[46] Eel serum anti-human blood-group H(O) Protein

[46]

EEL ANTI-H(O) GLOBULIN

383

specificity of the hemagglutinin tends to limit its usefulness in this respect. More useful may be the possibility to separate polysaccharides, glycoproteins, oligosaccharides or even cells with the aid of the hemagglutinin, made insoluble through polyeondensation or otherwise.

[46]

Eel S e r u m A n t i - H u m a n B l o o d - G r o u p H ( O ) P r o t e i n 1 B y PARIMAL R . DESAI a n d GEORG F. SPRINGER

Eel anti-human blood-group H(O) protein, a potent hemagglutinin of human blood-group 0 erythrocytes,2 is a 7 S globulin?,4 Originally it was thought that the combining sites of the eel antibody for bloodgroup H(O) specific structures are complementary to a-L-fucopyranose (6-deoxy-a-L-galactopyranose),~,6 but we found that monosaccharides with both L- and D-galactose configuration are complementary to these sites and function as inhibitory haptens provided they possess at least one methyl group on either C-3 or C-5. 7-1° Numerous other hexoses, pentoses and their derivatives are inactive. 7 The eel anti H(O) protein specifically precipitates not only with blood-group H(O) active macromolecules of human, animal, and plant origin, but surprisingly also with 3-O-methyl-D-fucose (D-digitalose) and 3-O-methyl-D-galactose.8,9,11 The minimum combining structure which shows inhibitory activity with the eel anti-H(O) antibody is smaller than a monosaccharide. It consists of a methyl substituent attached equatorially to a pyranose ring, an ether oxygen adjoining this methyl group, and an axial, oxygencarrying substituent cis to the methyl group on a contiguous C atom2 ,1° ' This investigation was supported by National Science Foundation Grant GB-8378. The Department is maintained by the Susan Rebecca Stone Fund for Immunochemistry Research. " S. Miyazaki, Nagasaki Idai Hoiqaku Gyoho 2, 542 (1930). A. Bezkorovainy, G. F. Springer, and P. R. Desai, Biochemistry 10, 3761 (1971). 4 G. F. Springer and P. R. Desai, Vox Sang. 18, 551 (1970). W. M. Watkins and W. T. J. Morgan, Nature (London) 169, 825 (1952). e R. Kuhn and H. G. Osman, Hoppe-Seyler's Z. Physiol. Chem. 303, 1 (1956). G. F. Springer and P. Williamson, Biochem. J. 85, 282 (1962). 8 G. F. Springer, P. R. Desai, and B. Kolecki, Biochemistry 3, 1076 (1964). 9 G. F. Springer, T. Takahashi, P. R. Desai, and B. J. Kolecki, Biochemistry 4, 2099

(1965). lop. R. Desai and G. F. Springer, Proc. lOth Congr. Int. Soc. Blood Trans]us., Stockholm, p. 500 (1965). 1~G. F. Springer and P. R. Desai, Biochemistry 10, 3749 (1971).

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PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS

[46]

While the overall molecular contours of the presumed combining sites of the inhibitors and the precipitinogens are closely similar, there are some additional specific requirements for monosaccharides to function as precipitinogens. They apparently consist, within this contour, of three vicinal oxygens protruding from a C1 pyranose ring. The oxygen at C-3 must carry an apolar group, and the two oxygens flanking this group must be capable of hydrogen bonding. One of these latter oxygens must be equatorial and trans to the oxygen at C-3 and the other axial and cis. Additional apolar groups are not compatible with the precipitating activity of the pyranose structure; they transform it into either an inhibitory hapten or an inactive compound. 11 Isolation Procedure

