6.17 10.30 1.31 3.65 6.74 4.03 0.13 3.15 4.31 5.09 1.08 5.38 3.17 10.95 9.05 15.85 0.94 99.02
of Data
HSA
(Kabi).
1.02 6.64 6.23 10.30 1.38 3.79 6.73 4.34 0.29 2.52 3.77 5.70 1.13 5.63 3.33 12.02 8.89 15.28 0.84 99.83
Spshr and Ed&l (1964)
acid residues/100
Comparison
Heimburger et ~2. (1964)
Grams
a Results for unfractionated b Not included in the total.
Glyeine Alanine Valme Leucine Isoleucine Proline Phenylalanine Tyrosine Tryptophan Serine Threonine Half-cystine Methionine Arginine Histidine Lysine Aspartic acid Glutamic acid Amide NH3 Total
-
Average
of five
1.23 6.33 5.69 9.34 1.38 3.69 6.45 4.20 Not determ. 2.95 4.15 4.88 1.10 5.43 3.16 10.67 8.66 15.19 1.02 95.73
a2. analysisa
11-12 62 41-42 60 8 24-25 30 16-17 1 23 26 34 5-6 23 15 56 ,5’2 79 36b :j66-6;1
This
of Human
analyses.
TABLE 1 Other Analyses
Potg$%te;L
gm protein
with of amino
Albumin
12 62 41 60 8 25 30 16 1 24 28 33 5 23 15 56 $2 81 38h ,570n
Heimburger et al. (1964)
No.
Serum renidlles/JI.W.
23 15 56 50 79 4W 557-560
78-79 3435b 56&578
16 det,erm. 23 27-28 32 6
17-18 1 19 24-25 37 6 L4 16 62 51
15 60 38-39
Not
Potg(i
66,OM)
12 62 41-Q 60 8 26 30
Spahrclsgngddsall
acid ul.
i! G
F
8 I f3
a: z
0
8
SAIFER
AND
PALO
sample and the number of amino acid residues per mole (66,000) in comparison with analyses of human albumin preparations reported by other investigators. Table 2 summarizes the percentage composition of the amino acids of the original albumin, monomeric fraction, and combined dimeric and trimeric fractions as well as the number of the amino acid residues per mole of these samples. RESULTS AND DISCUSSION
Disc gel electrophoresis, as developed by Ornstein and Davis (14), has proved to be a sensitive procedure capable of determining 20 or more separate protein components in human serum. While there is no unequivocal evidence for the presence of more than one albumin in whole human serum, crystalline human serum albumin samples, as prepared by the Cohn alcohol fractionation method (lo), have generally shown from 4 to 8 bands with this technique (Fig. 1). These results confirm our earlier reported findings (12) by means of starch gel electrophoresis in discontinuous buffer systems, as well as those of other investigators (23, 24), that these bands represent polymeric forms of an albumin monomer that are formed during the isolation procedure. It is evident from this illustration that the Kabi albumin contains the largest percentage of t.he monomeric form of the preparations commercially available and was thus the preferred sample for further investigation. Separation of the monomer from its polymeric forms was achieved by means of Sephadex gel chromatography and a typical elution pattern for one of the runs is illustrated in Figure 2. Because there is no clearcut separation between the peaks, it is not possible to obtain complete separation between the fractions, as is illustrated by their disc gel patterns in Figure 3. While the original Kabi albumin (Fig. 3A) shows the presence of 4 components, the separated monomer (Fig. 3B) is at least 95% pure and shows only a faint trace of the dimer. The dimer plus trimer fraction (Fig. 3C) is about 90% pure with only small amounts of monomer and tetramer. These results confirm those previously reported for albumin, isolated by means of starch gel electrophoresis (12)) and those separated with column chromatography (25)) that these components do not arise as a result of the electrophoretic or chromatographic procedure and represent distinct entities. The original albumin sample and the separated fractions were then subjected to immunoelectrophoresis as a sensitive technique for determining the possible presence of protein components other than albumin, and the patterns obtained are shown in Figure 4. When rabbit antihuman whole serum was used as the antiserum, no proteins besides albumin were visible at the 1% concentration level (Fig. 4A and 4B).
f 1.92
f 0.10 f 0.23 f 0.16 (mean)
amino
98.71
10.92 8.80 15.76 (0.95)
gm dry
iz 2.70
* 0.21 f 0.23 + 0.30 (mean)
kO.12 i 0.20 z!I 0.10 L!Z0.14 kO.17 kO.31 (mean) +0.07 * 0.13 + 0.20 310.04 f+_ 0.07 0.16
1.00 * 0.018 6.70 Z!Y0.23
Monomeric fraction
residues/100
5.98 10.30 1.35 3.80 6.77 4.07 0.28 2.98 3.90 5.36 1.06 5.54 3.19
acid dimeric fraction
dzO.12 kO.27 kO.09 + 0.13 zt0.37 f 0.41 (mean) f 0.12 +_ 0.04 (mean) (mean) 31 0.18 kO.04
and
100.06
f 2.66
10.70 _+ 0.23 8.71 (mean) 14.95 + 0.31 (0 93) (mean)
6.67 10.52 1.58 3.86 6.85 4.03 0.30 3.44 4.67 5.14 0.80 5.58 3.36
1.22 + 0.07 6.75 f0.28
Combined trimeric
protein
566-571
56 52 79 3&
41-42 60 8 24-25 30 16-17 1 ‘23 26 34 5-6 23 15
12 62
Original material (Ksbi-albumin)
of estimations
,569
56 51 81 37”
40 60 8 26 30 17 1 23 26 34 5-6 23 15
12 62
fractions 66,000)
of
has been less than
577
55 50 77 36”
44 61 9 26 31 16 1 26 30 33 4 23 16
14 63
Combiied dimeric and trimeric frm.tion
in various (for M.W.
