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BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the preparation and use of certain
insoluble polysaccharide derivatives comprising covalently linked
gangliosides, and, more especially, relates to the use of such derivatives
for the extraction and purification of cholera toxin by affinity
chromatography techniques.
Cross Reference to Related Applications
[1] Copending application, Ser. No. 475,305, filed May 31, 1974, now U.S.
Pat. No. 3,947,352; hereby expressly incorporated by reference and relied
upon.
[2] Copending application, Ser. No. 475,314, filed May 31, 1974, now
abandoned; hereby expressly incorporated by reference and relied upon.
Description of the Prior Art
It has recently been demonstrated that the enterotoxin from Vibrio
cholerae, which is responsible for the gastrointestinal manifestations of
clincial cholera, binds very strongly to gangliosides and less strongly to
certain glycoproteins such as fetuin and thyroglobulin. Cuatrecasas,
Biochemistry, 12, 3547 (1973a); Cuatrecasas, Biochemistry, 12, 3558
(1973b). Gangliosides block the biological effects of cholera toxin on
isolated fat cells [Cuatrecasas, 1973a, supra; Cuatrecasas, 1973b, supra;
van Heyningen et al, J. Infec. Dis., 124, 415 (1971)] and on the small
intestine [van Heyningen et al, supra; Pierce, J. Exp. Med., 137, 1009
(1973); Holmgren et al,Scand. J. Infec. Dis., 5, 77 (1973)], and they
prevent the binding of .sup.125 I-labeled cholera toxin to specific
receptors on the cell membranes of various tissues, such as adipose
tissue, liver erythrocytes, and intestinal epithelial cells; Cuatrecasas,
1973a, b, supra. There is considerable evidence indicating that
gangliosides, and specifically G.sub.M1 gangliosides, are the natural
membrane receptors with which cholera toxin specifically interacts to
elicit its biological effects in tissues; Cuatrecasas, 1973a, b, supra.
SUMMARY OF THE INVENTION
It has now been determined according to the invention that certain
insoluble polysaccharide, e.g., agarose, derivatives comprising covalently
linked gangliosides are useful for the extraction and purification of
cholera toxin by affinity chromatography [Cuatrecasas et al, Proc. Nat.
Acad. Sci. U.S., 61, 636 (1968); Cuatrecasas, Advan. Enzymol., 35, 29
(1972a); Cuatrecasas, Proc. Nat. Acad. Sci. U.S., 69, 1277 (1972b)]. The
utility of such insoluble biospecific adsorbents, as well as of soluble
polymers which contain covalently coupled gangliosides, in the therapeutic
approach to clinical cholera is demonstrated by evidencing that such
derivatives effectively block the binding and the metabolic effects of
cholera toxin in isolated adipocytes.
Briefly, according to the invention, columns of agarose derivatives [other
polysaccharide derivatives include cellulose, starch, and the cross-linked
polysaccharide gels, Sephadex and Sepharose] containing covalently
attached gangliosides quantitatively absorb the .sup.125 I-labeled cholera
toxin of chromatographed samples. The most effective derivatives are those
in which the gangliosides are coupled to "macromolecules" [native albumin,
denatured albumin, poly-L-lysine and poly(L-lysyl-DL-alanine) graft
copolymers] which are covalently linked to agarose. Ganglioside adsorbents
[1-ml columns] comprising such macromolecular "arms" can effectively
adsorb cholera toxin even after the adsorbent is diluted 200- to 600-fold
with unsubstituted agarose. Compare copending application, Ser. No.
475,314, supra. Selective adsorption is blocked if the toxin is incubated
with free gangliosides before chromatography. Quantitative elution is
achieved with buffers containing 5-7 M quanidine.HCl. The biological
activity of purified samples of cholera toxin is completely removed by
chromatography on small gangliosideagarose affinity columns, and this
activity can be quantitatively recovered upon elution with guanidine.HCl.
