Starburst Conjugates of Proteins with Carbon-Chain Polymers Containing Low Molecular Biologically Active Compounds: Synthesis and Immunogenicity

Article abstract of DOI:10.1023/A:1022226418497

The synthesis of starburst polymer-protein conjugates on the basis of bovine serum albumin and horseradish peroxidase was performed with the aim to study the possibilities of regulation of the immune response against the components of the conjugates. These polymers had one-point binding between the protein and the modifying carbon-chain polymer that contained 1-vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (a salsolinol analogue) or bradykinin (peptide hormone) residues in its carbon chain. Changes in the chemical nature of the carbon-chain part of the polymer-protein conjugate were shown to increase or decrease the level of antibody production both against the low-molecular compounds attached to the polymeric fragments and against the protein carrier.

Full text of DOI:10.1023/A:1022226418497

Russian Journal of Bioorganic Chemistry, Vol. 29, No. 1, 2003, pp. 33–42. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 1, 2003, pp. 38–48.
Original Russian Text Copyright © 2003 by Vlasov, Pankova, Nikonova, Antonov.
Starburst Conjugates of Proteins with Carbon-Chain Polymers
Containing Low Molecular Biologically Active Compounds:
Synthesis and Immunogenicity
1
G. P. Vlasov, G. A. Pankova, I. N. Nikonova, and N. G. Antonov
Institute of Macromolecular Compounds, Russian Academy of Sciences, Bol’shoi prosp. 31, St. Petersburg, 199004 Russia
Received October 18, 2000; in final form, January 30, 2002
Abstract—The synthesis of starburst polymer–protein conjugates on the basis of bovine serum albumin and
horseradish peroxidase was performed with the aim to study the possibilities of regulation of the immune
response against the components of the conjugates. These polymers had one-point binding between the protein
and the modifying carbon-chain polymer that contained 1-vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline
(a salsolinol analogue) or bradykinin (peptide hormone) residues in its carbon chain. Changes in the chemical
nature of the carbon-chain part of the polymer–protein conjugate were shown to increase or decrease the level
of antibody production both against the low-molecular compounds attached to the polymeric fragments and
against the protein carrier.
Key words: bradykinin, immunogenicity, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol),
starburst carbon-chain polymer–protein conjugates, 1-vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline
1
INTRODUCTION
molecules connected with them. As a result, specific
biological activity of the compounds immobilized on a
protein carrier can disappear [1, 5]. On the other hand,
this activation of the immune system by the protein car-
rier and the subsequent increase in the antibody produc-
tion against low molecular compounds attached to the
carrier (for example, fragments of antigenic determi-
nants of viral or bacterial glycoproteins, toxins, steroids
or the products of their metabolism) can be very useful
and is widely applied for the creation of diagnosticums
and the preparation of semisynthetic protective vac-
cines [1]. However, the production of antibodies
against the carrier itself and against the spacer that links
the low molecular hapten with the carrier is often
observed along with an increase in the immune
response toward the low molecular hapten when the
protein conjugates are used for such purposes. More-
over, the production of antibodies against the protein
carrier can be much higher than that against the hapten
[7]. The presence of antibodies against the carrier in the
antisera can result in both suppression of the immune
response to the hapten [8] and in the interactions of
these antibodies with other proteins [9].
Chemical modification of proteins, nucleic acids,
polysaccharides, and low molecular biologically active
compounds, such as antibiotics, cytostatics, and other
compounds, by water-soluble polymers is often used
for the preparation of bioconjugates with fundamen-
2
tally new and programmed properties [1]. Carbon
chain polymers [2] and heterochain polymers, such as
polysaccharides (e.g., dextran) [3], polyethylene glycol
[4], and polyethyleneimine [5], are most often used as
polymer carriers. Synthetic polypeptides are compara-
tively seldom used as modifiers of biologically active
substances [6], although polypeptides and proteins
offer some advantages over the carbon chain polymer
carriers. Conjugates prepared on their basis are capable
of biodegradation, and this property is important for
their application in medicine. At the same time, pro-
teins as carriers or modifiers of biologically active com-
pounds have immunogenic properties that distinguish
them from the carbon chain polymers or polyethylene
glycol; they induce an antibody production against
1
Please send correspondence to phone, (812) 323-1050 or e-mail,
gpvlasov@hq.macro.ru.