Principle. The isolation of the eel anti-H(O) serum protein is based on its ready and specific precipitability with the 2 monosaccharides mentioned above and the ease of dissociation of the precipitated monosaecharide-protein complex upon dialysis with simultaneous removal of the preeipitinogen. Procedure. Sera, 3-8 ml per fish, are obtained from live eels (Anguilla rostrata) weighing :>1.5 lb each, after anesthesis with urethane/These eels are caught in fresh or brackish water. We found that on the eastern seaboard of North America their serum has maximum anti-human bloodgroup H ( 0 ) protein content between September and December. The precipitinogen D-digitalose may be obtained from the cardiac glycosides strospesid or panstrosid as well as from the antibiotic chartreusin by repeated hydrolysis of 10% solutions with Kiliani's mixture 12 followed where necessary by preparative paper chromatography/,8 The anti-human blood-group H(O) antibody is precipitated from the eel serum with an equivalent amount of D-digitalose (2-6 ~moles per 0.5 ml of serum) as described under Assay Methods. The protein content of the precipitates of two tubes from each experiment is determined colorimetrically with Folin-Ciocalteau reagent 13 and used in calculation of percent recovery of the isolated anti-H(O) protein. The washed precipitates are either worked up immediately or stored at - 2 0 ° in the freeze-dried state. The precipitates are suspended at approximately double their original protein concentration in buffered saline (0.1M NaCI, 0.05 M P O ( 8, pH 7.3) and dialyzed on a mechanical shaker at 4 ° against 20 volumes of buffered saline which is gradually replaced by deionized, distilled water until no more Cl- is demonstrable with AgNO3. This stepwise electrolyte decrease minimizes antibody precipitation. In~l:I. Kiliani, Bet. Deut. Chem. Ges. 63, 2866 (1930). ~ M. Heidelberger and C. F. C. MacPherson, Science 97, 405 (1943); 98, 63 (1943).

[46]

~.EL

ANTI-H(O) GLOBULIN

385

soluble m a t t e r amounting on the average to 3.8% of the total recovered is removed by centrifugation at 1500 g and 4 ° for 1 hour. Both the soluble and the insoluble fractions are freeze-dried and then dried to constant weight at 22-25 ° over P205 at 10 -1 m m Hg. The average recovery of the soluble eel anti-blood-group H ( O ) protein, by weight, is about 94% of t h a t expected from the colorimetric analyses of the precipitates. Prior to chemical analyses the antibody is electrodialyzed at 4 ° through a dialysis membrane at 200V 9 and dried as described above.

Assay Methods Hemagglutination and Hemagglutination Inhibition Assays. 14-16 These are carried out at 22-25 ° in 10 × 75 m m tubes with buffered saline as diluent and solvent. The voulme of all reagents in a test is kept constant; either 0.05 or 0.02 ml is used throughout. Twofold serial dilutions of the test material are made using a different 0.1 ml serological pipette for each dilution. I n hemagglutination assays, 0.5% suspension of human 0 erythrocytes is added to the titrated solutions of the eel a n t i - H ( O ) serum; the mixtures are then shaken and allowed to stand for 90 minutes before reading. In hemagglutination inhibition assays, prior to addition of red cells 4 minimum hemagglutinating doses of eel anti-H (0) serum are added to the titrated solutions of the inhibitor; the mixtures are shaken, then incubated for 2 hours. In both assays agglutination is read microscopically by at least two individuals, and the materials are tested at least twice. I n hemagglutination assays an appropriately diluted suspension of h u m a n 0 erythrocytes serves as negative control; in hemagglutination inhibition assays, an additional positive control consisting of an eel serum standard diluted to 4 hemagglutinating doses and titrated in 2-fold serial dilutions is used. L-Fucose is included as a standard in all inhibition assays. Precipitin and Precipitation Inhibition Tests. These quantitative procedures, based on those of Heidelberger and Kendall, 17 are used with some modificationsS,9; 0.5-ml portions of all reagents are pipetted into graduated, glass-stoppered, 10-ml centrifuge tubes using Ostwald-Folin pipettes. The samples are incubated for 30 minutes in an ice bath and then for 7-10 days at 4 ° with daily agitation. I n inhibition studies haptens are added in 0.5-ml volume to 0.5 ml ~' G. F. Springer, R. E. Horton, and M. Forbes, J. Exp. Med. 110, 221 (1959). ~sG. F. Springer and R. E. Horton, J. Clin. Invest. 48, 1280 (1969). ~ G. F. Springer, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. IV. Academic Press, New York, in press, 1972. 1~E. A. Kabat, in "Kabat and Mayer's Experimental Immunochemistry," 2rid ed., pp. 22-96. Thomas, Springfield, Illinois, 1961.

