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Affinity chromatography of glycopeptides using Concanavalin A
ANALYTICAL
BIOCHEMISTRY
72, 389-399
(1976)
Affinity Chromatography of Glycopeptides Using Concanavalin A MILAN TOMANA, WILLIAM NIEDERMEIER, JIRI RALPH E. SCHROHENLOHER. AND SANDRA Division
of Clinical Immunology and Rheumatology, University of Alabama in Birmingham, Birmingham, Alabama
Received June 19, 1975; accepted A
technique
has
been
developed
that
MESTECKY, PORCH
and
The Institute of Dental University Station, 35294
November
utilizes
affinity
Research,
11, 1975 chromatography
on
Concanavalin A (Con A)-agarose column for isolation of glycopeptides from IgA protein. Three glycopeptides thus obtained were homogeneous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. One glycopeptide contained two oligosaccharides which were partially resolved following pronase digestion. Recovery of glycopeptides applied to the Con A column was virtually complete. The specificity of Con A for the glycopeptides present in the IgA, protein was demonstrated.
The preparation of glycopeptides from glycoproteins usually involves chemical or enzymatic cleavage of the polypeptide chains followed by isolation and purification of the individual glycopeptides by gel filtration, ion-exchange chromatography, partition chromatography, or high voltage electrophoresis. The isolation procedures are not specific for carbohydratecontaining peptides, they are time consuming and the yield of purified glycopeptides is usually low. If nonspecific enzymatic cleavage is used, the number of glycopeptides obtained bears no relationship to the number of oligosaccharides actually present in the glycoprotein. Each oligosaccharide usually appears in several glycopeptides that differ from each other only in the length of the polypeptide chains. Affinity chromatography using lectins covalently linked to Agarose appears to be a useful tool for separation of glycopeptides. The advantage of lectins is that they bind to specific monosaccharides, can discriminate between stereoisomers, and may give information about the type and position of substitution (1). The present study demonstrates the use of agarose-linked concanavalin A in isolating glycopeptides from an immunoglobulin A. MATERIALS
AND METHODS
The IgA, (Kni) myeloma protein was isolated from the serum of a patient with multiple myeloma by ammonium sulfate precipitation and gel 389 Copyright 0 1976 by Academic Pre\\. Inc. 411right\ of reproduction in any form reerved.
390
TOMANA
ETAL.
filtration as described earlier (2). Heavy and light chains were separated by gel filtration on Sephadex G-200 in 5 M guanidine-HCl after oxidative sulfitolysis (3). Pepsin digestion. IgA protein (200 mg) was incubated with pepsin (Worthington Biochemical Corp., Freehold, N.J.) at the enzyme:substrate ratio 1:100 at 37°C for 16 hr. The acidity of the solution was maintained at pH 1.9 for the first 2 hr of incubation. At the end of incubation the acidity was usually pH 2.2. The incubation mixture was then neutralized to pH 6 with 2 N NaOH and mixed in a ratio 5: 1 with 0.5 M acetate buffer, pH 6, containing0.75 M NaCl, 3 mM CaCI,, 5 mM MgC12, and 5 mM MnCI,. The pepsin digest was applied to a Concanavalin A-Agarose (Con A) column (Glycosilex, Miles Laboratories, Inc., Kankakee, Ill.), 29 x 1.6 cm, equilibrated with 0.1 M acetate buffer, pH 6.0, containing 0.15 M NaCl, 1 mM CaCl,, 1 mM MgC&, and 1 mM MnCl,. The glycopeptides retained in the column were eluted with a 10% solution of methyl (Y-Dmannoside (Sigma Chemical Co., St. Louis, MO.). Pronase digestion. The pepsin peptides were freeze-dried, redissolved in 0.1 M Tris-WC1 buffer, pH 8.0, containing 1 mM CaCl,, and incubated with pronase (Calbiochem, La Jolla, Calif.) at 37°C for 24 hr. The enzyme: substrate ratio was 1:50. The solution was maintained at pH 8 with 20 mM NaOH. The glycopeptides were isolated by exclusion chromatography on a column (88 x 1.4 cm) of Sephadex G-50 (fine grade). Labeling of siatic acid residues. Sialic residues in IgA or peptides derived from it were labeled with tritium by a modification of the method of Van Lenten and Ashwell (4). Proteins were dissolved in 0.1 M acetate buffer, pH 6, containing 0.15 M NaCl and mixed at a ratio of 5: 1 with 0.025 M sodium metape~odate. After incubation at 0°C for 10 min, the oxidation was stopped by the addition of a molar excess of ethylene glycol, and the solution was dialyzed overnight against 0.05 M sodium phosphate buffer, pH 7.5, containing 0.15 M NaCl. The protein solution in a total volume of l- 10 ml was cooled to 0°C and was reduced by addition of l-2.5 mCi of tritiated sodium borohydride (New England Nuclear, Boston, Mass.) with a specific activity of 200 mCi/mM dissolved in 0.1 ml ice-cold 0.01 M NaOH. The reaction mixture was warmed to room temperature and reduction was allowed to continue for 60 min. Then nonradioactive NaBH, (1% w/w in respect to protein) was added, and the incubation continued for another 60 min. The excess borohydride was destroyed by acidification with HCI and removed by dialysis first against 0.15 M NaCl, then against 0.1 M acetate buffer, pH 5.9, containing 0.15 M NaCI. When small peptides were labeled, the dialysis step was replaced with gel filtration using Sephadex G-25 columns. The radioactivity of tritium was measured in a Nuclear Chicago scintillation counter. Fractions (in lo- 100 ~1 aliquots) were measured after dispersion in 10 ml Aquasol (New England Nuclear).