Abbreviations: BK, bradykinin; BSA, bovine serum albumin;
In this connection, the main problem of the biocon-
jugate design that is independent of the aim of their
preparation is the urgent necessity to regulate the
immune response against the conjugate components.
This study is devoted to the synthesis of starburst con-
jugates of proteins with carbon chain polymers contain-
ing the residues of THIQ (an analogue of salsolinol),
VTHIQ, and bradykinin (a peptide hormone) using the
method that we previously developed [10–14]. We also
2
DEAA, acrolein diethylacetal; ELISA, enzyme linked immun-
osorbent assay; HRP, horseradish peroxidase; MAA, methacrylic
acid; MI, macroinitiator; MI-1, HPR-macroinitiator; MI-2, BSA-
macroinitiator; Ova, ovalbumin; SPPC, starburst polymer–protein
conjugate; Suc, succinyl; THIQ, 1-methyl-6,7-dihydroxy-1,2,3,4-
tetrahydroisoquinoline (salsolinol); TNBS, trinitrobenzene-
sulfonic acid; VMI, N-vinyl-2-methylimidazole; VP, N-vinylpyr-
rolidone; and VTHIQ, vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroiso-
quinoline.
1068-1620/03/2901-0033$25.00 © 2003 MAIK “Nauka /Interperiodica”

34
VLASOV et al.
investigated the possibility of regulating the immune
At the second stage, vinyl monomers were polymer-
response against both the biologically active low ized on MI to form the carbon-chain polymer–protein
molecular components of the conjugate and the protein conjugates in which one terminus of every molecule of
carrier.
the modifying polymer was bound to the protein in one
position. We somewhat modified this approach and
used it for the preparation of conjugates with synthetic
peptides. The C-terminus of a synthetic neuropeptide
analogue, enkephalin, was attached to the one terminus
of the modifying polymer [23]. A similar approach was
used for the preparation of biologically degradable
polymeric carriers, in particular, block-copolymers of
the A–B and A–B–A types, where A and B were
polypeptide and carbon-chain blocks, respectively [24,
25].
RESULTS AND DISCUSSION
Methods of Preparation of Polymer Conjugates
of Biologically Active Substances
Polymers containing preliminarily activated func-
tional groups are most often used for the synthesis of
conjugates with biologically active substances [1–3].
The drawbacks of such an approach is that many varia-
tions of binding between the activated polymer reagent
and polyfunctional reagent (protein) exist, and it is
impossible to obtain the conjugates of a standard com-
position and molecular mass [1–3]. Attempts to avoid
this variability (multi-point binding) of the conjugate
binding have been made for a long period of time. For
example, conjugates in which the C-terminus of every
molecule of the modifying peptide is bound to the one-
point modified protein can be prepared by the polymer-
ization of N-carboxyanhydrides of amino acids on
amino groups of a protein (that play role of the poly-
merization initiators) [15]. Another successful example
is the modification of proteins by polyethylene glycol
with an activated group on one of its termini [4].
This method was applied to the preparation of the
starburst conjugates of insulin with the following mod-
ifying polymers: poly(N-vinylpyrrolidone), poly(N-
vinylimidazole), polyacrylic acid, and polyacrylamide
[10, 12, 14]. The copolymer of N-vinylpyrrolidone and
acrolein diacetal was used as the modifying polymer
for the preparation of the starburst conjugates of trypsin
[11, 13] and horseradish peroxidase [26].
Advantages of SPPCs
The studies of physicochemical properties of such
polymer–protein conjugates revealed significant advan-
tages of them over the conjugates prepared on the basis
In recent years, the interest to preparation of conju- of preliminarily synthesized and activated polymers.
gates of the proteins to be modified with one-point For example, we demonstrated that the immunoreactiv-
attachment to the modifying carbon chain reagents has ity of the polypeptide component in the insulin star-
significantly been grown. The synthesis of such conju- burst conjugate significantly decreased and its hor-
gates most often involves the preparation of low-molec- monal activity was preserved [10]. It was also shown
ular oligomers containing one reactive group on one of that stability of the conjugate toward proteolysis could
the termini of the oligomeric chain and its covalent be regulated by changing the chemical nature of the
binding to a protein [16–20].
modifying polymer [10].