Monomeric fraction
No. of amino acid residues human serum albumin
TABLE 2 of Various Fractions of Human Serum Albumin
u Errors are t’he st,andard errors of the mean of five analyses. When S.E. is not given t,he number five. * Correct#ed for hydrolytic losses, tyrosine and serine values illcreased by 15%, t,hreonine by 107,. c Value not included in the total.
Total
10.91 9.08 15.50 (0.89) 98.81
Lysine Aspartic acid Glutamic acid Amide-NH3
kO.06 t 0.07 f 0.09 i0.14 kO.16 z!z 0.27 (mean) Ik 0.10 k 0.16 + 0.18 f0.03 f+ 0.06 0.03
6.23 10.30 1.34 3.60 6.70 4.06 0.40 3.04 3.96 5.30 1.10 5.54 3.14
Valine Leucine Isoleucine Prolme Phenylalanine Tyrosineb Tryptophan Serind Threonineb Half-cystine Methionine Arginine Histidine
Original material (K&i albumin)*
1.00 + 0.015 6.72 kO.06
acid
Glycine Alanine
Amino
Grams
Amino Acid Composition
(0
z 2
&
s 3 ;;
8
Et
0 r
ti iii
3
$2
10
SAIFER
AND
PAL6
However, a second faint band in the globulin region was visible for both the original albumin sample and the monomer fraction at the 6% level (Fig. 4C). These secondary bands may represent either denatured albumins or small amounts of globulin components. The additional faint bands obtained at the 6% level, when rabbit anti-human albumin is used as the antiserum (Fig. 4D), may represent poor antigens that did not produce antibodies when whole serum was used as the immunizing agent. The tryptophan content of the Kabi albumin and its fractions was determined on their perchloric acid hydrolyzates with the spectrophotometric procedure of Saifer and Gerstenfeld (20). Values reported here (0.30 to 0.40 gm/lOO gm protein) are in good agreement with those published by other investigators for human serum albumin (Table 1)) as well as that recently published by Spies (26), i.e., 0.347 gm/lOO gm protein. In any case, these results all lead to a value of 1 mole of tryptophan per mole (66,000) of human serum albumin, as is recorded in Table 2. Acrylamide gel electrophoresis is unquestionably a more sensitive technique than is ultracentrifugation for detecting small amounts of polymeric material in albumin preparations, and has also recently been employed to determine the molecular weights of many proteins, including the polymeric forms of bovine serum albumin (27). There is good agreement among moat recent investigators (27) that the molecular weight of monomeric human serum albumin is 67,000 + 2000. In this investigation a value of 66,000 was chosen in order to better relate our amino acid data to those of other investigators. While a large number of amino acid analyses have been recorded in the literature for bovine serum albumin (4, 8, 28)) very few analyses have been reported for the human protein. A number of these have been performed by either quantitative paper chromatographic or microbiological assay methods and these earlier data have been summarized by Wuhrmann and WunderIy (5). The results obtained by Brand and co-workers (3, 4) are those most frequently found in textbooks yet they gave no value for alanine although their total amino acid residues per 100 gm protein added up close to 100%. With the introduction of the ion-exchange automated column chromatographic techniques for amino acid analysis by Moore et at. (6), the accurate analysis (i.e., within rrt6% error) of the amino acid composition of proteins has become possible. In the present study the single column modification of Piez and Morris (7) was employed which gives excellent separation of all 18 amino acids found in protein hydrolysates. The only serious drawback in this procedure is that methionine sulfones, formed by oxidation during the hydrolysis step, may
AMINO
ACIDS
OF
MONOMERIC
ALBUMIh’
11
overlay or enhance the aspartic acid peak. Since it is possible to reproduccl standard runs on successive days, using norleucine as an internal standard, with a precision of about kl%, it is t,he hydrolysis step that makes the major contribution to the over-all error of the method. In part this is due to the fact that some amino acids, e.g., cystine, threonine, and tyrosine, are decomposed during hydrolysis while others, e.g., valine and isoleucine, may require more than 48 hr of hydrolysis for their maximum values. It was reasoned that the use of crystalline insulin, as a standard protein of known amino acid composition, would provide an additional estimate of the correction factors necessary to compensate for the changes occurring both during the analytical and the hydrolysis steps. Five such insulin analyses were performed at different hydrolysis times and t.hc average correct,ion factors applied to the albumin analyses. Additional small negative corrections, based on the insulin analysis, were made foi lysine, glutamic acid, alanine, phenylalanine, and leucine. The Sam<’ correction factors were used for all the albumin amino acid analyses. The results obtained for the original Kabi albumin preparation are listed in Table 1 in comparison with the data of other investigat’ors, all of whom used automated column chromatographic techniques for their analyses. The values of Potgieter et al. (19) are based on a single analysis of a 22 hr hydrolyzate and generally tend to be low as less than 96% of the amino acid nitrogen was accounted for. Examination of the amino acid data in Table 1 shows better agreement between our results am1 those of Heimburger et al. (9) than with those of Spahr and Edsall (8), with the possible exception of the glutamic acid values. In comparison with data of all other investigators, the serine values reported by Spahr and Edsall (8) are too low while those for half-cyst.ine and lysine are too high. However, there are no statistically significant differences between our data and those of the other two investigators (8, 9) for any amino acid. Our values for amide NH, are also in good agreement with those reported by other investigators. They have not been included in the total number of residues of the protein since they are derived from the glutamine and ssparagine residues of the intact protein (19). The amino acid analyses of Spahr and Edsall (8) show no detectable differences in amino acid composition between human serum mercaptalbumin and total human serum albumin. The former is precipitable with mercury salts and constitutes 60 to 70% of the total albumin so that small differences between the amino acid values of the two albumin fractions would generally fall within the experimental error of their det,ernnnations. Previous work from this laboratory (12) has shown that both human serum albumin and mercaptalbumin cont,ain a series of polymeric components the proportions of which depend upon the previous