386

PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS

[46]

of undiluted serum and incubated in an ice bath for 30 minutes; thereafter 0.5 ml of precipitinogen solution of concentration corresponding to that at the beginning of equivalence is added, and the procedure is continued as above. Inhibition is calculated as the percent decrease of prorein precipitated in presence of the inhibitory hapten as compared to that precipitated in its absence. Negative controls in all assays consist of eel serum or isolated antibody alone and antigen or hapten alone, each adjusted to appropriate volume. Precipitates obtained with antigens are recovered by centrifugation and washed twice with 1.5 ml of ice-cold buffered saline. Because of their greater solubility, precipitates obtained with monosaccharides are washed only once. Washed precipitates are dissolved in 2.5 ml of water containing 0.01 meq of NaOH, and their protein content is determined colorimetrically 13 with four different concentrations of human ),-globulin as standard. The extinction given by the eel antibody is the same as that given by the human ),-globulin between 15 and 200 ~g of protein with a deviation of _ 1% below and _+3% above 100 ~g protein. 11 Properties Appearance, Solubility, and Purity. 11 The isolated eel anti-bloodgroup H(O) protein is a white, fluffy powder that gives a clear or slightly opalescent solution up to at least 0.2% in buffered saline (pH 7.3) and up to at least 0.1% in buffers of pH 5.5-9.0. The average wet ash content of this antibody is 3.7% before and ~ 1 % after electrodialysis; maximum weight loss on drying to constant weight at 80 ° is 8%. Stability. 1~ Eel antibody solutions in buffered saline or deionized water are stable for at least 1 month at 4 °. Repeated freezing and thawing severely damages the antibody. A 12-day incubation of 0.1% solutions of eel antibody in buffers of pH 5.5-9.0 at 4 °, followed by adjustment of the solution pH to 7.3 by dialysis, does not alter the extent of their human 0 erythrocyte agglutinating activity. Immunochemical Reactivity and Specificity. 4,1~ The hemagglutinating activity of thc isolated antibody before and after electrodialysis is the same as that of the original eel serum. The isolated eel antibody also gives typical precipitin curves, in the same proportions as those when it is still in serum, with all the blood-group H(O) active macromolecules and the precipitating monosaccharides. Blood-group H(O) active ovarian cyst glycoprotein, Sassa]ras polysaccharide, D-digitalose and 3-O-methyln-galactose each precipitate in the equivalence zonc from 68.7 to 95.9% of the total antibody protein; the average precipitation from different pools is about 75%. An average of 2.3% protein is found in the washes while about 23% remains in the supernatants, whose agglutinin titers parallel

[46]