The most important advantage of SPPCs was the
possibility of modification of particular groups in a pro-
tein. We prepared the insulin conjugates containing
polymer fragments bound to one (A1), two (A1 and
B29), and three (A1, B1, and B29) amino groups [10,
12]. Not only the number of polymer chains grafted on
the protein component, but their molecular mass can be
changed as well. We found that the preservation of a
significant level of biological activity of the hormone
with simultaneous dramatic decrease in its immunore-
activity can be achieved even after the attachment of
polymeric chains with such low molecular mass as 3–
5 kDa [10, 12].
Starburst Polymer–Protein Conjugates
Previously, we developed a general method for the
synthesis of the starburst polymer–protein conjugates
with one-point binding of the modifying carbon chain
polymer to the modified protein [10–14]. The synthesis
involves two basic stages. At the first stage, a protein
was modified with an active derivatives of 2,2'-asobis-
isobutiric acid (dimethylimidate or diazide) with the
formation of the protein macroinitiator (MI). Amino
groups of the protein reacted to form the amidine
(−C(=NH2)+–NH–) and amide (–C(=O)–NH–) bonds
between the low-molecular fragment of the initiator
An advantage of the proposed SPPC method was
and the protein. The choice of one of the two variants of also the possibility of using such polymer as poly(N-
the initiator allowed us to preserve or change the total vinylpyrrolidone) [10, 11], which is widely used in
protein charge (pI). In the former case, the protein medicine for the protein modification without its pre-
charge remained practically unchanged after the forma- liminary chemical modification [27]. We were first who
tion of the amidine bond [21, 22], whereas, in the latter attached the polymeric chains on the basis of N-
case, pI was changed, because some of the amine vinylimidazole to proteins also without its modification
groups of the protein were converted into amide [10]. However, the SPPC containing low-molecular
groups.
biologically active compounds in the polymeric frag-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003

STARBURST CONJUGATES OF PROTEINS
35
Table 1. Characteristics of conjugates (Ia) and (Ib) containing a vinyl analogue of salsolinol
Number of
the polymeric
chains, m
Molecular mass
of polymeric
moiety
Conjugate
molecular mass
VTHIQ content,
mol %
Peroxidase
activity*, %
Number
Conjugate
(Ia) Poly(VPp,VTHIQq)-HRP
(Ib) The same
3
3
46600
49400
2600
5400
5
4
13
110
* The peroxidase activity was determined relative to the activity of the protein amount introduced into the conjugates.
ments have not been prepared until now. In this paper, cant decrease in the reactivity of vinyl bound in the
methacryloyl fragment of Nα-methacryloylbradykinin.
we report for the first time the synthesis of such conju-
gates and their use as an example of possibility of solu-
tion of the problem of direct regulation of the immune
response against separate components of the conju-
gates.
In this connection, the polycondensation approach
(method B, Scheme 1) was applied to the preparation of
SPPC on the basis of BSA. A special feature of this
approach was that the protein amino groups in MI-2
remained free after the modification by the fragments
of 2,2'-azobisisobutyric acid were additionally acylated
with succinic anhydride before the stage of grafting of
polymeric chains. This additional modification of free
amino groups converts the protein into a polyanion car-
rier capable of a significant activation of immune sys-
tem [29–31]. Polymeric conjugates (II), (III), and (IV)
containing the DEAA fragments along with the MAA,
VP, and VMI fragments in the polymeric part of the
conjugate were prepared by the copolymerization of
DEAA with MAA, VP, or VMI, respectively, on the
succinylated macroinitiator.