12
SAIFER
AND
PALO
history of the protein preparation. It can therefore be assumed that all amino acid analyses performed by previous investigators have contained appreciable amounts of dimer and trimer albumin molecules in addition to the preponderant monomeric form. With the possible exception of the glutamic acid results, there is a close correspondence of amino acid values between the original albumin sample and its monomeric fraction (Table 2). The lower glutamic acid polymer value may be due to its removal, or that of a bound glutamic acid containing peptide, during the column chromatographic process from the nonmercaptalbumin fraction which King (29) has reported to be a mixed disulfide of mercaptalbumin and cysteine or glutathione. Statistical analysis by the Students t test of the data for the dimer plus trimer fraction, in comparison with the monomer values, has shown significantly increased values at the 5% probability level for valine, serine, and threonine and a decreased value for glutamic acid. While it would appear unlikely that polymerization would produce a change in amino acid composition, such a possibility cannot be entirely excluded if peptide loss precedes polymerization. An alternative explanation for the observed amino acid differences of the polymer is that this fraction, which is first eluted from the column, may contain appreciable amounts of other proteins as impurities or of bound peptides. This matter is presently under investigation by means of N-terminal analysis and by peptide mapping after enzymic hydrolysis. Tanford (30) has published titration data for human serum albumin which gave about 190 moles of carboxyl groups per mole of protein. The Kabi albumin data gives a value of 95 plus 1-cr-carboxyl based upon 37 amide groups per mole. This value of 96 carboxyl groups checks well with the total of 104 groups for lysine, histidine, and arginine if one assumes that some of the histidine groups have lost their positive charge at pH 4.5, i.e., the isoelectric point. The presence of a number of genetic variants of albumin have been described in the literature with both faster and slower electrophoretic mobilities than normal albumin (13, 31). By analogy with the case of abnormal hemoglobins, it is to be expected that such differences in mobility reflect minor differences in the amino acid composition of these albumins. The accurate analysis of the amino acid composition of normal albumin is a necessary first step in the investigation of its structure, and that of its genetic variants, especially with respect to their unique ion binding properties (1, 2, 32, 33). SUMMARY
All previous amino
(HSA)
acid analyses of crystalline human serum albumin have been performed on preparations which have been shown by
AMINO
Actis OF
MONOMERIC
&suMlN
13
gel electrophoresis to contain a number of polymeric forms. Monomeric albumin was separated from its higher M.W. components by means of :I Sephadex G-150 column with Tris/NaCl buffer at pH 8.1 using crystalline human albumin (Kabi) as the starting material. The physicochemical characteristics of the monomeric and polymeric fractions were investigated by means of acrylamide gel electrophoresis and immunoelectrophoresis. Their amino acid composition was determined by automated ion-exchange chromatography. Although no significant differences were found between the original albumin sample and its monomer, some of the analytical data obtained differed from those of other investigators. The polymeric fraction showed some significant differences in amino acid composition from the monomer that require further verification. ACKNOWLEDGMENTS The authors wish to acknowledge the technical aid of Pod W. Hansen and Gilbert Lowenwirth in performng some of the experimental work presented here. We are grateful to Mrs. Lillian Salowitz for the typing and editing of the manuscript. REFERENCES J. F., in “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1, p. 179. Academic Press, New York, 1960. 2. SCHULTZE, H. E., AND HEREMANS, J. F., “Molecular Biology of Human Proteins.” Elsevier Publishing Co., Amsterdam, 1966. 3. BRAND, E., KASSEL, B., AND SAIDEL, L. J., J. Clin. Invest. 23, 437 (1944). 4. BRAND, E., Ann. N. Y. Acad. Sci. 4’7, 187 (1946). 5. WUHRMANN, F., AND WUNDERLY, C., “The Human Blood Proteins,” p. 24. Grunr & Stratton, New York, 1960. 6. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., ,4nal. Chem. 30, 1185 (1958). 7. PIEZ, K. A., AND MORRIS, L., Anal. Biochem. 1, 187 (1960). 8. SPAHR, P. F., AND EDSALL, J. T., J. Biol. Chem. 911, 8.50 (1964). 9. HEIMBURGIER, N., HEIDE, K., HAUPT, N., AND SCHULTZE, H. E., Clin. Chim. A& 10, 293 (1964). 10. PENNELL, R. B., in “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1, p. 9. Academic Press, New York, 1960. 11. HUGHES, W. L., JR., Cold spring Harbor Symp. Quant. Bid. 14, 79 (1950). 12. SAIFER, A., ROBIN, M., AND VENTRICE, M., Arch. Biochem. Biophys. 92, 409 (1~1). 13. MELARTIN, L., BLUMBERE, B. S., AND LISKER, R., Nature 215, 1288 (1967). 14. ORNSTEIN, M., AND DAVIS, B., Ann. N. Y. Acad. Sci. 121, 404 (1964). 15. PEDERSEN, K. O., Arch. Biochem. Biophys., Suppl. 1, 157 (196‘2). 16. SCHEIDECICIER, J. J., Intern. Arch. Allergy ‘7, 103 (1965). 17. HARFENIST, E. J., J. Am. Chem. Sot. 75, 5528 (1953). 18. CORNFIELD, M. C., AND ROBSON, A., Biochem. J. 84, 146 (1962). 19. POTQIETER, G. M., HINES, M., AND KENCH, J. E., Chin. Chim. Acta 18, 107 (1~7). 20. SAIFER, A., AND GERSTENFELD, S., CZin. Chem. 10, 970 (1964). 21. STEOEMANN, H., 2. Physiol. Chem. 312, 255 (1958). 22. MOOBE, S., J. Biol. Chem. 238, 235 (1963). 23. Somu, H. A,, GUTER, F. J., WYCKOFF, M., AND PETERSON, E. A., J. Am, C&m. Sot. 78, 756 (1956). 1. FOSTER,
14
SAIFER
AND
PALO
S., AND NAKAMICHI, M., Anal. Biochem. 7, 225 (1964). TISELIUS, A., HJERTEN, S., AND LEVIN, O., Arch. B&hem. Biophys. 65, 132 (1956). SPIEB, J. R., Anal. Chem. 39, 1412 (1967). ZWAAN, J., Anal. Biochem. 21, 155 (1967). STEIN, W. H., AND MOORE, S., J. Biol. Chesm. 178, 79 (1949). KING, T. P., J. Biol. Chem. 236, PC5 (1961). TANFORD, C., J. Am. Chem. Sot. 72, 441 (1950). MELARTIN, L., AND BLUMBERG, B. S, Clin. Res. 14, 482 (1966). KLOTZ, I. M., in “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. I, Part B, p. 727. Academic Press, New York, 1953. 33. SAIEER, A., AND STEIOMAN, J., J. Phys. Chem. 65, 141 (1961).