Small [5-ml] affinity columns can remove virtually all [more than 99%] of
the cholera toxin activity and .sup.125 I-labeled toxin present as a
tracer in preparations of filtrates of Vibrio cholerae derived from about
41. of crude culture medium. Gangliosideagarose beads can block the
lipolytic effects of cholera toxin on isolated fat cells. Soluble
ganglioside polymers, prepared by covalently attaching the glycolipids to
branched copolymers of lysine and alanine (see again copending
application, Ser. No. 475,314, supra), can prevent the binding of .sup.125
I-labeled toxin to liver membranes, as well as block completely the
lipolytic activity of cholera toxin on fat cells. These polymeric
ganglioside derivatives thus are useful in the management of the
manifestations of clinical cholera. Studies with sodium dodecyl sulfate
disc gel electrophoresis indicate, albeit applicant does not wish to be
bound by this theory, that cholera toxin is composed of two major subunits
having molecular weights of about 66,000 and 36,000. Reduction and
alkylation convert the larger subunit into components having a molecular
weight of about 8,000, and the smaller subunit is converted to two
components having molecular weights of about 27,000 and 8,000; the role of
disulfide bonds in maintaining or stabilizing the oligomeric structure of
the two major subunits is uncertain. The larger subunit [molecular weight
66,000] appears to be very similar to or identical with choleragenoid, a
toxin derivative which is antigenically very similar to toxin, which is
biologically inactive, and which competitively inhibits the binding and
biological activity of cholera toxin. The smaller subunit [molecular
weight 36,000] does not appear to bind to cells. Hence, it is proposed
that the ability to cholera toxin to bind specifically to cells is
governed solely by the larger subunit, but that the ability to elicit a
specific biological response resides in the smaller subunit. Cholera toxin
is thus suggested to consist of one subunit which acts to deliver to the
cell membrane, in a highly specific manner, another molecule [subunit]
which in turn is capable of inducing subsequent changes which lead to the
biological response.
DETAILED DESCRIPTION OF THE INVENTION
General Properties of Affinity Adsorbents
Whereas [.sup.125 I]-cholera toxin does not bind to columns containing
unsubstituted agarose, a substantial portion of the radioactivity does
absorb to columns containing fetuin-agarose or A-DADA-gang.sup.1 ; compare
FIG. 1, Table I and the methodology appurtenant thereto, Cuatrecasas et
al, Biochemistry, 12, No. 21, 4253 at page 4255 (1973e), hereby expressly
incorporated by reference and relied upon. About 80% of the radioactivity
in the iodinated toxin preparation can bind specifically to liver
membranes before chromatography. About 30% of the radioactive material
applied to a fetuin-agarose column is not adsorbed, and about 20% of this
material can still bind selectively to liver membranes. The
ganglioside-agarose adsorbent appears to be more effective than that which
contains fetuin since 15-20% of the radioactive material which is applied
to the column appears in the breakthrough of the column, and virtually
none of this material can bind selectively to liver membranes. It appears
that about 15-20% of the total radioactive content of [.sup.125 I]cholera
toxin represents radioactivity on denatured or contaminating protein. The
specificity of the adsorptive process is further illustrated by
demonstrating that incubation of the [.sup.125 I]cholera toxin with
gangliosides before chromatography effectively prevents the subsequent
adsorption of radioactivity to the column.