ISOLATION
OF GLYCOPEPTIDES
391
Immunoefectrophoresis . LKB 68OOA immunoelectrophoresis apparatus was used with 1% lonagar No. 2 in Verona1 buffer. Antisera to L-chains and H-chains were prepared and characterized as described earlier (5). Ultracentri~~gatjon . Molecular weight values were dcte~ined by sedimentation equilibrium in a Beckman Model E ultracentrifuge equipped with an electronic speed control, absorption optics, and a photoelectric scanner (6). An An-F rotor and 12-mm cells were used in all experiments. The monochromator was set at 280 nm, and the temperature was regulated at 20°C. The sample column was 3-mm high. Equilibrium was judged to be established when there was no change in the slope of the logarithm of the absorbance plotted against the square of the distance from the center of rotation during a minimum period of 4 hr. Prior to analysis, the proteins were dissolved in and dialyzed against 5 M guanidine-HCI. The partial specific volumes for glycopeptides (0.695-0.712) were calculated from their amino acids and carbohydrate compositions (7). Apparent molecular weight of glycopeptides was estimated also from their elution volume from calibrated Sephadex columns. All columns were calibrated with a mixture of ovalbumin, ribonuclease (both from Pharmacia Fine Chemicals, Piscataway, N.J.), insulin, adrenoco~icotrophic hormone (ACTH), oxytocin (hormone peptides were obtained by Dr. Roy Mundy, University of Alabama), and glutathione (Sigma Chemical Co.). Disc efectrophoresis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was used as a criterion of purity. Each peptide was subjected to electrophoresis in three gel systems: (I) 5% gel at ph 7.2 (8); (II) 15% gel at pH 8.9 (9); (III) 30% gel at pH 8.9 (9). For molecular weight determination, the polyacrylamide gels were calibrated with L-chains, H-chains, ribonuclease, and insulin by the method of Fairbanks et al. (10). Carbohydrate analyses. Fucose, mannose, and galactose were released from their glycosidic linkages by hydrolysis for 4 hr in 1 N HCI at lOO”C, converted to alditol acetates, and subjected to gas-liquid chromatog~phy using 6 ft x % in. glass columns packed either with 1% OV-225 on Varaport 30,70/80 mesh (Varian Aerograph, Walnut Creek, Calif.) or with 3% SP-2340 on Supelcoport, 1001120 mesh (Supelco, Inc., Belefonte, Pa.). From the initial 180°C the temperature was increased at the rate of 5”Cimin to 260°C. Arabinose was used as internal standard. To liberate N-acetyl glucosamine and N-acetylgalactosamine the hydrolysis was done either with 1 N HCl for 10 hr or with 6 N HCl for 2 hr at 100°C. The conditions of gas chromatography for analysis of neutral sugars (I 1) and amino hexoses (12) were described earlier. Gas-chromatographic analyses were done on an F & M Model 402 dual-column gas chromatograph with flame ionization detectors (Hewlett- Packard Corp., Avondale, Pa.) and an Infotronics Model CRS 104 electronic integrator (Infotronics, Inc., Houston, Tex.). Sialic acid was determined by the method of Warren
392
TOMANA
ET AL.
d, IO 20
30 40 50 60 FRACTION NUMBER
‘\
70
---. so
90
FIG. 1. Separation of pepsin peptides of IgA, on a 1.6 x 29 cm concanavalin A-agarose column equilibrated with 0.1 M acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM CaCI,, 1 mM MgCl*, and 1 mM MnCI,. Fraction volume 4.8 ml. Full line: optical density at 230 nm. Broken line: [3H]sialic acid radioactivity of lOGpI fractions.
(13) following 45 min.
hydrolysis of the glycoprotein
in 0.08 M H,SO, at 80°C for
Amino acid analyses. Samples were hydrolyzed for 24 hr in 6 N HCl at 105°C. Amino acid analyses were done on a Durrum Model DSOO amino acid analyzer with a single high-pressure cation-exchange column.
RESULTS Sedimentation analyses showed that the IgA, protein (Kni) was a monomer with a sedimentation constant of 6.2 s. Assuming a molecular weight of 158,000, the oligosaccharides of this glycoprotein, all of which were located on the alpha chains, consisted of 2-3 residues of fucose, 13- 14 of mannose, 16 of galactose, 14- 15 of N-acetyl glucosamine, 9- 10 of N-acetyl galactosamine, and 4-5 of sialic acid. The peptides resulting from pepsin hydrolysis of the tritiated IgA, protein were separated into two fractions on the concanavalin A-agarose column as shown in Fig. 1. Fraction A contained peptides that did not interact with the lectin. It represented about 90% of the starting material. Fraction B contained peptides that were retained on the column and were eluted with 10% methyl a--D-mannoside (about 10% of the hydrolysate). Both fractions were freeze-dried, redissolved in a minimal
ISOLATION
OF GLYCOPEPTIDES
393
FIG. 2. Gel filtration of fractions A (upper graph) and B (lower graph) from concanavalin A-agarose column. Column (2.5 x 96 cm): Sephadex G-SO (medium grade) in 0.1 M acetic acid. Fraction volume 4.8 ml. Full line: optical density at 280 nm (upper graph) and 230 nm (lower graph). Broken line: [3H]sialic acid radioactivity of lOO-~1 fractions.
volume of 0.1 M acetic acid, desalted by dialysis or by gel filtration on a Sephadex G-25 column, and analyzed for carbohydrate content. Both fractions contained fucose, mannose, galactose, N-acetyl glucosamine, Nacetyl galactosamine, and sialic acid. Fraction A contained a lower content of carbohydrate/mg of protein than fraction B. Fractions A and B were subjected to elution chromatography on a Sephadex G-50 column. Five fractions were obtained from the IgA, protein. Three, designated Al, AZ, and A, were isolated from fraction A, and two, designated B1 and Bz, were isolated from fraction B as shown in Fig. 2. Examination by polyacrylamide gel electrophoresis indicated that fractions A, and Bz were homogeneous. Fraction A, was homogeneous only when prepared from IgA that was not subjected to radiolabeling. Fraction B, contained more than one peptide. Fraction A, contained low molecular weight radioactive compounds that were not completely re-
394
TOMANA
ETAL.
TABLE
1
CARBOHYDRATE COMPOSITION~ANDAPPARENT MOLECULAR WEIGHT OF PEPSIN PEPTIDESFROM IgAl PROTEIN ISOLATEDBY CONCANAVALIN A-AGAROSE COLUMN AND GEL FILTRATION
Protein
k.41
Fraction
FUCO%
Aid
0
A* BI BZ
0.42 0.20 0.47
Galactose
Mannose
N-Acetyl glucD samine
7.55 I .59 4.7 0.95
0 2.34 2.80 2.60
0 3.26 I.59 3.01
N-Acetyl galactosamine 8.89 0 2.10 0
Sialic acid
HOlllOgeneityb
Apparent moleculalf weight (dakons)
o.s7 0.37 0.35 0.28
+ + +
14.w (UC) 4,7w {CCL) l2,OOO (DG)” 4.800 (Cob)
a Concentrations expressed in moles/mole of glycopeptide. ’ (+) Homogeneous, (-) heterogeneous peptide. c (U.C.) Determined by sedimentation equilibrium; (Cal.) by Sephadex chromatography: (D.G.) by polyacryiamide electrophoresis. d Molecular weights and carbohydrate compositions were determined on preparations that were not radiolabeled. c Apparent molecular weight of the main component.