24. RAYMOND,
25. 26. 27. 28. 29. 30. 31. 32.
BIOCHEMISTRY
Amino
27, 1-14 (1969)
Acid Composition of Monomeric Polymeric Human Serum Albumin’ ABRAHAM
Biochemistry of the Kingsbrook
SAIFER
AND
JORMA
and
PALO”
Department, Isaac Albert Research Znstitute Jew&h Medical Center, Brooklyn, New York 11203 Received December
18, 1967
Although crystalline human serum albumin (HSA) is among the best studied of all “purified” proteins (1, 2), few accurate analyses of its amino acid composition have been performed. The most widely quoted values in the literature are those of Brand and his co-workers (3, 4). Since these investigators found no alanine, which is present in HSA to at least 6 amino acid residues (gm/lOO gm protein), many of their other amino values are of questionable validity. Replacement of the relatively inaccurate microbiological and paper chromatographic techniques (5) with automated ion-exchange column chromatographic methods (6, 7) has made possible accurate amino acid analysis of protein hydrolyzates. Only two significant reports of the amino acid composition of HSA have a,ppeared in the lit,erature (8, 9) and there are appreciable differences between them for several amino acids. There is definitive experimental evidence for at least two kinds of heterogeneity in crystalline HSA preparations as obtained by the Cohn low-temperature alcohol fractionation method (10). The first is due to the presence of a fraction reacting with mercuric ions, i.e., mercaptalbumin (11)) and an unreactive fraction, i.e., nonmercaptalbumin. The second is the presence of polymeric forms in both the Yotal” crystallized albumin preparation and in the mercaptalbumin fraction, as was first demonstrated by means of starch gel electrophoresis (12). The work of Spahr and Edsall (8) has shown that there are no significant differences in the amino acid composition of human serum mercaptalbumin as compared to the “total” crystallized albumin. The present study was undertaken to determine whether the amino acid composition of “monomeric” ‘This investigation was supported by grants from the John A. Hartford Foundation, New York, N. Y., and by Grant NB-285C14 of the U. S. Public Health Service. *On leave from Department of Neurology, University of Helsinki, Helsinki, Finland. Recipient of a Fulbright travel grant. @ 1968 by Academic
Press, Inc.
2
SAIFER
AND
PALO
HSA is the same or different from that of the “polymeric” form and to furnish additional analytical data for this important protein that might clarify existing discrepancies among published values. Such analyses are important in the interpretation of the physical chemical properties of HSA (such as ion binding, acid-base equilibria, molecular weight determinations, etc.) and for the study of the inherited variants of albumin which differ in their electrophoretic mobility in starch gel media (13). EXPERIMENTAL
Gel electrophoresis: Commercially available crystalline human serum albumin is usually prepared from outdated blood plasma by some modification of the Cohn cold-ethanol fractionation procedure (10). Five albumin preparations, obtained from different manufacturers, were subjected to acrylamide gel disc electrophoresis with a modification of the technique described by Ornstein and Davis (14). The modification consisted of changing the composition of solutions C and D so as to give the small-pore gel an acrylamide concentration of lo%, as follows: Solution C: Acrylamide 40.0 gm Bisacrylamide 0.12 gm Hz0 to 100 ml
Solution D: Acrylamide 14.0 gm Bkacrylamide 0.25 gm Hz0 to 100 ml
The sample gel was replaced by a mixture of the sample and a buffer (pH 8.3) plus 40% sucrose solution (7: 1 v/v). For a 1% protein solution, 0.1 volume of protein to 0.4 volume of buffer/sucrose was found to be suitable. After filling the buffer reservoirs with buffer, this mixture was introduced into the electrophoresis tubes by means of a capillary pipet, through the buffer solution. The sample was gently expelled and permitted to layer between the top of the surface of the spacer gel and the less dense buffer solution above. Electrophoresis was carried out for 1.5 hr at about 2.5 mA per column. Staining with amido black and electrophoretic destaining were carried out as described by Ornstein and Davis (14). Disc electrophoresis showed that all five samples of human albumin analyzed (Fig. 1) consisted of one major monomeric component together with 2 to 7 polymeric fractions. Sample No. 1 (Kabi albumin) contained the highest percentage of the monomeric form and was employed in all subsequent experiments. In order to separate the albumin preparation into its monomeric and polymeric forms, exclusion chromatography on Sephadex G-150 columns was utilized with 0.1 M Tris/O.2 M NaCl buffer (pH 8.1) as the eluting fluid, as proposed by Pedersen (15). The optical density of the outflow from the column was continuously monitored at 280 rnp and recorded by
AMINO
ACIDS
OF
MONOMERIC
ALBUMIN
3
means of a Gilford model 2000 spectrophotometer. The elution curve obtained is shown in Figure 2. The fractions containing the monomer and the combined dimer plus trimer were collected, dialyzed against water at 4O, and freeze dried. Disc gel electrophoretic patterns of the separated fractions as compared to the original albumin preparation are shown in Figure 3 and substantiate the adequacy of the separation into relatively pure monomer and dimer plus trimer fractions. Blue dextran 2000 wac run on the column prior to each sample in order to determine void volume and as a check on the operation of the column. The homogeneity of each
FIG. 1. Disc gel electrophoresis available human serum albumin. monomeric form. Kabi albumin sample A. Albumin samples (B to
patterns obtained for 5 samples of commercially The fastest moving component (bottom) is the (Tu‘o. RD030, AB Kabi, Stockholm, Sweden) is E) are those of other manufacturers.