.sup.1 ganglioside-diaminodipropylamine-agarose
In the experiments described in said FIG. 1, Cuatrecasas et al, 1973e,
supra, elution of the adsorbed [.sup.125 I]-toxin was achieved with 7 M
guanidine.HCl. Experiments were performed to determine whether milder
conditions could be utilized to elute the toxin from such columns. The
strength with which the toxin is adsorbed is evident from the inability to
achieve elution with 0.1 M acetic acid, 2 N NaCl, and 3 M guanidine.HCl
containing 1 N NaCl. Even 4 M guanidine.HCl results in the elution of only
about one-third of the bound toxin. Nearly quantitative elution, however,
can be obtained with higher concentrations (5 M) of guanidine.HCl or with
0.1 N HCl.
Samples which had been chromatographed on A-DADA-gang columns such as that
described in the aforesaid Cuatrecasas et al, 1973e, FIG. 1 contained
detectable amounts of free gangliosides. Because the presence of this
compound in the samples can interfere with assays of the breakthrough
material, and it can also potentially interfere with adsorption of the
toxin to the column, experiments were performed to determine if other
derivatives were less susceptible to this "leakage" phenomenon [Table II,
Cuatrecasas et al, 1973e, supra, at page 4256]. Since the presence of free
gangliosides in the column breakthrough samples is meaningful only when
considered in relation to the concentration and effectiveness of the
selective adsorbent, the various derivatives were diluted serially with
unsubstituted agarose and their ability to extract [.sup.125 I]cholera
toxin was compared. As predicted from the considerations described
earlier, it is clear that the leakage of free gangliosides from the
adsorbents which contain macromolecular spacers [native albumin, denatured
albumin, poly-L-lysine and poly(L-lysyl-DL-alanine) graft copolymer] is
much less marked than that which occurs with A-DADA-gang. Of equal
importance, however, is the fact that the adsorbents containing the
polymeric spacers are inherently much more effective in extracting the
toxin. It is notable that these derivatives are quite effective even when
diluted 50-fold with unsubstituted agarose. The most preferred derivative
appears to be A-NatAlb-gang.sup.2 ; with this adsorbent some adsorption is
detectable even after a 600-fold dilution. Furthermore, leakage is not a
problem in this case since no significant free ganglioside is detectable
in effluents of columns containing a 10-fold diluted adsorbent.
Experiments of this type, which are quite useful in comparing the relative
effectiveness of a variety of adsorbents, indicate that fetuin-agarose is
quite inferior to any of the ganglioside-agarose derivatives since
virtually no adsorption occurs to adsorbents diluted 1:10 with
unsubstituted agarose.
.sup.2 ganglioside-native albumin-agarose
The chromatographic behavior of a sample of purified cholera toxin
containing a tracer quantity of [.sup.125 I] toxin on a column containing
A-Alb-gang is presented in FIG. 2 and Table III of said Cuatrecasas et al,
1973e, supra, at page 4257. Adsorption is prevented by incubating the
toxin with gangliosides before chromatography. In the absence of
gangliosides the column extracts 70% of the protein and more than 95% of
the lipolytic activity. The protein which does not adsorb to the column is
virtually without dipolytic activity and the radioactivity in this peak
does not bind to liver membranes. Nearly 90% of the lipolytic activity
applied to the column is recovered upon elution with 7 M guanidine.HCl.
These experiments demonstrate that the behavior of .sup.125 I-labeled and
native cholera toxin on such affinity columns is very similar.
Chromatography of Crude Vibrio cholerae Filtrates on Affinity Columns
The total material obtained from 3.4 l. of crude culture medium of V.
cholerae was chromatographed on a 5-ml column of A-NatAlb-gang (FIG. 3 and
Table IV of said Cuatrecasas et al, 1973e, supra, at pages 4257-4258).
More than 99% of the lipolytic activity and 80% [or virtually all of the
active form] of the tracer .sup.125 I-labeled toxin present in this
material disappeared after passage through this column, and no free
gangliosides could be detected in the effluent samples. After very
thorough and prolonged washing, elution with 5 M guanidine.HCl resulted in
the recovery of about 1 mg of protein and at least 70% of the .sup.125
I-labeled toxin which had adsorbed to the column. On the basis of
radioactivity, the toxin was purified more than 90-fold by this procedure.
The material eluted from this column was virtually devoid of biological
activity. The lack of activity in this material is not explained by the
presence of gangliosides or of residual guanidine [which inhibits
lipolysis] since it did not alter the lipolytic response of native cholera
toxin when these were incubated together before assay. The lack of
activity is similarly not explained by the presence of biologically
inactive choleragenoid, which can block the binding and activity of
cholera toxin [Cuatrecasas, Biochemistry, 12, 3577 (1973d)], since
preincubation of cells with the eluted material did not block the binding
of .sup.125 I-labeled toxin or the lipolytic response to native toxin.
Data presented suggests that the loss of activity may have resulted from
dissociation of the toxin into subunits, as aforesaid; this process is
essentially irreversible when the concentration of cholera toxin is very
low. In some experiments it has been possible to elute about 10-20% of the
lipolytic activity adsorbed, although the reasons for such recovery in
certain experiments is not known.
In the experiment described in the noted Cuatrecasas et al, 1973e, FIG. 3,
supra, it was estimated on the basis of lipolytic activity that the entire
material applied to the column contained about 3.5 mg of cholera toxin.