gel
movedin previousdialysisanda traceof free sialic acidthat was apparently cleavedfrom the immunoglobulin duringpepsinhydrolysis. The molecular weightsand the carbohydratecompositions of fractions Al, AZ, B,, and B2 are shown in Table I. Tritium-labeled fraction A, was subjectedto proteolytic treatment with pronase. Two peptides with identical carbohydratecompositions were resolved by gel filtration on SephadexG-50. The larger peptide had an apparent molecular weight approximately twice as great as that of the smaller one, indicating that these two glycopeptidesrepresenteddimeric and monomeric forms of the sameglycopeptide. The partial cleavageof interchain disulfide bonds was apparently causedby oxidation or reduction during the processof radiolabeling. Fraction B, was heterogeneousas judged from polyacrylamide gel electrophoresis. The presenceof both N-acetyl galactosamineand Nacetyl glucosamine indicated that it contained at least two different oligosaccharides.Fraction B, was subjectedto further proteolytic treatment with pronase. Two glycopeptides were resolved by gel filtration. The larger of these (B,J contained galactose,N-acetyl galactosamine, sialic acid, and trace amountsof mannoseand N-acetyl glucosamine.The smaller one (BIB) contained mannose,galactose,N-acetyl glucosamine, and trace amounts of N-acetyl galactosamine,fucose, and sialic acid. Largeamountsof proline andcysteinein glycopeptideB,,and thepresence of galactosamineand galactoseindicated that it was identical with glycopeptideA,. GlycopeptidesA, and B2 had almost identical carbohydratecompositions but interacted differently with Con A. To determine whether the interaction of glycopeptidesA, and B, with Con A were specific, purified tritium-labeled glycopeptide A2 was added to a nonlabeled pepsin
ISOLATION
OF GLYCOPEPTIDES
395
FIG. 3. Separation of the mixture of pepsin peptides of IgA, and tritium-labeled glycopeptide A2 (upper graph) or B, (lower graph) on concanavalin A-agarose column. IgA, protein (40 mg) was hydrolyzed with pepsin under conditions described in Material and Methods. After neutralization, 4 mg of tritium-labeled glycopeptide AZ or B, was added. Column (1.6 X 29 cm) equilibrated with 0.1 M acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM CaCl,, 1 mM MgCl,, and 1 mM MnCl,. Fraction volume 4.8 ml. Full line: optical density at 230 nm. Broken line: 3H activity of 100 /*I fractions.
hydrolysate of IgA,, and the mixture was reapplied to the concanavalin A column. As shown in Fig. 3a, practically all of the tritium activity again appeared in fraction A. When tritium-labeled glycopeptide Bz was added to a nonlabeled pepsin hydrolysate of IgA,, most of the radioactivity appeared in fraction B, as shown in Fig. 3b. Both glycopeptides A2 and B, were further subjected to proteolytic treatment with pronase. One glycopeptide was recovered from A2 and one from B,. The amino acid composition of pronase peptides A,, AZ, Blu, BIP, and B2 is shown in Table 2. The carbohydrate compositions of the IgA, protein and the alpha chains prepared from it are compared in Table 3 with the sum of the carbohydrate found in the four glycopeptides. The calculation was made on the assumption that glycopeptide A, was a dimer and that glycopeptide B, contained the same galactosamine containing oligosaccharide found in A, in addition to a second oligosaccharide that was responsible for retention of this glycopeptide in the Con A column.
396
TOMANA TABLE
ET AL. 2
AMINO ACID COMPOSITION OF IgA GLYCOPEFTIDES AFTER P~ONASE DIGESTION Moles/100 moles Amino Acid Amino acid
A,
A2
ha
Bli?
B2
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Leucine Histidine Lysine Arginine Cysteine
19.4 16.7 44.7 7.5 11.7*
25.7 8.2 12.4 4.0 6.1 5.9 5.3 19.6 2.7 4.9 3.5 1.7 -
5.3 17.6 20.9 2.5 40.0 1.9 1.2 5.4 2.2 3.v
23.8 10.7 14.5 7.9 4.9 7.5 9.2 2.5 12.1 1.0 1.6 4.2
36.4 9.1 16.3 7.2 2.8 4.0 5.9 7.9 4.6 2.7 3.1 -
LIThe presence of glucosamine in B,, and galactosamine in BIB indicate that these two glycopeptides were not completely separated. b Calculated as cysteic acid after performic acid oxidation. c Not oxidized with performic acid.
DISCUSSION Four glycopeptides were isolated from pepsin digests of IgA, using affinity chromatography on a concanavalin-agarose column and gel filtration. Three of these were homogeneous when examined by disc gel electrophoresis in the presence of SDS. The observation that glycopeptides A, and Bz had almost identical carbohydrate compositions suggested initially that the quantity of peptides applied to the Concanavalin A column exceeded its capacity, or that the interaction of Con A with glycopeptides of the IgA, protein were nonspecific. Similar results, however, were obtained when the quantity of pepsin hydrolysate applied to the column was reduced by a factor of 10 or 100. This observation indicated that the Con A column had not been overloaded. That the interaction of Con A with glycopeptides from the IgA protein was indeed specific was verified by the observation that the tritiated glycopeptides that were initially retained on the Con A column were again retained when they were reapplied to the column after mixing with an unlabeled pepsin digest of the whole IgA molecule. Likewise, tritiated glycopeptides that were not initially retained on the Con A column failed to be retained when mixed with an unlabeled pepsin digest of the whole IgA molecule and reapplied to the column. Results of amino acid analyses of glycopeptides A, and B, (Table 2) further support the sug-
ISOLATION
TABLE CARBOHYDRATE
397
OF GLYCOPEPTIDES
COMPOSITION OF IgA, AND PEPSIN GLYCOPEPTIDES
3 (Kni)
IMMUNOGLOBULIN. (RESIDUES/MOLE)
N-Acetyl ghlcosamine
H-CHAIN. N-Acetyt gatactosamine
Siaiic acid
Fucose
Mannose
Galactose
2.2
12.5
16.0
14.3
9.5
4.6
H-Chain (calculated from bYV H-Chain (analysis) (MW: 56,300)
1.1
6.3
8.0
7.2
4.8
2.3
1.0
6.0
8.0
7.6
4.1
2.3
Glycopeptides”
0.9
7.7
9.3
7.9
4.5
0.93
w, (MW: 158,000)
(1 Calculated on the assumption that glycopeptide A, was a dimer and that glycopeptide B, contained the same oligosaccharide present in A, in addition to a glucosamine containing oligosaccharide.