The further synthesis involved the conversion of a
part of diethylacetal groups [–CH2–CH(OC2H5)2–] in
conjugates (II)–(IV) into aldehyde groups of acrolein
fragments [–CH2–CH(CH=O)–], which proceeded dur-
ing the isolation at pH 3.5. The BSA conjugates con-
taining aldehyde groups in polymeric moiety were fur-
ther used for the attachment to the N-terminus of brady-
kinin with the formation of aldimine bond (Schiff base)
between the polymer and the peptide [–CH2–
CH(CH=BK)–]. The reduction of the aldimine bond by
NaBH4 resulted in SPPCs (V), (VI), and (VII) (Table 2,
Scheme 1) that contained bradykinin in the form of
fragments [–CH2–CH(CH2–BK)–].
The Synthesis of SPPC Containing Low-Molecular
Biologically Active Compounds in Polymeric
Fragments
Two approaches (A and B) were applied to the syn-
thesis of the conjugates (Scheme 1). The conjugates
containing the model heterocyclic biologically active
compound (VTHIQ) were synthesized by the method
A. The MI-1 macroinitiator on the basis of HRP was
prepared by the enzyme modification with 2,2'-azobi-
sisobutiric acid dimethylimidate dihydrochloride at the
first stage and was further used for the copolymeriza-
tion of VP and VTHIQ on it. Conjugates (Ia) and (Ib)
with various compositions of co-monomers were pre-
pared. Salsolinol that is formed in human organism by
condensation of endogenous dopamine with acetalde-
hyde (the product of oxidation of ethanol by alcohol
dehydrogenase) exhibits strong teratogenic effect [28].
Our interest in the synthesis of conjugates (Ia) and (Ib)
on the basis of HRP is due to the necessity of optimiza-
tion of the process of antibody production against sal-
solinol and the use of these antibodies as the antigen
containing enzymic label in the enzyme immunoassay
of the antisalsolinol antibodies, as was performed for
takrin [26].
VTHIO was synthesized like salsolinol (see the
Experimental section) by the condensation of dopam-
ine with acrolein (Scheme 2). The substitution of vinyl
for methyl group allowed the use of original polymer-
ization approach (methodA) for the introduction of sal-
solinol residue into polymer–protein conjugates (Ia)
and (Ib). The characteristics of the starburst polymer-
protein conjugates (Ia) and (Ib) prepared on the basis
of MI-1 and containing VTHIQ are given in Table 1.
We tried to synthesize a series of conjugates con-
taining bradykinin bound to BSA in polymeric frag-
ments by a similar scheme (method A, Scheme 1).
However, the copolymerization of the preliminarily
prepared Nα-methacryloylbradykinin with N-vinylpyr-
The characteristics of polymeric conjugates (VIII)–
(X) that contain BK but not BSA and are similar to the
of SPPCs on the basis of BSA (V)–(VII) in their poly-
meric structure are also given in Table 2. They were
synthesized with the goal to study the effect of the BSA
protein component on the immunogenicity of conju-
gates and the immunoadjuvant activity of the polymeric
part of the conjugates. The synthesis of these conju-
gates, their isolation, and binding of bradykinin were
performed according to Scheme 3, which was similar to
Scheme 1 and differed from it by the use of commercial
2,2'-azobisisobutonitrile as an initiator of the polymer-
ization.