Since all of the activity and all of the active radioactivity were removed
by the column, and since the recovery of adsorbed radioactivity upon
elution was about 70%, it was anticipated that elution should have yielded
about 2.4 mg of protein had the purification been complete. However, only
1.3 mg of protein was present in the eluted sample. The reason for the
slight but significant disparity between the quantity of protein actually
eluted and that anticipated is not apparent. It is possible that
alterations of the native toxin, not reflected in the .sup.125 I-labeled
material, occur during the step of concentration of the crude toxin since
this exposes the protein to high ionic strength.
Since in the experiment depicted all of the cholera toxin applied on the
column was extracted from the sample, the binding capacity of such columns
was examined. When the quantity of sample applied was increased by 2.5
times and the adsorbent was diluted 5-fold with unsubstituted agarose,
only one-third of the cholera toxin applied was adsorbed to the column
[FIG. 4, Cuatrecasas et al, 1973e, supra, at page 4259]. The toxin was
effectively extracted from the first effluent fractions while virtually no
extraction occurred in the last fractions. As in the other experiments,
only a very small proportion of the total protein was adsorbed to the
column, and elution of the radioactivity labeled toxin was satisfactory.
There was excellent correspondence between the appearance of radioactivity
and lipolytic activity in the breakthrough fractions, pointing again to
the similarity in the behavior of the labeled and native toxins. The use
of .sup.125 I-labeled tracers in these experiments greatly facilitates
monitoring and quantitation of the chromatographic experiments.
Reversible Denaturation of Cholera Toxin
The binding of cholera toxin to the affinity columns is so strong that to
achieve elution it is necessary to use buffers which are likely to unfold,
and possibly denature, the protein. Because of this, and because the
protein eluted from columns on which crude samples were chromatographed
yielded essentially inactive toxin preparations, the ability of cholera
toxin to renature after removal of denaturants was examined [Table V,
Cuatrecasas et al, 1973e, supra, at page 4259]. Brief exposure of
[.sup.125 I]cholera toxin to acidic and basic conditions, and to
relatively low concentrations of urea and guanidine.HCl, diminishes
profoundly the ability of the iodoprotein to bind to liver membranes upon
dilution or neutralization of the denaturant; similar effects are observed
if urea and guanidine.HCl are removed by dialysis. The .sup.125 I-labeled
toxin which is eluted from affinity columns such as those depicted in the
aforesaid noted FIG. 1 and Tables I and II does not bind at all to liver
membranes if tested after removal of guanidine.HCl. These results suggest
that the .sup.125 I-labeled toxin is undergoing an irreversible unfolding
or denaturation. The conditions which cause this irreversible effect occur
with concentrations of urea and guanidine.HCl which are lower than those
which are required to elute the toxin which is adsorbed to a
ganglioside-agarose column. This suggests that ganglioside binding greatly
stabilizes the tertiary or quaternary structure of the protein.
The effects described immediately above suggest results contradictory to
those of the experiments described in the noted FIG. 2, where
guanidine.HCl elution of a chromatographed sample of purified toxin
yielded active toxin. The possibility was examined that the irreversible
denaturation described is dependent on the concentration of toxin used in
such experiments. Samples of native cholera toxin [0.1-0.5 mg/ml]
containing a tracer of .sup.125 I-labeled toxin were exposed for 25 min.
at 24.degree. to (a) 0.1 M phosphate buffer [pH 7.4], (b) distilled water,
(c) 0.1 N HCl, (d) 7 M guanidine.HCl, and (e) 7 M urea under conditions
similar to those described in the Cuatrecasas et al, 1973e, Table V. The
samples were then diluted fivefold and dialyzed overnight against large
volumes of Krebs-Ringer-bicarbonate buffer. Virtually no radioactivity was
lost during the period of dialysis, and the lipolytic activity of all the
samples was equal to that of the sample exposed only to phosphate buffer.
It is clear that at these concentrations of cholera toxin, which are about
10,000 times higher than those described in said Table V, denaturation of
the toxin by exposure of these solvents is readily reversible.