gestion that IgA, (Km) contains two glycopeptides of similar carbohydrate composition but with different internal structures that were derived from different regions of the alpha chains. It has been shown that Con A combines with glycopeptides that contain a-D-mannopyranoside or cr-D-glucopyranoside residues with unmodified hydroxyl groups at the C-3, C-4 and C-6 positions (14). Inhibition studies indicate that ~-acetylglucosamine can also bind Con A although less effectively than mannose (15). Kornfeld and Ferris (16) have shown that relatively small changes in the structure of the oligosaccharide can strongly influence the binding capacity of Con A. Thus, for example, the cleavage of one terminal galactose residue from the branched oligosaccharide of IgE resulted in tenfold increase in the binding capacity of the ~y~opeptide with Con A. A glycopeptide has been isolated from IgA that contains three mannose residues located in the core of the oligosaccharide unit. Two mannose residues are linked to the third one by al-3 or al-6 glycosidic linkages. The hydroxyl group on C-2 in two of the mannose residues is involved in a glycosidic linkage with N-acetyl glucosamine (19). The interaction of glycopeptide Bz with Con A is consistent with such a structure. Glycopeptides that exhibit similar interactions with Con A were found also in IgA,, secretory component, J-chain, and in IgG (unpublished data). In glycopeptide A,, the mannose residues appear to be either panomers, or some of their hydroxyl groups that are critical for interactions with Con A are involved in glycosidic linkages with neighboring
398
TOMANA
ET
AL.
monosaccharides. The possibility, however, cannot be excluded that AZ, that has a slightly higher content of galactose, represents a more completed carbohydrate unit that interacts less strongly with Con A than the uncompleted unit in glycopeptide Bz. Glycopeptide A, which contained neither mannose nor glucosamine predictably did not interact with Con A. This glycopeptide appeared to be a dimer, composed of fragments of two adjacent alpha chains. The carbohydrate and amino acid analyses of the monomer that was isolated following pronase digestion indicated that this glycopeptide was identical to the hinge glycopeptide described earlier (18). Fraction B, was heterogeneous and contained two oligosaccharides. Pronase digestion followed by gel filtration on Sephadex G-25 permitted the partial separation of two glycopeptides, one of which (B,,J was identical to A,, the hinge peptide (18). The other peptide (BID) did not contain fucose or sialic acid, and was similar to the leucine reach glycopeptide which Putnamet al. (19) found near the hinge region of an IgA, (19) protein. The results suggest that these two oligosaccharides (A, and B,,) are located in close proximity on the heavy chain. Apparently some o-chains were cleaved by pepsin between these two oligosaccharides releasing glycopeptide A, (hinge peptide) which was not bound to Con A and the fucose free peptide that was bound to Con A. In other molecules, the a! chain was not cleaved between these two oligosaccharides releasing a glycopeptide that contained both oligosaccharides. This explanation was supported by the results of polyacrylamide gel electrophoresis of fraction B, that showed multiple bands. The use of Con A as well as other lectin columns thus appears to present a useful tool for the isolation, purification, and characterization of glycopeptides. Con A allows recovery of high yields of glycopeptides, and it appears to possess a high degree of specificity. Essentially all of the carbohydrate found in the IgA, protein was accounted for in the four glycopeptides isolated by affinity chromatography on Con A and gel filtration. When similar Con A preparations purchased from alternate commercial sources were used, difficulty was sometimes encountered in obtaining quantitative recovery of the fraction that was retained on the column. ACKNOWLEDGMENTS The authors wish to express their thanks to Mrs. Rose Kulhavy for preparation of human serum IgA used in this study. These studies were supported by grants AM-03555 and AI-10854 from the National Institutes of Health, Bethesda, Maryland and the John A. Hartford Foundation, New York.
REFERENCES 1. Sharon, N.. and Lis, H. (1972) Science 177, 949. 2. Tomana, M., Niedermeier, W., Mestecky, J., and Hammack, W. (1972) chemistry
9, 933.
Immuno-
ISOLATION
OF GLYCOPEPTIDES
399
3. Edelman, G. M., and Marchalonis, J. J. (1967) in Methods in Immunology and Immunochemistry (Williams, A. C.. and Chase, M. W., eds.), Vol. 1, p. 405, Academic Press, New York. 4. Van Lenten, L., and Ashwell, G. (1971) J. Biol. Chem. 246, 1889. 5. Mestecky, J.. Kulhavy, R., and Kraus, F. W, (1972) J. Immunol. 108, 738. 6. Schrohenloher, R. E., Mestecky. J.. and Stanton, T. H. (1973) Biochim. Biophys. Acta 295, 576. 7. Cohn, E. J., and Edsall, J. T. (1943) in Proteins, Amino Acids, and Peptides (Cohn, E. J., and Edsall, J. T., eds.), p. 370, Hafner Publishing Co., New York. 8. Maizel. J. V., Jr. (1966) Science 151, 988. 9. Maurer, H. R. (1971) in Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, (de Gruyter, W.. ed.), 2nd ed., p. 44, Berlin New York. 10. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10, 2606. 11. Niedermeier, W. (1971) Anal. Biochem. 40, 465. 12. Neidermeier, W., and Tomana, M. (1974) Anal. Biochem. 57, 363. 13. Warren, L. J. (1959)J. Biol. Chem. 234, 1971. 14. Goldstein, I. J., Reichert, C. M., Misaki, A., and Gorin. A. J. (1973) Biochem. Biophys. Acta 317, 500. 15. Iyer, R. N., and Goldstein, I. J. (1973) Immunochemistry 10, 313. 16. Komfeld, R.. and Ferris, C. (1975)J. Biol. Chem. 250, 2614. 17. Baenziger, J., and Komfeld, S. (1974) J. Biol. Chem. 249, 7260. 18. Frangione, B., and Wolfenstein-Todel, C. (1972)Proc. Nat. Acad. Sci. USA 69,3673. 19. Putnam, F. W., Low, T., Liu, V., Huser, H., Raff, E., Wong, F. C., and Clamp, J. R. (1973) in Advances in Experimental Medicine and Biology (Mestecky, J., and Lawton, A. R., III, eds.). Vol. 45, pp. 177- 189, Plenum Press, New York/London.