The characteristics of the standard conjugate BK
rolidone gave the conjugate that contained almost no with BSA (ïI) prepared by the coupling of succiny-
bradykinin. This failure can be explained by a signifi- lated BSA with BK in the presence of a water-soluble
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003

2HCl × CH3O–C(=NH2)+–C(CH3)2–N=N–C(CH3)2–C(=NH2)+–OCH3 + (NH2)n
HRP, BSA
(NH2)n – m
pH 10–11
Macroinitiator
MI-1 MI-2
HRP, BSA
[HO–C(=O)–C(CH3)2–N=N–C(CH3)2–C(=NH2)+–NH]m
Protein
HRP BSA
MI-1, MI-2
+ (M1 + M2)
(NH2)n – m
Macroinitiator
Ä
+Succinic anhydride/pH
B
(NH2)n – m
HRP
BSA
BSA
[HO–C(=O)–C(CH3)2–N=N–C(CH3)2–C(=NH2)+–NH]m
[{(M1)p, (M2)q}–C(CH ) –C(=NH )+–NH]
3
2
2
m
MI-2S
Macroinitiator
(Ia), (Ib)
Conjugate
M1
(Ia), (Ib)
VP
(NH-Suc)n – m
+ (M1 + M2)
M2
VTHIQ
[[(M1)k, (M2)l]–C(CH ) –C(=NH )+–NH]
m
(II), (III), (IV)
3
2
2
+ H+/H2O;
+ BK
+ NaBH4
(NH-Suc)n – m
[[(M1)k, (M2)1, (M3)h]–C(CH ) –C(=NH )+–NH]
m
BSA
(V), (VI), (VII)
3
2
2
(VI)
(VII)
Conjugate
(V)
M1
M2
M3
MAA
DEAA
BK
VP
DEAA
BK
VMI
DEAA
BK
Scheme 1. Preparation of SPPCs on the basis of horseradish peroxidase and bovine serum albumin.

STARBURST CONJUGATES OF PROTEINS
37
carbodiimide [32] are also given in Table 2. Conjugate
HO
HO
NH2
HO
HO
RCH=O
(XI) was synthesized in order to compare the immuno-
genic properties of SPPCs (V)–(VII) and the polymeric
conjugates (VIII)–(X) with that prepared by the con-
ventional method.
NH
R
Dopamine
R=CH3 THIQ
R=CH=CH2 VTHIQ
Immunogenic Properties of Polymeric Conjugates
Containing Salsolinol and Bradykinin
The immunogenic properties of the polymeric con-
jugates containing salsolinol (Table 3) and bradykinin
(Table 4) were determined as a titer of the antihapten
antibodies in blood of the rabbits immunized with the
corresponding conjugates in the presence and in the
absence (for the BK-containing conjugates) of Freund’s
adjuvant. ELISA was used for the determination of the
antibody titers.
Scheme 2. The synthesis of salsolinol (THIQ) and its
1-vinyl analogue (VTHIQ)
standard conjugates gave the antisera enriched with the
antibodies against the carrier. A significant increase in
the content of antibodies against salsolinol in the case
of SPPCs and a decrease in the titer of antibodies
against the carrier is probably due to a shielding of the
antigenic determinants of protein carrier by polymeric
chains and a more effective presentation of the low-
molecular hapten to immunocompetent cells. In this
case, the polymeric fragment plays the role of spacer.
The advantages of SPPCs as semisynthetic immuno-
gens is more obvious when inspecting Table 4.
A Comparison of SPPC Immunogenicity
The comparative data on the immunogenic proper-
ties of SPPCs (Ia) and (Ib) prepared on the basis of
HRP and containing salsolinol in their polymeric parts
and the salsolinol-containing conjugates (XIIa) and
(XIIb) prepared on the basis of Suc-BSA using a water-
Table 4 clearly shows that, in the presence of Fre-
soluble carbodiimide are given in Table 3. One can see und’s adjuvant, the antiserum produced against SPPC
that the antibody titers against salsolinol were signifi- (V) containing polymethacrylic acid in its polymeric
cantly higher for SPPC (Ia) and (Ib) than those for con- part gave the maximal antibody titer against both BK
jugates (XIIa) and (XIIb) although their salsolinol and BSA in comparison with that against conjugates
content was lower. The rabbit immunization with the (VI), (VII), and (XI). The reason of such increase in the
Table 2. Characteristics of the bradykinin-containing conjugates
(V)
Poly(MAAk, DEAAl,
BKh)-BSA(Suc)
30
1
30
–
170000 5600 MAA /DEAA, 75/25*
30000 MAA/DEAA, 70/30**
760000 40000 VP/DEAA, 90/10*
40000 VP/DEAA, 80/20***
760000 19000 VMI/DEAA, 93/7*
6.5
4.8
4.0
5.0
3.5
2
46
2
(VIII) Poly(MAAk,DEAAl,
–
BKh)
(VI) Poly(VPk, DEAAl,
19
1
41
–
12
2
BKh)-BSA(Suc)
(IX) Poly(VPk, DEAAl,
–
BKh)
(VII) Poly(VMIk, DEAAl,
40
1
20
–
23
1
BKh)-BSÄ(Suc)
(X)
Poly(VMIk, DEAAl,
BKh)
30000
–
–
–
VMI/DEAA, 75/ 25***
–
(XI) BKh-BSA(Suc)****
–
60
–
16
* NMR data.