There is some evidence that the denaturation described above involves a
process of dissociation of cholera toxin into subunits. As suggested
above, exposure of high concentrations [0.5 mg/ml] of toxin to 7 M
guanidine.HCl followed by dialysis does not result in the loss of the
.sup.125 I-labeled toxin which is added as a tracer. In contrast, exposure
of tracer quantities [10 ng/ml] of .sup.125 I-labeled toxin to 7 M
guanidine.HCl results in the rapid loss of radioactivity upon dialysis,
even when 0.1% albumin is added to the sample to prevent adsorption to the
dialysis membrane. Under these conditions 60% of the radioactivity is lost
after dialysis for 2 hours at 24.degree., and about 85% is lost after
dialysis for 24 hours at 4.degree.. Further evidence for the dissociation
into subunits comes from disc gel electrophoretic experiments in 0.5%
sodium dodecyl sulfate, Cuatrecasas et al, 1973e, supra, at pages
4260-4262. These results are consistent with findings of LoSpalluto and
Finkelstein, Biochim. Biophys. Acta, 257, 158 (1972), who described
reversible dissociation of cholera toxin into subunits of about 15,000
molecular weight upon exposure to 6 M urea or to a pH 3.6; these
experiments were performed at concentrations of toxin varying from 2.5 to
4 mg per ml.
Ganglioside-Agarose and the Lipolytic Response to Cholera Toxin
The ganglioside-agarose derivatives described herein are quite effective in
removing .sup.125 I-labeled toxin from buffer solutions when the
derivatized beads are added and incubated in suspension. The derivatives
can be diluted with unsubstituted agarose, and adsorption is generally
complete after incubating for 15 min. at 24.degree.. The derivatives are
also quite potent in protecting fat cells against the metabolic effects of
the toxin provided that the beads are added to the cells before the toxin
[Table VI, Cuatrecasas et al, 1973e, supra, at page 4260]. Addition of the
adsorbent 10 min. after addition of the toxin has no effect on the
lipolytic response to the toxin. These results are consistent with the
nearly irreversible nature of the binding of cholera toxin to cell
membranes [Cuatrecasas, 1973a, b, supra].
Effect of Water-soluble Polymers Containing Gangliosides
The water-soluble copolymer of poly(L-lysine) (backbone) and -(DL-alanine)
(side branches) which contains covalently linked gangliosides effectively
inhibits the binding of .sup.125 I-labeled cholera toxin to liver
membranes [Table VII, Cuatrecasas et al, 1973e, supra, at page 4260]. This
ganglioside polymer is effective in concentrations which in the final
incubation medium are as low as 0.1 .mu.g/ml.
The ganglioside-containing polymer is also quite effective in blocking the
lipolytic effect of cholera toxin or fat cells [Table VIII, Cuatrecasas et
al, 1973e, supra, at page 4260]. However, as with the insoluble
ganglioside derivatives, marked effects are observed only if the polymer
is added to the cells before the addition of cholera toxin. These soluble
derivatives appear to be more potent than the comparable insoluble agarose
derivatives. Nearly complete inhibition of activity is achieved with
concentrations of the polymer is low as 0.5 .mu.g/ml.
Disc gel electrophoretic characterization of cholera toxin and
choleragenoid consistent with the invention too is described at
Cuatrecasas et al, 1973e, supra, at pages 4260-4262.
Experimental
Materials
The crude culture filtrate cholera toxin from Vibrio cholerae was lot 022
[Wyeth], provided by the SEATO Cholera Research Program, NIAID. This
material was prepared by lyophilization of culture filtrate of V. cholerae
strain 569B, grown in Richardson's [Richardson and Noftle, J. Infec. Dis.,
121, Suppl. 73 (1970)] TRY medium; 100 g of this material represented the
lyophilized filtrate of about 8.45 l. of crude culture medium. Cholera
toxin [lot 1071], purified by the method of Finkelstein and LoSpalluto, J.
Infec. Dis., 121, Suppl., 563 (1970), was obtained from SEATO Cholera
Research Program; it was prepared under contract for the National
Institute of Allergy and Infectious Diseases by Dr. R. A. Finkelstein, The
University of Texas Southwestern Medical School, Dallas, Texas.