BIOCHEMISTRY
72, 389-399
(1976)
Affinity Chromatography of Glycopeptides Using Concanavalin A MILAN TOMANA, WILLIAM NIEDERMEIER, JIRI RALPH E. SCHROHENLOHER. AND SANDRA Division
of Clinical Immunology and Rheumatology, University of Alabama in Birmingham, Birmingham, Alabama
Received June 19, 1975; accepted A
technique
has
been
developed
that
MESTECKY, PORCH
and
The Institute of Dental University Station, 35294
November
utilizes
affinity
Research,
11, 1975 chromatography
on
Concanavalin A (Con A)-agarose column for isolation of glycopeptides from IgA protein. Three glycopeptides thus obtained were homogeneous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. One glycopeptide contained two oligosaccharides which were partially resolved following pronase digestion. Recovery of glycopeptides applied to the Con A column was virtually complete. The specificity of Con A for the glycopeptides present in the IgA, protein was demonstrated.
The preparation of glycopeptides from glycoproteins usually involves chemical or enzymatic cleavage of the polypeptide chains followed by isolation and purification of the individual glycopeptides by gel filtration, ion-exchange chromatography, partition chromatography, or high voltage electrophoresis. The isolation procedures are not specific for carbohydratecontaining peptides, they are time consuming and the yield of purified glycopeptides is usually low. If nonspecific enzymatic cleavage is used, the number of glycopeptides obtained bears no relationship to the number of oligosaccharides actually present in the glycoprotein. Each oligosaccharide usually appears in several glycopeptides that differ from each other only in the length of the polypeptide chains. Affinity chromatography using lectins covalently linked to Agarose appears to be a useful tool for separation of glycopeptides. The advantage of lectins is that they bind to specific monosaccharides, can discriminate between stereoisomers, and may give information about the type and position of substitution (1). The present study demonstrates the use of agarose-linked concanavalin A in isolating glycopeptides from an immunoglobulin A. MATERIALS
AND METHODS
The IgA, (Kni) myeloma protein was isolated from the serum of a patient with multiple myeloma by ammonium sulfate precipitation and gel 389 Copyright 0 1976 by Academic Pre\\. Inc. 411right\ of reproduction in any form reerved.
390
TOMANA
ETAL.
filtration as described earlier (2). Heavy and light chains were separated by gel filtration on Sephadex G-200 in 5 M guanidine-HCl after oxidative sulfitolysis (3). Pepsin digestion. IgA protein (200 mg) was incubated with pepsin (Worthington Biochemical Corp., Freehold, N.J.) at the enzyme:substrate ratio 1:100 at 37°C for 16 hr. The acidity of the solution was maintained at pH 1.9 for the first 2 hr of incubation. At the end of incubation the acidity was usually pH 2.2. The incubation mixture was then neutralized to pH 6 with 2 N NaOH and mixed in a ratio 5: 1 with 0.5 M acetate buffer, pH 6, containing0.75 M NaCl, 3 mM CaCI,, 5 mM MgC12, and 5 mM MnCI,. The pepsin digest was applied to a Concanavalin A-Agarose (Con A) column (Glycosilex, Miles Laboratories, Inc., Kankakee, Ill.), 29 x 1.6 cm, equilibrated with 0.1 M acetate buffer, pH 6.0, containing 0.15 M NaCl, 1 mM CaCl,, 1 mM MgC&, and 1 mM MnCl,. The glycopeptides retained in the column were eluted with a 10% solution of methyl (Y-Dmannoside (Sigma Chemical Co., St. Louis, MO.). Pronase digestion. The pepsin peptides were freeze-dried, redissolved in 0.1 M Tris-WC1 buffer, pH 8.0, containing 1 mM CaCl,, and incubated with pronase (Calbiochem, La Jolla, Calif.) at 37°C for 24 hr. The enzyme: substrate ratio was 1:50. The solution was maintained at pH 8 with 20 mM NaOH. The glycopeptides were isolated by exclusion chromatography on a column (88 x 1.4 cm) of Sephadex G-50 (fine grade). Labeling of siatic acid residues. Sialic residues in IgA or peptides derived from it were labeled with tritium by a modification of the method of Van Lenten and Ashwell (4). Proteins were dissolved in 0.1 M acetate buffer, pH 6, containing 0.15 M NaCl and mixed at a ratio of 5: 1 with 0.025 M sodium metape~odate. After incubation at 0°C for 10 min, the oxidation was stopped by the addition of a molar excess of ethylene glycol, and the solution was dialyzed overnight against 0.05 M sodium phosphate buffer, pH 7.5, containing 0.15 M NaCl. The protein solution in a total volume of l- 10 ml was cooled to 0°C and was reduced by addition of l-2.5 mCi of tritiated sodium borohydride (New England Nuclear, Boston, Mass.) with a specific activity of 200 mCi/mM dissolved in 0.1 ml ice-cold 0.01 M NaOH. The reaction mixture was warmed to room temperature and reduction was allowed to continue for 60 min. Then nonradioactive NaBH, (1% w/w in respect to protein) was added, and the incubation continued for another 60 min. The excess borohydride was destroyed by acidification with HCI and removed by dialysis first against 0.15 M NaCl, then against 0.1 M acetate buffer, pH 5.9, containing 0.15 M NaCI. When small peptides were labeled, the dialysis step was replaced with gel filtration using Sephadex G-25 columns. The radioactivity of tritium was measured in a Nuclear Chicago scintillation counter. Fractions (in lo- 100 ~1 aliquots) were measured after dispersion in 10 ml Aquasol (New England Nuclear).