albumin fraction, as determined with the sensitive acrylamide gel electrophoretic procedure, also indicates the absence of appreciable amounts of serum components other than albumin. Immuno~electrophoresis: The samples were run with the micromethod of Scheidegger (16). The antigen wells were 3 mm in diameter, the serum trenches were 5.5 cm in length, and the distance between their edges was 3 mm. A potential of approximately 5 V/cm was applied for 2 hr. Antiserum used was rabbit anti-human or rabbit anti-albumin. The results obtained are shown in Figure 4. Hydrolysis: For every 3 mg of protein, 0.5 ml of constant-boiling HCI
4
SAIFER
AND
PALO
Albumin ( KABI) Sephodex G-150 TRIS 0.1 M NoCl 0.2M pH *” FRACTION Monomeric Dlmerlc Trimerac Tubes areas
TUBE
NO
34-43 25-30 IS-22
under Ihe hatched were discarded
c TUBE
NUMBER
FM. 2. Elution curves of human serum albumin monomeric,
dimeric, and trimeric
-
(Kabi)
showing
separation
into
fractions.
was used. Usually, 6 mg of protein was weighed out for one analysis, transferred into a Pyrex glass tube together with the acid solution, flushed three times with nitrogen, and sealed under vacuum. The samples were hydrolyzed in an oven maintained at 110 * 1°C for 24,48, 72, and 96 hr. After cooling, the tubes were opened and the samples were dried in a vacuum desiccator over NaOH at room temperature. The remaining HCl was removed by dissolving the dry residue in distilled H,O and this procedure was repeated three times. The final residue was dissolved in 1.0 ml of 0.1 N HCl and stored at -20°C. Usually an aliquot of 100 J.J of this solution (corresponding to about 600 pg of the original sample) was anaiyzed with the Technicon amino acid analyzer. Chromatography: The amino acid analysis was performed with the method described by Piez and Morris (7) using the Technicon automated amino acid system. However, two modifications were made. First, methanol was incorporated into the buffer system in order to improve the separation of the acidic amino acids. Second, the ninhydrin solution was prepared without using 4 N acetate buffer and ammonia was removed from the solution by shaking with acid-washed Permutit. The stock solution was found to be stable for at least two weeks. The color produced
AMINO
ACIDS
OF
MONOMERIC
ALBUMIN
FIG. 3. Disc gel electrophoretic patterns of (A) human serum albumin (Kabi), (B) monomeric albumin component, and (C) dimer plus trimer fraction. These fractions were separated by Sephadex G-150 column chromatography.
in this reaction was measured at two wavelengths, 440 rnp for proline and 570 rnp for other amino acids. Calibration: The chromatographic system was calibrated by means of repeated runs with amino acid standard solutions. The reliability of the hydrolytic procedure was checked by heating the amino acid standard solutions with 6 N HCl and hydrolyzing the protein samples for 24, 48, 72, and 96 hr. The final check on both of these procedures was made by analyzing the composition of crystallized beef insulin (Burroughs Wellcome & Co., Inc., Tuckahoe, N. Y.) and comparing the results obtained with some earlier data (17, 18). Calculations: Norleucine served as an internal standard in each run and the amount most commonly used was 0.25 pmole. For human serum albumin, the molecular weight of 66,000 was used in order to make the final data comparable to those of other investigators (8, 9, 19). Special methods: Tryptophan was determined by the micromethod published earlier from this laboratory (20). For amide NH,, a modified procedure of Stegemann (21) was employed in which the liberated ammonia was measured with the sensitive Berthelot indophenol reaction. The half-cystine content was determined as cysteic acid (22) on some
6
SAIFER
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Fm. 4. Immunoelectrophoretic patterns of the original (Kabi) albumin preparation and the isolated monomer and polymer fractions as separated electrophoretically in buffered agar gel and the precipitin bands visualized with rabbit anti-human whole serum (A, B, and C) and with rabbit anti-human albumin (D). (A) Original 1% albumin (top) and 1% monomer (bottom). (B) 1% dimer plus trimer (top and bottom). (C) Original 6% albumin (top) and 6% monomer (bottom). (D) Original 6% albumin (top) and 6% monomer (bottom).
oxidized protein samples; but on other samples, because of the small amounts available, it was estimated on the same chromatograms as the other amino acids. The water content of the Kabi albumin sample was 5.0% by gravimetric analysis. Results of the chromatographic analyses: Five analyses were carried out on each protein sample at different hydrolysis times. Because of the good reproducibility of the method, usually only one amino acid run was performed for each hydrolyzate. Table 1 lists the percentage composition of the amino acids of the original Kabi human serum albumin
Amino
acid
1.00 6.72 6.23 10.30 1.34 3.60 6.70 4.06 0.40 3.04 3.96 5.30 1.10 5.54 3.14 10.91 9.08 15.50 0.89 98.81
This analysisO
amino
crystalline
1.05 6.67 6.17 10.30 1.31 3.65 6.74 4.03 0.13 3.15 4.31 5.09 1.08 5.38 3.17 10.95 9.05 15.85 0.94 99.02
of Data
HSA
(Kabi).
1.02 6.64 6.23 10.30 1.38 3.79 6.73 4.34 0.29 2.52 3.77 5.70 1.13 5.63 3.33 12.02 8.89 15.28 0.84 99.83
Spshr and Ed&l (1964)
acid residues/100
Comparison
Heimburger et ~2. (1964)
Grams
a Results for unfractionated b Not included in the total.