EEI, ANTI-H(O) GLOBULIN

387

their ~)rotein content. After adjustment of the protein content of dialyzed supernatants of monosaccharide precipitin tests to its preprecipitation concentration, the same proportion of protein is precipitable by the precipitinogens as from the original antibody solution. The results are the same when whole eel serum is used. At equivalence the precipitation of eel antibody with I)-digitalose is optimal between pH 6.70 and 7.15. It should be noted that > 9 8 % of the D-digitalose remains in the supernatant in all areas of the precipitin curve. Immunochemical homogeneity of the isolated eel antibody is indicated by the shape of the precipitin curves and by the single symmetric arc obtained in immunoelectrophoresis using rabbit anti-isolated eel antiH(O) protein. Also, agar gel diffusion studies on the eel anti-H(O) protein with the H(O)-specific human ovarian cyst glycoprotein, Taxus, and Sassa]ras antigens as well as with the blood-group H (O) -specific antigen from E. coli O128is show only one sharp band; all these bands fuse completely with one another. Removal, in the equivalence area, of >85% of the eel anti-H (O) antibody by monosaccharide precipitation and inhibition of this monosaccharide precipitation to >80% by H(O) specific haptens such as L-fucose, methyl a-L-fucopyranoside and 2,3-di-O-methyl-n-galactose but not by the H(O) inactive 3-O-methyl-D-glucose attest to the specificity of the reaction. Chemical Characteristics. 11 The isolated electrodialyzed eel anti-H (0) protein contains ca. 15.7% N (corrected). Asp, Gly, Glu, Ala, Ser, and Thr are the predominant amino acids, and there is a scarcity of Met, Trp, and Phe. No significant amounts of carbohydrate are demonstrable except GlcN, of which 0.39% (3 moles per mole of antibody) is found. End-group analyses show approximately equal quantities of Set and Ala at the NH2-termini, and Ser and Gly in the same ratio at the COOHtermini and suggest the presence of two types of polypeptide chains in the eel anti-H(O) protein. Physicochemical Characteristics2 ,~9 The isolated eel anti-H(O) protein is usually homogeneous by ultracentrifugal and electrophoretic criteria. Ultracentrifugal analyses of some preparations show a more rapidly migrating component, apparently a multimer of the eel antibody molecules, which may account for up to 10% of the total material; it disappears in presence of 8 M urea. The eel antibody migrates like a human a2-globulin at pH 8.6 and its isoelectric point is at pH 5.25 ___0.05. Its s o20.w is 7.2 S, D °20,w is 5.0 × 10-7 cm~/sec. Its partial specific 5, G. F. Springer, P. Williamson, and W. C. Brandes, J. Exp. Med. 113, 1077 (1961). ~B. Jirgensons, G. F. Springer, and P. R. Desai, Comp. Biochem. Physiol. 34, 721 (1970).

388

PURIFICATION OF CARBOHYDRATE-BINDING PROTEINS

[47]

volume is 0.705 ml/g and its intrinsic vieosity is 3.4 ml/g. Its molecular weight is 123,000 and its fl-value is 2.0 × 10-8 suggesting a nearly spherical shape. Its ,~sAI~nm is 12.696 in water and its [a][~ is - 7 7 ° (C 0.5, water, 1 dm). The anti-H(O) antibody preparations from individual eels give closely similar circular dichroism spectra between 185 and 310 nm which differ significantly from those of individual human 7 S immunoglobulins. Suceinylation and/or reduetion-alkylation of the eel anti-H(O) antibody followed by sedimentation and diffusion studies as well as disc electrophoresis in polyacrylamide-sodium dodeeyl sulfate indicate that this protein consists of three physically bonded subunits of apparently identical molecular weight of 40,000 each of which in turn consists of four polypeptide chains of identical size; these chains, whose molecular weight is about 10,000, are joined by disulfide linkages.

[47] H o m o g e n e o u s M o u s e I m m u n o g l o b u l i n s ( M y e l o m a Proteins) That Bind Carbohydrates

By MICHA~ POTTER and C. P. J. GLAUDEMANS Plasma cell tumors are derived from highly specialized cells that are restricted to making single molecular species of immunoglobulin {homogeneous immunoglobulin, myeloma proteins). These tumors can be induced in strain BALB/c mice by the intraperitoneal injection of mineral oil (0.5 ml given 3 times when the mouse is 2, 4, 6 months of age). Although these homogeneous immunoglobulins are synthesized and secreted by tumors, a modest percentage (ca. 5%) of them can be shown to bind antigens. This is usually demonstrated by a random screening process in which a myeloma protein is reacted with a battery of test antigens. The antigens are macromolecules that contain multiple haptenic repeating units, e.g., derivatized proteins, or polysaccharides. The multivalent IgA and IgM myeloma proteins can be tested for precipitating activity to polyvalent antigens in agar-gel diffusion tests. This permits the rapid screening of large numbers of samples. When a myeloma protein-antigen system is identified, it can be further studied to establish the chemistry of the hapten, specificity of related haptens, and the binding affinity of the myeloma protein for the hapten (KA). When large numbers of mouse myeloma proteins are screened (i.e., several hundred) often more than one protein can be found that binds the same antigen. Immunochemieal characterization of myeloma proteins that bind the same antigen usually