ISOLATION
OF GLYCOPEPTIDES
391
Immunoefectrophoresis . LKB 68OOA immunoelectrophoresis apparatus was used with 1% lonagar No. 2 in Verona1 buffer. Antisera to L-chains and H-chains were prepared and characterized as described earlier (5). Ultracentri~~gatjon . Molecular weight values were dcte~ined by sedimentation equilibrium in a Beckman Model E ultracentrifuge equipped with an electronic speed control, absorption optics, and a photoelectric scanner (6). An An-F rotor and 12-mm cells were used in all experiments. The monochromator was set at 280 nm, and the temperature was regulated at 20°C. The sample column was 3-mm high. Equilibrium was judged to be established when there was no change in the slope of the logarithm of the absorbance plotted against the square of the distance from the center of rotation during a minimum period of 4 hr. Prior to analysis, the proteins were dissolved in and dialyzed against 5 M guanidine-HCI. The partial specific volumes for glycopeptides (0.695-0.712) were calculated from their amino acids and carbohydrate compositions (7). Apparent molecular weight of glycopeptides was estimated also from their elution volume from calibrated Sephadex columns. All columns were calibrated with a mixture of ovalbumin, ribonuclease (both from Pharmacia Fine Chemicals, Piscataway, N.J.), insulin, adrenoco~icotrophic hormone (ACTH), oxytocin (hormone peptides were obtained by Dr. Roy Mundy, University of Alabama), and glutathione (Sigma Chemical Co.). Disc efectrophoresis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was used as a criterion of purity. Each peptide was subjected to electrophoresis in three gel systems: (I) 5% gel at ph 7.2 (8); (II) 15% gel at pH 8.9 (9); (III) 30% gel at pH 8.9 (9). For molecular weight determination, the polyacrylamide gels were calibrated with L-chains, H-chains, ribonuclease, and insulin by the method of Fairbanks et al. (10). Carbohydrate analyses. Fucose, mannose, and galactose were released from their glycosidic linkages by hydrolysis for 4 hr in 1 N HCI at lOO”C, converted to alditol acetates, and subjected to gas-liquid chromatog~phy using 6 ft x % in. glass columns packed either with 1% OV-225 on Varaport 30,70/80 mesh (Varian Aerograph, Walnut Creek, Calif.) or with 3% SP-2340 on Supelcoport, 1001120 mesh (Supelco, Inc., Belefonte, Pa.). From the initial 180°C the temperature was increased at the rate of 5”Cimin to 260°C. Arabinose was used as internal standard. To liberate N-acetyl glucosamine and N-acetylgalactosamine the hydrolysis was done either with 1 N HCl for 10 hr or with 6 N HCl for 2 hr at 100°C. The conditions of gas chromatography for analysis of neutral sugars (I 1) and amino hexoses (12) were described earlier. Gas-chromatographic analyses were done on an F & M Model 402 dual-column gas chromatograph with flame ionization detectors (Hewlett- Packard Corp., Avondale, Pa.) and an Infotronics Model CRS 104 electronic integrator (Infotronics, Inc., Houston, Tex.). Sialic acid was determined by the method of Warren
392
TOMANA
ET AL.
d, IO 20
30 40 50 60 FRACTION NUMBER
‘\
70
---. so
90
FIG. 1. Separation of pepsin peptides of IgA, on a 1.6 x 29 cm concanavalin A-agarose column equilibrated with 0.1 M acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM CaCI,, 1 mM MgCl*, and 1 mM MnCI,. Fraction volume 4.8 ml. Full line: optical density at 230 nm. Broken line: [3H]sialic acid radioactivity of lOGpI fractions.
(13) following 45 min.
hydrolysis of the glycoprotein
in 0.08 M H,SO, at 80°C for
Amino acid analyses. Samples were hydrolyzed for 24 hr in 6 N HCl at 105°C. Amino acid analyses were done on a Durrum Model DSOO amino acid analyzer with a single high-pressure cation-exchange column.
RESULTS Sedimentation analyses showed that the IgA, protein (Kni) was a monomer with a sedimentation constant of 6.2 s. Assuming a molecular weight of 158,000, the oligosaccharides of this glycoprotein, all of which were located on the alpha chains, consisted of 2-3 residues of fucose, 13- 14 of mannose, 16 of galactose, 14- 15 of N-acetyl glucosamine, 9- 10 of N-acetyl galactosamine, and 4-5 of sialic acid. The peptides resulting from pepsin hydrolysis of the tritiated IgA, protein were separated into two fractions on the concanavalin A-agarose column as shown in Fig. 1. Fraction A contained peptides that did not interact with the lectin. It represented about 90% of the starting material. Fraction B contained peptides that were retained on the column and were eluted with 10% methyl a--D-mannoside (about 10% of the hydrolysate). Both fractions were freeze-dried, redissolved in a minimal
ISOLATION
OF GLYCOPEPTIDES
393
FIG. 2. Gel filtration of fractions A (upper graph) and B (lower graph) from concanavalin A-agarose column. Column (2.5 x 96 cm): Sephadex G-SO (medium grade) in 0.1 M acetic acid. Fraction volume 4.8 ml. Full line: optical density at 280 nm (upper graph) and 230 nm (lower graph). Broken line: [3H]sialic acid radioactivity of lOO-~1 fractions.
volume of 0.1 M acetic acid, desalted by dialysis or by gel filtration on a Sephadex G-25 column, and analyzed for carbohydrate content. Both fractions contained fucose, mannose, galactose, N-acetyl glucosamine, Nacetyl galactosamine, and sialic acid. Fraction A contained a lower content of carbohydrate/mg of protein than fraction B. Fractions A and B were subjected to elution chromatography on a Sephadex G-50 column. Five fractions were obtained from the IgA, protein. Three, designated Al, AZ, and A, were isolated from fraction A, and two, designated B1 and Bz, were isolated from fraction B as shown in Fig. 2. Examination by polyacrylamide gel electrophoresis indicated that fractions A, and Bz were homogeneous. Fraction A, was homogeneous only when prepared from IgA that was not subjected to radiolabeling. Fraction B, contained more than one peptide. Fraction A, contained low molecular weight radioactive compounds that were not completely re-
394
TOMANA
ETAL.
TABLE
1
CARBOHYDRATE COMPOSITION~ANDAPPARENT MOLECULAR WEIGHT OF PEPSIN PEPTIDESFROM IgAl PROTEIN ISOLATEDBY CONCANAVALIN A-AGAROSE COLUMN AND GEL FILTRATION
Protein
k.41
Fraction
FUCO%
Aid
0
A* BI BZ
0.42 0.20 0.47
Galactose
Mannose
N-Acetyl glucD samine
7.55 I .59 4.7 0.95
0 2.34 2.80 2.60
0 3.26 I.59 3.01
N-Acetyl galactosamine 8.89 0 2.10 0
Sialic acid
HOlllOgeneityb
Apparent moleculalf weight (dakons)
o.s7 0.37 0.35 0.28
+ + +
14.w (UC) 4,7w {CCL) l2,OOO (DG)” 4.800 (Cob)
a Concentrations expressed in moles/mole of glycopeptide. ’ (+) Homogeneous, (-) heterogeneous peptide. c (U.C.) Determined by sedimentation equilibrium; (Cal.) by Sephadex chromatography: (D.G.) by polyacryiamide electrophoresis. d Molecular weights and carbohydrate compositions were determined on preparations that were not radiolabeled. c Apparent molecular weight of the main component.