** Data of conductometric titration.
*** Data of elemental analysis.
**** Prepared from Suc-BSA and BK in the presence of water-soluble carbodiimide [32].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003

38
VLASOV et al.
+ NC–C(CH3)2–N=N–C(CH3)2–CN
1) H+/H2O; 2) BK; 3) NaBH4
M1 + M2
Poly[(M1)k, (M2)l]
Poly[(M1)k(M2)l, (M3)h]
(IX)
VP
(X)
VMI
DEAA
BK
Conjugate
(VIII)
MAA
DEAA
BK
M1
M2
M3
DEAA
BK
Scheme 3. The preparation of bradykinin-containing polymeric conjugates
immune response against the components of SPPC on (V), (VI) and (VII) (the antisera against these conju-
the basis of BSA containing polymethacrylic acid can gates were produced in the absence of the Freund’s
probably be explained by the activation of immune sys- adjuvant) and the polymeric BK conjugates (VIII),
tem [29–31] by the polyanion structure of the carrier (IX), and (X) containing no BSA.
and the resulting induction of the thymus-independent
immune response [31].
Immunoadjuvant Activity of the BSA Polymeric
The antiserum against conjugate (VI) with poly(N-
Conjugates
vinylpyrrolidone) fragments produced in the presence
The contribution of the polymeric fragments of the
conjugates into the immunoadjuvant activity can be
determined from the data for the antisera produced after
the immunization with conjugates (V), (VI), and (VII)
without Freund’s adjuvant (Table 4). Conjugate (V)
containing polymethacrylic acid gave the antibody titer
against BK and BSA in the absence of Freund’s adju-
vant that was comparable with the similar titers against
the standard conjugate on the basis of Suc-BSA (XI)
also produced without Freund’s adjuvant.
of Freund’s adjuvant had the antibody titer against
bradykinin comparable with that obtained after the
immunization with the standard conjugate (XI) but
lower than that against SPPC (V) with polymethacrylic
acid. Presumably, the introduction of the uncharged
poly(N-vinylpyrrolidone) chains into the polymeric
immunogen results in a decrease in the total negative
charge of the conjugate, which possibly decreases its
immunogenicity.
This proposal can be confirmed by the unusual (at
the first face) immunogenic properties of conjugate
The immunization with conjugate (VI) without Fre-
(VII) with poly(N-vinyl-2-methylimidazole) chains. und’s adjuvant gave a significantly lower antibody titer
We could expect that the polycation structure of poly- against BSA than those obtained after the immuniza-
meric fragments would activate the immune system in tion with conjugates (V) and (XI) (also without Fre-
this case [29–31] like the polyanion structure of poly- und’s adjuvant). It is noteworthy that the antibodies
methacrylic fragments of conjugate (V) discussed against BK were practically absent in the pool of anti-
above. However, we received a significant decrease in bodies produced against conjugate (VI). We can con-
the titer of antibodies both against bradykinin and BSA clude that, in contrast to polymethacrylic acid [29–31],
(Table 4). The explanation of this fact was found after poly(N-vinylpyrrolidone) exhibits no immunoadjuvant
the comparison of immunogenicity of BK conjugates activity.
Table 3. The titers of antibodies to THIQ, VTHIQ, and BSA
Conjugate
Immunogen
Antigen
BSA
Titer (ELISA)
(XIIa)
THIQ10-BSA(Suc)*
204800
not determined
192400
160
THIQ13-Ova**
BSA
(XIIb)
THIQ20-BSA(Suc)*
THIQ13-Ova**
THIQ13-Ova**
THIQ13-Ova**
(Ia)
(Ib)
Poly(VPp, VTHIQq)–HRP
the same
640
2560
* This immunogen was prepared for comparison from Suc-BSA and THIQ by the carbodiimide method [32]; the content of THIQ was
10 mol % for (XIIa) and 20 mol % for (XIIb).