Choleragenoid was a gift from Dr. Finkelstein. Bovine brain gangliosides
[grades II and III] were purchased from Sigma. Procine thyroglobulin was
obtained from Miles, fetuin from Calbiochem, bovine albumin [grade A] from
Pentex, guanidine.HCl [Ultra Pure] and urea from Schwartz-Mann, Sepharose
4B from Pharmacia, dicyclohexylcarbodiimide and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide from Pierce, and
N-hydroxysuccinimide from Aldrich. The multichain copolymer,
poly(L-lysyl-DL-alanine), which consists of a polylysyl backbone and has
an alanine to lysine ratio of 15 to 1, was purchased from Miles; the
molecular weight of this compound was about 37,500. Poly(L-lysine).HCl
[mol wt 160,000] was obtained from Schwarz-Mann.
Procedures
].sup.125 I]Cholera toxin [5-20 .mu.Ci/.mu.g] was prepared from toxin
samples chromatographed on Sephadex G-75 by procedures described at
Cuatrecasas, 1973a, supra. About 80% of the radioactive material prepared
in this way bound specifically to liver membranes. The molecular weight of
cholera toxin was assumed to be 84,000 and the A.sup. 1%.sub.1 cm [280 nm]
11.41 [LoSpalluto and Finkelstein, supra]. Isolated fat cells were
prepared from male Sprague-Dawley rats [90-120 g] by the method of
Rodbell, J. Biol. Chem., 241, 140 (1966). Liver membranes were prepared by
homogenization in 0.25 M sucrose followed by differential centrifugation
[Cuatrecasas, 1972b, supra]. Protein content was determined by the method
of Lowrey et al, J. Biol. Chem., 193, 265 (1951) using bovine albumin as
the standard.
The specific binding of [.sup.125 I] cholera toxin to liver membranes were
performed as described previously [Cuatrecasas, 1973a, b, supra]. Liver
membranes [20-100 .mu.g of protein] were incubated for 20 min. at
24.degree. in Krebs-Ringer-bicarbonate buffer [pH 7.4], containing 0.1%
albumin and the iodinated toxin [5-10.times.10.sup.4 cpm]; binding was
determined by filtration on cellulose acetate (EGWP, Millipore Corp.)
filters. For every determination nonspecific binding was determined by
including control samples in which native toxin [5 .mu.g/ml] was added to
the membranes before adding [.sup.125 I]toxin. The presence of free
gangliosides in column effluents was determined by measuring the ability
of these samples to block the binding of [.sup.125 I]cholera toxin to
liver membranes [Cuatrecasas, 1973a, b, supra]. The iodoprotein was
incubated with the sample for 50 min. at 24.degree. in
Krebs-Ringer-bicarbonate buffer [pH 7.4], containing 0.1% albumin before
determining specific binding. By these methods it is possible to detect
less than 50 ng/ml of crude bovine brain gangliosides [type III, Sigma].
Lipolysis by fat cells was studied by determining the concentration of
glycerol in the medium by the method of Ryley, Biochem. J., 59, 353
(1955). The bioassay of cholera toxin was based on the potent lipolytic
action of the toxin on fat cells. Fat cells [2-8.times.10.sup.5 cells/ml]
were incubated at 37.degree. in Krebs-Ringer-bicarbonate buffer containing
3.degree. albumin; samples [0.1 ml] were analyzed for glycerol at various
time periods between 90 and 160 min. [Cuatrecasas, Biochemistry, 12, 3567
(1973c)]. Since the absolute lipolytic responses varied between
experiments, the activity of unknown toxin samples were expressed on the
basis of comparisons with standard curves obtained with native cholera
toxin.
Protein was analyzed by electrophoresis in 7.5% polyacrylamide disc gels
[7.5.times.0.5 cm] at pH 7.0 in 0.1 M sodium phosphate buffer, both in the
presence and absence of sodium dodecyl sulfate [Weber and Osborn, J. Biol.
Chem., 244, 4406 (1969)]. Protein was detected by staining 1-2 hours with
Coomassic Brilliant Blue [0.25% in methanol water-acetic acid (5:5:1,
v/v)], and gels were destained overnight in water-acetic acid-methanol
[35:3:2, v/v]. Molecular weights were estimated from electrophoretic
mobilities of standard proteins [cytochrome c, ovalbumin, serum albumin]
in gels containing 0.1% sodium dodecyl sulfate.
Preparation of Ganglioside-Agarose Derivatives
Poly(L-lysine) and the branched, multichain copolymer of L-lysine
("backbone") and DL-alanine ("side arms") were coupled to cyanogen bromide
activated agarose by the methods of Sica et al, Nature (London), New
Biol., 244, 36 (1973a); Sica et al, J. Biol. Chem., in press (1973b);
Cuatrecasas, J. Biol. Chem., 245, 3059 (1970); copending application, Ser.