gel
movedin previousdialysisanda traceof free sialic acidthat was apparently cleavedfrom the immunoglobulin duringpepsinhydrolysis. The molecular weightsand the carbohydratecompositions of fractions Al, AZ, B,, and B2 are shown in Table I. Tritium-labeled fraction A, was subjectedto proteolytic treatment with pronase. Two peptides with identical carbohydratecompositions were resolved by gel filtration on SephadexG-50. The larger peptide had an apparent molecular weight approximately twice as great as that of the smaller one, indicating that these two glycopeptidesrepresenteddimeric and monomeric forms of the sameglycopeptide. The partial cleavageof interchain disulfide bonds was apparently causedby oxidation or reduction during the processof radiolabeling. Fraction B, was heterogeneousas judged from polyacrylamide gel electrophoresis. The presenceof both N-acetyl galactosamineand Nacetyl glucosamine indicated that it contained at least two different oligosaccharides.Fraction B, was subjectedto further proteolytic treatment with pronase. Two glycopeptides were resolved by gel filtration. The larger of these (B,J contained galactose,N-acetyl galactosamine, sialic acid, and trace amountsof mannoseand N-acetyl glucosamine.The smaller one (BIB) contained mannose,galactose,N-acetyl glucosamine, and trace amounts of N-acetyl galactosamine,fucose, and sialic acid. Largeamountsof proline andcysteinein glycopeptideB,,and thepresence of galactosamineand galactoseindicated that it was identical with glycopeptideA,. GlycopeptidesA, and B2 had almost identical carbohydratecompositions but interacted differently with Con A. To determine whether the interaction of glycopeptidesA, and B, with Con A were specific, purified tritium-labeled glycopeptide A2 was added to a nonlabeled pepsin
ISOLATION
OF GLYCOPEPTIDES
395
FIG. 3. Separation of the mixture of pepsin peptides of IgA, and tritium-labeled glycopeptide A2 (upper graph) or B, (lower graph) on concanavalin A-agarose column. IgA, protein (40 mg) was hydrolyzed with pepsin under conditions described in Material and Methods. After neutralization, 4 mg of tritium-labeled glycopeptide AZ or B, was added. Column (1.6 X 29 cm) equilibrated with 0.1 M acetate buffer, pH 6, containing 0.15 M NaCl, 1 mM CaCl,, 1 mM MgCl,, and 1 mM MnCl,. Fraction volume 4.8 ml. Full line: optical density at 230 nm. Broken line: 3H activity of 100 /*I fractions.
hydrolysate of IgA,, and the mixture was reapplied to the concanavalin A column. As shown in Fig. 3a, practically all of the tritium activity again appeared in fraction A. When tritium-labeled glycopeptide Bz was added to a nonlabeled pepsin hydrolysate of IgA,, most of the radioactivity appeared in fraction B, as shown in Fig. 3b. Both glycopeptides A2 and B, were further subjected to proteolytic treatment with pronase. One glycopeptide was recovered from A2 and one from B,. The amino acid composition of pronase peptides A,, AZ, Blu, BIP, and B2 is shown in Table 2. The carbohydrate compositions of the IgA, protein and the alpha chains prepared from it are compared in Table 3 with the sum of the carbohydrate found in the four glycopeptides. The calculation was made on the assumption that glycopeptide A, was a dimer and that glycopeptide B, contained the same galactosamine containing oligosaccharide found in A, in addition to a second oligosaccharide that was responsible for retention of this glycopeptide in the Con A column.
396
TOMANA TABLE
ET AL. 2
AMINO ACID COMPOSITION OF IgA GLYCOPEFTIDES AFTER P~ONASE DIGESTION Moles/100 moles Amino Acid Amino acid
A,
A2
ha
Bli?
B2
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Leucine Histidine Lysine Arginine Cysteine
19.4 16.7 44.7 7.5 11.7*
25.7 8.2 12.4 4.0 6.1 5.9 5.3 19.6 2.7 4.9 3.5 1.7 -
5.3 17.6 20.9 2.5 40.0 1.9 1.2 5.4 2.2 3.v
23.8 10.7 14.5 7.9 4.9 7.5 9.2 2.5 12.1 1.0 1.6 4.2
36.4 9.1 16.3 7.2 2.8 4.0 5.9 7.9 4.6 2.7 3.1 -
LIThe presence of glucosamine in B,, and galactosamine in BIB indicate that these two glycopeptides were not completely separated. b Calculated as cysteic acid after performic acid oxidation. c Not oxidized with performic acid.
DISCUSSION Four glycopeptides were isolated from pepsin digests of IgA, using affinity chromatography on a concanavalin-agarose column and gel filtration. Three of these were homogeneous when examined by disc gel electrophoresis in the presence of SDS. The observation that glycopeptides A, and Bz had almost identical carbohydrate compositions suggested initially that the quantity of peptides applied to the Concanavalin A column exceeded its capacity, or that the interaction of Con A with glycopeptides of the IgA, protein were nonspecific. Similar results, however, were obtained when the quantity of pepsin hydrolysate applied to the column was reduced by a factor of 10 or 100. This observation indicated that the Con A column had not been overloaded. That the interaction of Con A with glycopeptides from the IgA protein was indeed specific was verified by the observation that the tritiated glycopeptides that were initially retained on the Con A column were again retained when they were reapplied to the column after mixing with an unlabeled pepsin digest of the whole IgA molecule. Likewise, tritiated glycopeptides that were not initially retained on the Con A column failed to be retained when mixed with an unlabeled pepsin digest of the whole IgA molecule and reapplied to the column. Results of amino acid analyses of glycopeptides A, and B, (Table 2) further support the sug-
ISOLATION
TABLE CARBOHYDRATE
397
OF GLYCOPEPTIDES
COMPOSITION OF IgA, AND PEPSIN GLYCOPEPTIDES
3 (Kni)
IMMUNOGLOBULIN. (RESIDUES/MOLE)
N-Acetyl ghlcosamine
H-CHAIN. N-Acetyt gatactosamine
Siaiic acid
Fucose
Mannose
Galactose
2.2
12.5
16.0
14.3
9.5
4.6
H-Chain (calculated from bYV H-Chain (analysis) (MW: 56,300)
1.1
6.3
8.0
7.2
4.8
2.3
1.0
6.0
8.0
7.6
4.1
2.3
Glycopeptides”
0.9
7.7
9.3
7.9
4.5
0.93
w, (MW: 158,000)
(1 Calculated on the assumption that glycopeptide A, was a dimer and that glycopeptide B, contained the same oligosaccharide present in A, in addition to a glucosamine containing oligosaccharide.