Glyeine Alanine Valme Leucine Isoleucine Proline Phenylalanine Tyrosine Tryptophan Serine Threonine Half-cystine Methionine Arginine Histidine Lysine Aspartic acid Glutamic acid Amide NH3 Total
-
Average
of five
1.23 6.33 5.69 9.34 1.38 3.69 6.45 4.20 Not determ. 2.95 4.15 4.88 1.10 5.43 3.16 10.67 8.66 15.19 1.02 95.73
a2. analysisa
11-12 62 41-42 60 8 24-25 30 16-17 1 23 26 34 5-6 23 15 56 ,5’2 79 36b :j66-6;1
This
of Human
analyses.
TABLE 1 Other Analyses
Potg$%te;L
gm protein
with of amino
Albumin
12 62 41 60 8 25 30 16 1 24 28 33 5 23 15 56 $2 81 38h ,570n
Heimburger et al. (1964)
No.
Serum renidlles/JI.W.
23 15 56 50 79 4W 557-560
78-79 3435b 56&578
16 det,erm. 23 27-28 32 6
17-18 1 19 24-25 37 6 L4 16 62 51
15 60 38-39
Not
Potg(i
66,OM)
12 62 41-Q 60 8 26 30
Spahrclsgngddsall
acid ul.
i! G
F
8 I f3
a: z
0
8
SAIFER
AND
PALO
sample and the number of amino acid residues per mole (66,000) in comparison with analyses of human albumin preparations reported by other investigators. Table 2 summarizes the percentage composition of the amino acids of the original albumin, monomeric fraction, and combined dimeric and trimeric fractions as well as the number of the amino acid residues per mole of these samples. RESULTS AND DISCUSSION
Disc gel electrophoresis, as developed by Ornstein and Davis (14), has proved to be a sensitive procedure capable of determining 20 or more separate protein components in human serum. While there is no unequivocal evidence for the presence of more than one albumin in whole human serum, crystalline human serum albumin samples, as prepared by the Cohn alcohol fractionation method (lo), have generally shown from 4 to 8 bands with this technique (Fig. 1). These results confirm our earlier reported findings (12) by means of starch gel electrophoresis in discontinuous buffer systems, as well as those of other investigators (23, 24), that these bands represent polymeric forms of an albumin monomer that are formed during the isolation procedure. It is evident from this illustration that the Kabi albumin contains the largest percentage of t.he monomeric form of the preparations commercially available and was thus the preferred sample for further investigation. Separation of the monomer from its polymeric forms was achieved by means of Sephadex gel chromatography and a typical elution pattern for one of the runs is illustrated in Figure 2. Because there is no clearcut separation between the peaks, it is not possible to obtain complete separation between the fractions, as is illustrated by their disc gel patterns in Figure 3. While the original Kabi albumin (Fig. 3A) shows the presence of 4 components, the separated monomer (Fig. 3B) is at least 95% pure and shows only a faint trace of the dimer. The dimer plus trimer fraction (Fig. 3C) is about 90% pure with only small amounts of monomer and tetramer. These results confirm those previously reported for albumin, isolated by means of starch gel electrophoresis (12)) and those separated with column chromatography (25)) that these components do not arise as a result of the electrophoretic or chromatographic procedure and represent distinct entities. The original albumin sample and the separated fractions were then subjected to immunoelectrophoresis as a sensitive technique for determining the possible presence of protein components other than albumin, and the patterns obtained are shown in Figure 4. When rabbit antihuman whole serum was used as the antiserum, no proteins besides albumin were visible at the 1% concentration level (Fig. 4A and 4B).
f 1.92
f 0.10 f 0.23 f 0.16 (mean)
amino
98.71
10.92 8.80 15.76 (0.95)
gm dry
iz 2.70
* 0.21 f 0.23 + 0.30 (mean)
kO.12 i 0.20 z!I 0.10 L!Z0.14 kO.17 kO.31 (mean) +0.07 * 0.13 + 0.20 310.04 f+_ 0.07 0.16
1.00 * 0.018 6.70 Z!Y0.23
Monomeric fraction
residues/100
5.98 10.30 1.35 3.80 6.77 4.07 0.28 2.98 3.90 5.36 1.06 5.54 3.19
acid dimeric fraction
dzO.12 kO.27 kO.09 + 0.13 zt0.37 f 0.41 (mean) f 0.12 +_ 0.04 (mean) (mean) 31 0.18 kO.04
and
100.06
f 2.66
10.70 _+ 0.23 8.71 (mean) 14.95 + 0.31 (0 93) (mean)
6.67 10.52 1.58 3.86 6.85 4.03 0.30 3.44 4.67 5.14 0.80 5.58 3.36
1.22 + 0.07 6.75 f0.28
Combined trimeric
protein
566-571
56 52 79 3&
41-42 60 8 24-25 30 16-17 1 ‘23 26 34 5-6 23 15
12 62
Original material (Ksbi-albumin)
of estimations
,569
56 51 81 37”
40 60 8 26 30 17 1 23 26 34 5-6 23 15
12 62
fractions 66,000)
of
has been less than
577
55 50 77 36”
44 61 9 26 31 16 1 26 30 33 4 23 16
14 63
Combiied dimeric and trimeric frm.tion
in various (for M.W.
Monomeric fraction
No. of amino acid residues human serum albumin
TABLE 2 of Various Fractions of Human Serum Albumin
u Errors are t’he st,andard errors of the mean of five analyses. When S.E. is not given t,he number five. * Correct#ed for hydrolytic losses, tyrosine and serine values illcreased by 15%, t,hreonine by 107,. c Value not included in the total.