No. 475,365, filed May 31, 1974, now U.S. Pat. No. 3,947,352; and
copending application, Ser. No. 475,314, filed May 31, 1974, now
abandoned. These polymers were used to increase the number of potentially
modifiable functional groups [.alpha.-amino groups in the copolymer,
.epsilon.-amino groups on the homopolymer] on the agarose, to place these
groups at a considerable distance from the agarose backbone, and to
enhance the likelihood of multipoint attachment of the soluble polymer on
the agarose, which would increase the stability of linkage of subsequently
substituted ligands. The derivatives used contained about 1.2 mg of
copolymer/ml of agarose and about 1.4 mg of poly(L-lysine)/ml of agarose.
Albumin was also used as a macromolecular spacer for the same reasons
described above. Albumin was coupled to cyanogen bromide activated agarose
in the absence [native] or presence [denatured] of 10 M urea, as described
recently [Sica et al, 1973a, b, supra]; these derivatives contain 2-3 mg
of albumin/ml of gel. 3,3'-Diaminodiproplamine, fetuin, and thyroglobulin
were coupled to agarose with cyanogen bromide [ Cuatrecasas, 1970, supra];
these agarose derivatives contained about 10 .mu.mol. 6 mg, and 8 mg,
respectively, of the ligand and proteins per ml of packed gel.
Gangliosides were coupled through carboxy groups of the terminal sialic
acid residues to amino groups of the derivatized agaroses by utilizing a
water-soluble carbodiimide reagent or dicyclohexylcarbodiimide, by
preparing an active N-hydroxysuccinimide ester of the ganglioside, or by
preparing an activated, mixed anhydride of the ganglioside.
Coupling with Carbodiimides
Ther derivatized agarose [containing amino groups] [25 ml] was washed and
suspended in 50 ml of 50% (v/v) aqueous dioxane. Brain gangliosides [type
III, Sigma] [50 mg] were added and the suspension was gently shaken at
24.degree. for 15 min. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [100
mg] was added and the suspension was shaken for 6 hours at 24.degree., and
another 100 mg portion of the carbodiimide was added. After shaking for
another 12 hours, the gel was washed with 500 ml of water, 500 ml of 75%
(v/v) aqueous methanol, 250 ml of 6 M guanidine.HCl, and 500 ml of water.
The content of ganglioside, as judged by the recovery of unreacted
ganglioside, was about 0.5 mg/ml of gel. Coupling was also performed in an
organic solvent by reacting 20 mg of ganglioside and 5 mg of
dicyclohexylcarbodiimide in 10 ml of dioxane for 30 minutes at 15.degree..
This was added to 20 ml of albumin-agarose suspended in dioxane in a total
volume of 40 ml. After reacting for 15 hours at 24.degree., the gel was
washed with 500 ml of dioxane, 500 ml of 90% (v/v) methanol, and 250 ml of
6 M guanidine.HCl.
N-Hydroxysuccinimide Ester
Ganglioside [20 mg] was reacted with 2.5 mg of N-hydroxysuccinimide and 2.5
mg of dicyclohexylcarbodiimide for 30 min. at 15.degree. in 10 ml of
dioxane. The solution was then added to 20 ml of albumin agarose suspended
in dioxane [total volume, 40 ml]. After shaking for 15 hours at
24.degree., the gel was washed as above described [water-soluble
carbodiimide reaction].
Mixed Anhydride
A 100-.mu.l portion of 0.1 M N-methylmorpholine in tetrahydrofuran was
added to a solution of anhydrous tetrahydrofuran containing 20 mg of
ganglioside. After stirring the solution for 10 min. at 0.degree., 100 l
of 0.1 M isobutyl chloroformate [Vaughan and Osato, J. Amer. Chem. Soc.,
74, 676 (1952)] in tetrahydrofuran was added and the reaction was allowed
to continue for 20 min. at 0.degree.. The reaction mixture was added to 20
ml of the amino agarose derivative suspended in dioxane [total volume, 40
ml]. After reacting for 15 hours at 24.degree. the gel was washed as above
described.