** This antigen was prepared from Ova and THIQ using glutaraldehyde [9].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003

STARBURST CONJUGATES OF PROTEINS
Table 4. Titer of antibodies against BK and BSA
39
Antigen
immunization
with Freund’s adjuvant
immunization
without Freund’s adjuvant
Conjugate
Immunogen
BK-Ova***
BSA
BK-Ova***
BSA
(V)
Poly(MAAk, DEAAlBKh)-BSA(Suc)
Poly(MAAk, DEAAlBKh)
Poly(VPkDEAAlBKh)-BSA(Suc)
Poly(VPk, DEAAlBKh)
5120
1280
2560
*
102400
*
320
**
*
2560
**
(VIII)
(VI)
(IX)
(VII)
(X)
3200
*
160
**
**
*
Poly(VMIk, DEAAlBKh)-BSA(Suc)
Poly(VMIk, DEAAlBKh)
40
640
*
*
320
2560
**
320
**
(XI)
BKh-BSA(Suc)***
6400
2560
* Titer was not determined.
** Immunization was not carried out.
*** This immunogen was prepared from Ova and BK using glutaraldehyde [9].
The antisera in which antibodies against both brady- conjugates (VII) and (X) were compared. The BK con-
kinin and BSA were absent were obtained after the jugate on the basis of poly(N-vinyl-2-methylimidazole)
immunization with conjugate (VII) without Freund’s (X) ranked below the polymethacryl BK conjugate
adjuvant.
(VIII), whereas it ranked far above the SPPC (VII).
Conjugate (VII) includes both polycation poly(N-
vinyl-2-methylimidazole) fragments and polyanion
fragments of Suc-BSA; each of them could separately
activate the immune system. However, the immunoge-
nicity of this conjugate decreases due to significant
neutralization of its total charge.
The immunization with the polymeric conjugates of
bradykinin (VIII), (IX), and (X) containing no protein
component (BSA) that could activate the immune sys-
tem itself was carried out, in order to find all the reasons
for the decrease in the antibody production against the
components of conjugate (VII) in the presence of Fre-
und’s adjuvant and its complete absence after the
immunization in the absence of it.
CONCLUSIONS
As expected [29–31], polycation conjugate (X) as
well as the polyanion conjugate (VIII) can activate the
immune system and induce the immune response
against BK, which distinguish them from conjugate
(IX), obtained on the basis of uncharged poly(N-
vinylpyrrolidone).
We developed two approaches to the synthesis of
SPPCs containing low molecular biologically active
compounds in the polymeric fragments one-point
bound to protein. The first approach (polymerization,
methodA), considered by the example of the salsolinol-
containing conjugates, involved the copolymerization
of 1-vinyl derivative of salsolinol (VTHIQ) with the
corresponding vinyl monomer (VP) with the use of the
preliminarily synthesized macroinitiator on the basis of
HRP. The second approach (polycondensation, method
B) involved the preliminary preparation of SPPCs on
the basis of Suc-BSA. The polymeric fragment in these
SPPC was subjected to the acidic hydrolysis, and a part
of diacetal groups was converted into aldehyde groups
that were further used for coupling with bradykinin.
Contribution of Suc-BSA into the Immunogenicity
of Its Conjugates
The titers of antibodies produced on the basis of
polyanion BK conjugate (VIII) should be compared
with those produced against SPPC (V) containing poly-
mer of the same structure and Suc-BSA in order to
determine the contribution of the protein component
into the immunogenicity of the conjugates. In the case
of (V), the antibody titer against BK was obviously
We studied the immunogenic properties of SPPCs
higher than that against BK on the polymer carrier containing the salsolinol analogue and bradykinin in
(VIII). Similar results were obtained for the pair of the polymeric fragment and demonstrated that changes
conjugates (VI) and (IX); the only difference was that in the chemical nature of the polymeric component
conjugate (IX) was practically nonimmunogenic. could, to some extent, regulate the immune response
These results unambiguously confirm a certain contri- against both the low molecular hapten and the protein
bution of Suc-BSA in the immunogenicity of conju- component. The immune response against the conju-
gates (V) and (VI). However, the reversed relationship gate can be enhanced by the use of polymethacrylic
was observed when the immunogenic properties of acid fragments as modifying polymers. On the other
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003

40
VLASOV et al.