Total
10.91 9.08 15.50 (0.89) 98.81
Lysine Aspartic acid Glutamic acid Amide-NH3
kO.06 t 0.07 f 0.09 i0.14 kO.16 z!z 0.27 (mean) Ik 0.10 k 0.16 + 0.18 f0.03 f+ 0.06 0.03
6.23 10.30 1.34 3.60 6.70 4.06 0.40 3.04 3.96 5.30 1.10 5.54 3.14
Valine Leucine Isoleucine Prolme Phenylalanine Tyrosineb Tryptophan Serind Threonineb Half-cystine Methionine Arginine Histidine
Original material (K&i albumin)*
1.00 + 0.015 6.72 kO.06
acid
Glycine Alanine
Amino
Grams
Amino Acid Composition
(0
z 2
&
s 3 ;;
8
Et
0 r
ti iii
3
$2
10
SAIFER
AND
PAL6
However, a second faint band in the globulin region was visible for both the original albumin sample and the monomer fraction at the 6% level (Fig. 4C). These secondary bands may represent either denatured albumins or small amounts of globulin components. The additional faint bands obtained at the 6% level, when rabbit anti-human albumin is used as the antiserum (Fig. 4D), may represent poor antigens that did not produce antibodies when whole serum was used as the immunizing agent. The tryptophan content of the Kabi albumin and its fractions was determined on their perchloric acid hydrolyzates with the spectrophotometric procedure of Saifer and Gerstenfeld (20). Values reported here (0.30 to 0.40 gm/lOO gm protein) are in good agreement with those published by other investigators for human serum albumin (Table 1)) as well as that recently published by Spies (26), i.e., 0.347 gm/lOO gm protein. In any case, these results all lead to a value of 1 mole of tryptophan per mole (66,000) of human serum albumin, as is recorded in Table 2. Acrylamide gel electrophoresis is unquestionably a more sensitive technique than is ultracentrifugation for detecting small amounts of polymeric material in albumin preparations, and has also recently been employed to determine the molecular weights of many proteins, including the polymeric forms of bovine serum albumin (27). There is good agreement among moat recent investigators (27) that the molecular weight of monomeric human serum albumin is 67,000 + 2000. In this investigation a value of 66,000 was chosen in order to better relate our amino acid data to those of other investigators. While a large number of amino acid analyses have been recorded in the literature for bovine serum albumin (4, 8, 28)) very few analyses have been reported for the human protein. A number of these have been performed by either quantitative paper chromatographic or microbiological assay methods and these earlier data have been summarized by Wuhrmann and WunderIy (5). The results obtained by Brand and co-workers (3, 4) are those most frequently found in textbooks yet they gave no value for alanine although their total amino acid residues per 100 gm protein added up close to 100%. With the introduction of the ion-exchange automated column chromatographic techniques for amino acid analysis by Moore et at. (6), the accurate analysis (i.e., within rrt6% error) of the amino acid composition of proteins has become possible. In the present study the single column modification of Piez and Morris (7) was employed which gives excellent separation of all 18 amino acids found in protein hydrolysates. The only serious drawback in this procedure is that methionine sulfones, formed by oxidation during the hydrolysis step, may
AMINO
ACIDS
OF
MONOMERIC
ALBUMIh’
11
overlay or enhance the aspartic acid peak. Since it is possible to reproduccl standard runs on successive days, using norleucine as an internal standard, with a precision of about kl%, it is t,he hydrolysis step that makes the major contribution to the over-all error of the method. In part this is due to the fact that some amino acids, e.g., cystine, threonine, and tyrosine, are decomposed during hydrolysis while others, e.g., valine and isoleucine, may require more than 48 hr of hydrolysis for their maximum values. It was reasoned that the use of crystalline insulin, as a standard protein of known amino acid composition, would provide an additional estimate of the correction factors necessary to compensate for the changes occurring both during the analytical and the hydrolysis steps. Five such insulin analyses were performed at different hydrolysis times and t.hc average correct,ion factors applied to the albumin analyses. Additional small negative corrections, based on the insulin analysis, were made foi lysine, glutamic acid, alanine, phenylalanine, and leucine. The Sam<’ correction factors were used for all the albumin amino acid analyses. The results obtained for the original Kabi albumin preparation are listed in Table 1 in comparison with the data of other investigat’ors, all of whom used automated column chromatographic techniques for their analyses. The values of Potgieter et al. (19) are based on a single analysis of a 22 hr hydrolyzate and generally tend to be low as less than 96% of the amino acid nitrogen was accounted for. Examination of the amino acid data in Table 1 shows better agreement between our results am1 those of Heimburger et al. (9) than with those of Spahr and Edsall (8), with the possible exception of the glutamic acid values. In comparison with data of all other investigators, the serine values reported by Spahr and Edsall (8) are too low while those for half-cyst.ine and lysine are too high. However, there are no statistically significant differences between our data and those of the other two investigators (8, 9) for any amino acid. Our values for amide NH, are also in good agreement with those reported by other investigators. They have not been included in the total number of residues of the protein since they are derived from the glutamine and ssparagine residues of the intact protein (19). The amino acid analyses of Spahr and Edsall (8) show no detectable differences in amino acid composition between human serum mercaptalbumin and total human serum albumin. The former is precipitable with mercury salts and constitutes 60 to 70% of the total albumin so that small differences between the amino acid values of the two albumin fractions would generally fall within the experimental error of their det,ernnnations. Previous work from this laboratory (12) has shown that both human serum albumin and mercaptalbumin cont,ain a series of polymeric components the proportions of which depend upon the previous
12
SAIFER
AND
PALO