Although all of the methods above described resulted in effective
adsorbents, the most preferred results were consistently obtained with
derivatives prepared with the water-soluble carbodiimide and with the
mixed anhydride.
Preparation of Water-Soluble Polymers Containing Gangliosides
Gangliosides were coupled to the branched-chain copolymer of lysine and
alanine with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; 20 mg of
poly(L-lysyl-DL-alanine) was dissolved in 7 ml of water and 5 ml of
methanol and 40 mg of ganglioside [dissolved in 10 ml of 50% aqueous
methanol] were added. The mixture was stirred at 24.degree. for 15 min.
and two 40 mg portions of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
were added at 6-hour intervals. After stirring for an additional 12 hours,
the reaction mixture was dialyzed against 4 l. of H.sub.2 O for 15 hours
and lyophilized. The sample was then chromatographed on a column
[2.4.times.70 cm] of Sephadex G-75 equilibrated with 25% aqueous methanol
containing 0.005 M HCl; the flow rate was 15 ml/hr, 4 ml fractions were
collected and the elution was monitored by continuously recording the
absorbance at 256 nm. The peak in the void volume was collected,
lyophilized, and rechromtographed on a column [1.6.times.26 cm] of
Sephadex G-100 equilibrated with 6 M guanidine.HCl [12 ml/hr, 4
ml/fraction]. The material present in the first peak was dialyzed against
four changes of H.sub.2 O (4 l) for 24 hours and lyophilized. The yield
was 22 mg.
Thus, the present invention demonstrates that columns containing
ganglioside-agarose derivatives can selectively extract cholera toxin even
when the toxin is present in concentrations as low as 10.sup.-11 M. The
most preferred adsorbents are those which contain macromolecular spacers
[i.e., poly(amino acid) polymers, albumin] interposed between the agarose
bakbone and the covalently attached ganglioside. Some of these derivatives
are still effective after 600-fold dilution with unsubstituted agarose.
Such macromolecular adsorbents have recently proved most useful in the
purification of estrogen receptors from the uterus [Sica et al, 1973a, b,
supra]. It has been possible to purify estrogen receptors about
100,000-fold in a single step by using 20-fold diluted
poly(L-lysyl-DL-alanine)-agarose derivatives containing covalently
attached estradiol.
Among the specific advantages of the derivatives having "macromolecular"
spacers is a high degree of ligand substitution which permits the use of
the adsorbent in dilute form, and this results in a decrease in the
nonspecific protein adsorptive properties of the gel. Furthermore, the
ligand is separated from the agarose backbone by large distances, which in
the case of the branched amino acid copolymer may be as great as 150 A.
The high probability that the interposing macromolecule is anchored to the
insoluble polymer by multiple points greatly increases the overall
chemical stability of the attached ligand by stabilizing the basic
cyanogen bromide attachment of the unit to the agarose. This enhanced
stability may be quite important in very high-affinity systems such as the
present one in which leakage of even small quantities of the ligand may
seriously interfere with the specific adsorptive behavior of the proteins.
These derivatives, particularly those prepared with albumin, may in
addition present a more favorable microenvironment for the specific
ligand-protein interaction. This appears to be the case with the
estradiol-agarose derivatives [Sica et al, 1973b, supra].
The ability of gangliosides to bind extremely tightly to cholera toxin, and
thus to prevent the binding and the biological effects of the toxin in
various tissues [Cuatrecasas, 1973a, b, supra], evidenced that
gangliosides are useful therapeutic agents for clinical cholera. However,
the demonstration that free gangliosides may be incorporated spontaneously
into cell membranes, and that this can ultimately result in increased
binding of the toxin and in enhanced biological effects [Cuatrecasas,
1973b, supra], suggests that the in vivo use of such agents may be
dangerous if not ineffective. For this reason the insoluble and soluble
polymeric ganglioside derivatives described herein are more rational
agents for the therapeutic value of gangliosides in clinical cholera.
While the invention has been shown and described and pointed out with
reference to certain preferred embodiments thereof, those skilled in the
art will appreciate that various changes, modifications, substitutions,
and omissions can be made by those skilled in the art without department
from the spirit of the invention. It is intended, therefore, that the
invention be limited only by the scope of the claims which follow.
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