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Concanavalin A Reaction with Human Normal Immunoglobulin G
ready described, except that 10, 30, or
60 mg of concanavalin A were added
and Myeloma Immunoglobulin G
per milliliter of each serum.
Abstract. Concanavalin A precipitated less than 5 percent of immunoglobulin G
from human serum. It reacted with all of 42 myeloma serums of the immunoglobulin G type tested, but no more than approximately 50 percent of the total
myeloma protein was ever precipitated. The fact that not all of the protein was
precipitated and that the amounts precipitated varied from serum to serum may
be interpreted as demonstrating heterogeneity of the carbohydrate in these myeloma proteins. Other glycoproteins precipitated by concanavalin A were iden-
fractions were analyzed electrophoret-
tified, and subsequently separated from concanavalin A by chromatography.
Concanavalin A, the globulin from
jack-bean meal, reacts with serum gly-
coproteins (7, 2}, presumably because
of its specificity for their nonreducing
mannosyl- and N-acetylglucosaminy]
end groups (3). Harris and Robson
demonstrated that crude jack-bean meal
extracts precipitated oa,-macroglobulin,
yi-macroglobulin (IgM), @-lipoprotein,
and ceruloplasmin; as judged by elec-
trophoresis haptoglobins were also precipitated (2). Since no reaction with
7S y-globulin (IgG) was noted, I attempted to use concanavalin A to sep-
arate IgM from IgG. I now present
data demonstrating that, depending on
the serum, variable amounts of IgG
from normal and myeloma serums
precipitated with concanavalin A.
Concanavalin A was prepared by a
modification of Agrawal and Goldstein’s procedure (4). The crude saline
extract of commercial, defatted jack-
bean meal wasclarified by centrifugation at 25,000g (0°C) for 1 hour. After
removal of the lipid layer, the supernatant was applied directly to a Sephadex G-75 column. Subsequent procedures were as described (4). The purified product was dialyzed against 0.14
phosphate buffer, pH 6.2, and kept in
small vials at —70°C to prevent the
canavalin A with glycoproteins. Components in the dissociated precipitate,
confirmed by immunodiffusion tests
with specific antiserums or by specific
biological activities, were IgM, IgA, aj1-
antitrypsin, qo-macroglobulin, transferrin, 8.-lipoprotein, f2-glycoprotein, haptoglobin, ceruloplasmin, group-specific
components, the serum inhibitor of C’l
esterase (5) and all the components of
complement.
Although, by immunoelectrophoresis,
no IgG was observed in the dissociated
precipitate, both immunodiffusion and
Gm typing (6) showed that a small
serum, the quantity of IgG precipitated by concanavalin A varied from
approximately 50 percent to mere traces
of the total present.
Immunoelectro-
phoretic patterns of the most reactive,
an IgG myeloma serum of heavy-chain
subtype y3, are shown in Fig. 1. Our
limited data do not yet permit correla-
tion of heavy-chain subtype with reactivity towards concanavalin A. In all
reactive serums, the dissociated myeloma protein showed the same elec-
trophoretic mobility as the myeloma
protein of the original serum. Increasing the ratio of concanavalin to
serum above 30 mg/ml did notsignifi-
cantly increase the amount of myeloma
protein precipitated. If one accepts the
theory that monoclonal myeloma pro-
tein in an individual serum is homo-
geneous
with
respect to
amino
acid
sequence, then all molecules of mye-
wliin carbohydrate (7) or (ii) differences in availability of IgG carbohydrate for reaction with concanavalian A
as a result of conformational differences
in IgG molecules with different amino
acid sequences. Since homogeneous IgG
of both reactive and nonreactive types
would facilitate testing of these hypo-
theses, 42 IgG myeloma serums were
examined for reactivity with concanavalin A (8). Precipitation, washing,
and dissociation were performedas al-
conformational possibilities in equilibrium.
Carbohydrate
chains
attached
that the carbohydrate chains are identi-
cal and are attached at the same position in the peptide chain. The partial
precipitation of IgG myeloma proteins
by concanavalin A, demonstrates existence of some type of heterogeneity of
each IgG myeloma carbohydrate. The
data further suggest that a given se-
quence of amino acids either does not
uniquely determine the structure, or
does not uniquely determine the location, of the carbohydrate attached to
the peptide chain. These findings ex-
tend, by entirely different techniques,
those reported of carbohydrate heterogeneity in an IgA myeloma globulin
(7).
,
Despite the observation that small
amounts of IgG are precipitated by con-
canavalin A, the preparation of IgM is
cubation at 4°C prior to centrifugation
gave only slightly increased yields of
glycoproteins. The washed concanaval-
8 DECEMBER 1967
ically. Depending on the IgG myeloma
to these molecules should show similar
reactivity with concanavalin A provided
These data may beattributed to (i) the
known heterogeneity of immunoglob-
fuged at 4°C and then washed three
times with cold saline. Overnight in-
coside to prevent recombination of con-
nonprecipitable
loma protein have the same range of
7 to 8 mg of concanavalin A were
added to 1 ml of serum, and the mixture was incubated at 37°C for 1 hour.
The flocculent precipitate was centri-
terial were performed in agarose gels
which contained 0.25M methyl e-D-glu-
and
amount (<5 percent) of IgG had precipitated with the concanavalin A.
precipitation noted on storage at 4°C.
To precipitate serum glycoproteins,
in-glycoprotein complexes were dissociated at 37°C with 1 ml of saline containing 0.5M methyl! a-D-glucoside.
Immunoelectrophoretic and immunodiffusion tests on the dissociated ma-
Precipitable
Fig. 1. Immunoelectrophoresis of precipitable (top) and nonprecipitable (bottom)
fractions obtained from reaction of concanavalin A with serum of a patient with
IgG myeloma of heavy-chain subtype ys.
Both fractions are diluted 1 to 3 with respect to original serum. The antiserum is
horse antiserum to human IgG.
greatly facilitated by a preliminary
precipitation of the serum with concanavalin A. The chromatographic behavior of concanavalin A makes it readily
separable from IgM and from most
other serum glycoproteins. When the
dissociated mixture of concanavalian A
and glycoproteins is applied to diethylaminoethyl Sephadex at ionic strength
above 0.02, in tris buffer at pH 8.0. the
coacanavalian A is not bound. A com1325