gestion that IgA, (Km) contains two glycopeptides of similar carbohydrate composition but with different internal structures that were derived from different regions of the alpha chains. It has been shown that Con A combines with glycopeptides that contain a-D-mannopyranoside or cr-D-glucopyranoside residues with unmodified hydroxyl groups at the C-3, C-4 and C-6 positions (14). Inhibition studies indicate that ~-acetylglucosamine can also bind Con A although less effectively than mannose (15). Kornfeld and Ferris (16) have shown that relatively small changes in the structure of the oligosaccharide can strongly influence the binding capacity of Con A. Thus, for example, the cleavage of one terminal galactose residue from the branched oligosaccharide of IgE resulted in tenfold increase in the binding capacity of the ~y~opeptide with Con A. A glycopeptide has been isolated from IgA that contains three mannose residues located in the core of the oligosaccharide unit. Two mannose residues are linked to the third one by al-3 or al-6 glycosidic linkages. The hydroxyl group on C-2 in two of the mannose residues is involved in a glycosidic linkage with N-acetyl glucosamine (19). The interaction of glycopeptide Bz with Con A is consistent with such a structure. Glycopeptides that exhibit similar interactions with Con A were found also in IgA,, secretory component, J-chain, and in IgG (unpublished data). In glycopeptide A,, the mannose residues appear to be either panomers, or some of their hydroxyl groups that are critical for interactions with Con A are involved in glycosidic linkages with neighboring
398
TOMANA
ET
AL.
monosaccharides. The possibility, however, cannot be excluded that AZ, that has a slightly higher content of galactose, represents a more completed carbohydrate unit that interacts less strongly with Con A than the uncompleted unit in glycopeptide Bz. Glycopeptide A, which contained neither mannose nor glucosamine predictably did not interact with Con A. This glycopeptide appeared to be a dimer, composed of fragments of two adjacent alpha chains. The carbohydrate and amino acid analyses of the monomer that was isolated following pronase digestion indicated that this glycopeptide was identical to the hinge glycopeptide described earlier (18). Fraction B, was heterogeneous and contained two oligosaccharides. Pronase digestion followed by gel filtration on Sephadex G-25 permitted the partial separation of two glycopeptides, one of which (B,,J was identical to A,, the hinge peptide (18). The other peptide (BID) did not contain fucose or sialic acid, and was similar to the leucine reach glycopeptide which Putnamet al. (19) found near the hinge region of an IgA, (19) protein. The results suggest that these two oligosaccharides (A, and B,,) are located in close proximity on the heavy chain. Apparently some o-chains were cleaved by pepsin between these two oligosaccharides releasing glycopeptide A, (hinge peptide) which was not bound to Con A and the fucose free peptide that was bound to Con A. In other molecules, the a! chain was not cleaved between these two oligosaccharides releasing a glycopeptide that contained both oligosaccharides. This explanation was supported by the results of polyacrylamide gel electrophoresis of fraction B, that showed multiple bands. The use of Con A as well as other lectin columns thus appears to present a useful tool for the isolation, purification, and characterization of glycopeptides. Con A allows recovery of high yields of glycopeptides, and it appears to possess a high degree of specificity. Essentially all of the carbohydrate found in the IgA, protein was accounted for in the four glycopeptides isolated by affinity chromatography on Con A and gel filtration. When similar Con A preparations purchased from alternate commercial sources were used, difficulty was sometimes encountered in obtaining quantitative recovery of the fraction that was retained on the column. ACKNOWLEDGMENTS The authors wish to express their thanks to Mrs. Rose Kulhavy for preparation of human serum IgA used in this study. These studies were supported by grants AM-03555 and AI-10854 from the National Institutes of Health, Bethesda, Maryland and the John A. Hartford Foundation, New York.
REFERENCES 1. Sharon, N.. and Lis, H. (1972) Science 177, 949. 2. Tomana, M., Niedermeier, W., Mestecky, J., and Hammack, W. (1972) chemistry
9, 933.
Immuno-
ISOLATION
OF GLYCOPEPTIDES
399
3. Edelman, G. M., and Marchalonis, J. J. (1967) in Methods in Immunology and Immunochemistry (Williams, A. C.. and Chase, M. W., eds.), Vol. 1, p. 405, Academic Press, New York. 4. Van Lenten, L., and Ashwell, G. (1971) J. Biol. Chem. 246, 1889. 5. Mestecky, J.. Kulhavy, R., and Kraus, F. W, (1972) J. Immunol. 108, 738. 6. Schrohenloher, R. E., Mestecky. J.. and Stanton, T. H. (1973) Biochim. Biophys. Acta 295, 576. 7. Cohn, E. J., and Edsall, J. T. (1943) in Proteins, Amino Acids, and Peptides (Cohn, E. J., and Edsall, J. T., eds.), p. 370, Hafner Publishing Co., New York. 8. Maizel. J. V., Jr. (1966) Science 151, 988. 9. Maurer, H. R. (1971) in Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, (de Gruyter, W.. ed.), 2nd ed., p. 44, Berlin New York. 10. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10, 2606. 11. Niedermeier, W. (1971) Anal. Biochem. 40, 465. 12. Neidermeier, W., and Tomana, M. (1974) Anal. Biochem. 57, 363. 13. Warren, L. J. (1959)J. Biol. Chem. 234, 1971. 14. Goldstein, I. J., Reichert, C. M., Misaki, A., and Gorin. A. J. (1973) Biochem. Biophys. Acta 317, 500. 15. Iyer, R. N., and Goldstein, I. J. (1973) Immunochemistry 10, 313. 16. Komfeld, R.. and Ferris, C. (1975)J. Biol. Chem. 250, 2614. 17. Baenziger, J., and Komfeld, S. (1974) J. Biol. Chem. 249, 7260. 18. Frangione, B., and Wolfenstein-Todel, C. (1972)Proc. Nat. Acad. Sci. USA 69,3673. 19. Putnam, F. W., Low, T., Liu, V., Huser, H., Raff, E., Wong, F. C., and Clamp, J. R. (1973) in Advances in Experimental Medicine and Biology (Mestecky, J., and Lawton, A. R., III, eds.). Vol. 45, pp. 177- 189, Plenum Press, New York/London.