history of the protein preparation. It can therefore be assumed that all amino acid analyses performed by previous investigators have contained appreciable amounts of dimer and trimer albumin molecules in addition to the preponderant monomeric form. With the possible exception of the glutamic acid results, there is a close correspondence of amino acid values between the original albumin sample and its monomeric fraction (Table 2). The lower glutamic acid polymer value may be due to its removal, or that of a bound glutamic acid containing peptide, during the column chromatographic process from the nonmercaptalbumin fraction which King (29) has reported to be a mixed disulfide of mercaptalbumin and cysteine or glutathione. Statistical analysis by the Students t test of the data for the dimer plus trimer fraction, in comparison with the monomer values, has shown significantly increased values at the 5% probability level for valine, serine, and threonine and a decreased value for glutamic acid. While it would appear unlikely that polymerization would produce a change in amino acid composition, such a possibility cannot be entirely excluded if peptide loss precedes polymerization. An alternative explanation for the observed amino acid differences of the polymer is that this fraction, which is first eluted from the column, may contain appreciable amounts of other proteins as impurities or of bound peptides. This matter is presently under investigation by means of N-terminal analysis and by peptide mapping after enzymic hydrolysis. Tanford (30) has published titration data for human serum albumin which gave about 190 moles of carboxyl groups per mole of protein. The Kabi albumin data gives a value of 95 plus 1-cr-carboxyl based upon 37 amide groups per mole. This value of 96 carboxyl groups checks well with the total of 104 groups for lysine, histidine, and arginine if one assumes that some of the histidine groups have lost their positive charge at pH 4.5, i.e., the isoelectric point. The presence of a number of genetic variants of albumin have been described in the literature with both faster and slower electrophoretic mobilities than normal albumin (13, 31). By analogy with the case of abnormal hemoglobins, it is to be expected that such differences in mobility reflect minor differences in the amino acid composition of these albumins. The accurate analysis of the amino acid composition of normal albumin is a necessary first step in the investigation of its structure, and that of its genetic variants, especially with respect to their unique ion binding properties (1, 2, 32, 33). SUMMARY
All previous amino
(HSA)
acid analyses of crystalline human serum albumin have been performed on preparations which have been shown by
AMINO
Actis OF
MONOMERIC
&suMlN
13
gel electrophoresis to contain a number of polymeric forms. Monomeric albumin was separated from its higher M.W. components by means of :I Sephadex G-150 column with Tris/NaCl buffer at pH 8.1 using crystalline human albumin (Kabi) as the starting material. The physicochemical characteristics of the monomeric and polymeric fractions were investigated by means of acrylamide gel electrophoresis and immunoelectrophoresis. Their amino acid composition was determined by automated ion-exchange chromatography. Although no significant differences were found between the original albumin sample and its monomer, some of the analytical data obtained differed from those of other investigators. The polymeric fraction showed some significant differences in amino acid composition from the monomer that require further verification. ACKNOWLEDGMENTS The authors wish to acknowledge the technical aid of Pod W. Hansen and Gilbert Lowenwirth in performng some of the experimental work presented here. We are grateful to Mrs. Lillian Salowitz for the typing and editing of the manuscript. REFERENCES J. F., in “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1, p. 179. Academic Press, New York, 1960. 2. SCHULTZE, H. E., AND HEREMANS, J. F., “Molecular Biology of Human Proteins.” Elsevier Publishing Co., Amsterdam, 1966. 3. BRAND, E., KASSEL, B., AND SAIDEL, L. J., J. Clin. Invest. 23, 437 (1944). 4. BRAND, E., Ann. N. Y. Acad. Sci. 4’7, 187 (1946). 5. WUHRMANN, F., AND WUNDERLY, C., “The Human Blood Proteins,” p. 24. Grunr & Stratton, New York, 1960. 6. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., ,4nal. Chem. 30, 1185 (1958). 7. PIEZ, K. A., AND MORRIS, L., Anal. Biochem. 1, 187 (1960). 8. SPAHR, P. F., AND EDSALL, J. T., J. Biol. Chem. 911, 8.50 (1964). 9. HEIMBURGIER, N., HEIDE, K., HAUPT, N., AND SCHULTZE, H. E., Clin. Chim. A& 10, 293 (1964). 10. PENNELL, R. B., in “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1, p. 9. Academic Press, New York, 1960. 11. HUGHES, W. L., JR., Cold spring Harbor Symp. Quant. Bid. 14, 79 (1950). 12. SAIFER, A., ROBIN, M., AND VENTRICE, M., Arch. Biochem. Biophys. 92, 409 (1~1). 13. MELARTIN, L., BLUMBERE, B. S., AND LISKER, R., Nature 215, 1288 (1967). 14. ORNSTEIN, M., AND DAVIS, B., Ann. N. Y. Acad. Sci. 121, 404 (1964). 15. PEDERSEN, K. O., Arch. Biochem. Biophys., Suppl. 1, 157 (196‘2). 16. SCHEIDECICIER, J. J., Intern. Arch. Allergy ‘7, 103 (1965). 17. HARFENIST, E. J., J. Am. Chem. Sot. 75, 5528 (1953). 18. CORNFIELD, M. C., AND ROBSON, A., Biochem. J. 84, 146 (1962). 19. POTQIETER, G. M., HINES, M., AND KENCH, J. E., Chin. Chim. Acta 18, 107 (1~7). 20. SAIFER, A., AND GERSTENFELD, S., CZin. Chem. 10, 970 (1964). 21. STEOEMANN, H., 2. Physiol. Chem. 312, 255 (1958). 22. MOOBE, S., J. Biol. Chem. 238, 235 (1963). 23. Somu, H. A,, GUTER, F. J., WYCKOFF, M., AND PETERSON, E. A., J. Am, C&m. Sot. 78, 756 (1956). 1. FOSTER,
14
SAIFER
AND
PALO
S., AND NAKAMICHI, M., Anal. Biochem. 7, 225 (1964). TISELIUS, A., HJERTEN, S., AND LEVIN, O., Arch. B&hem. Biophys. 65, 132 (1956). SPIEB, J. R., Anal. Chem. 39, 1412 (1967). ZWAAN, J., Anal. Biochem. 21, 155 (1967). STEIN, W. H., AND MOORE, S., J. Biol. Chesm. 178, 79 (1949). KING, T. P., J. Biol. Chem. 236, PC5 (1961). TANFORD, C., J. Am. Chem. Sot. 72, 441 (1950). MELARTIN, L., AND BLUMBERG, B. S, Clin. Res. 14, 482 (1966). KLOTZ, I. M., in “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. I, Part B, p. 727. Academic Press, New York, 1953. 33. SAIEER, A., AND STEIOMAN, J., J. Phys. Chem. 65, 141 (1961).
24. RAYMOND,
25. 26. 27. 28. 29. 30. 31. 32.