hand, the application of poly(N-vinyl-2-methylimid- Sephadex G-75 at a flow rate of 20 ml/h. MI-2 was
azole) fragments as polymeric modifiers of the protein eluted with dilute hydrochloric acid (pH 3.2–3.7) and
carrier provides a decrease in the immune response lyophilized. MI-1 was eluted with 0.15 M NaCl. The
against both low molecular hapten and polymer-protein products were detected at 280 nm. The MI-1 eluate was
carrier. Thus, we can directionally regulate the immu- dialyzed against water for 24 h and lyophilized. The
nogenicity of SPPCs containing low molecular biolog- content of the modified residues of 2,2'-azobisisobutiric
ically active compounds in the polymeric fragments in acid (m) was determined as a difference between the
dependence on the aim of their further application.
content of residual amino groups in the starting (n) and
modified protein by the titration of amino groups with
TNBS [35]. The activity of horseradish peroxidase was
determined by the method [36] in PCB with the addi-
tion of hydrogen peroxide using Ó-phenylenediamine
as a substrate.
EXPERIMENTAL
Bovine serum albumin, ovalbumin, dopamine, and
N-vinylpyrrolidone (Merck, Germany); N-vinyl-2-
methylimidazole (Fluka, Switzerland); bradykinin
(Saxon Biochemical GMBH, Germany); 1-ethyl-3-(3'-
dimethylaminopropyl)carbodiimide (Sigma, United
States); glutaraldehyde, trinitrobenzenesulfonic acid,
and methacrylic acid (Serva, Germany); horseradish
peroxidase (Biolar, Lithuania); and acrolein diethylac-
etal and 2,2'-azobisisobutyronitrile (Reakhim, Russia)
were used in this study. The compounds were purified
by distillation in a vacuum or by recrystallization if
necessary. Visking dialysis tubes (Serva) were used for
dialysis. The amino acid analysis of the conjugates was
performed on a AAA T339M Mikrotecna Praha (Czech
Republic) after acidic hydrolysis. Potentiometric titra-
tions were carried out on an automatic titrator (Institute
of High-molecular Compounds, Russian Academy of
Sciences). ELISA was performed on anAKI-Ts-01 col-
orimetric immunoenzyme analyzer (OKBA, Russia).
1H NMR spectra were recorded on an AC 200 spec-
trometer (Bruker) with the working frequency of
200.13 MHz. Chemical shifts were measured using the
residual protons resonances of the deuterated solvent
(4.8 ppm for HDO). Standard impulse sequences were
used for the spectra with inhibition of the solvent reso-
nances and without the inhibition.
Succinylated macroinitiator on the basis of BSA
(MI-2S). Succinic anhydride (0.8 g per g of BSA) was
portionwise added to a 1% solution of MI-2 in 1 M
sodium bicarbonate buffer (pH 8.0) for 1 h at 25°ë. The
value of pH (8.0) was maintained by addition of 1 M
NaOH. After two hours, the reaction mixture was
diluted with twofold volume of water, dialyzed against
water for 24 h, and lyophilized. The absence of free
amino groups was determined by the reaction with
TNBS [35].
Starburst polymer–protein conjugates on the
basis of HRP (Ia) and (Ib) (method A). A mixture of
VP and VTHIG was polymerized using MI-1 (the ratio
of MI-1 to the monomer mixture was 1 : 300) in
ampoules in the argon atmosphere in 0.1 M phosphate
buffer (pH 7.7) at 65°C for 2–2.5 h. The VP/VTHIQ
molar ratios were 70 : 30 and 90 : 10, for (Ia) and (Ib),
respectively. After the polymerization was completed,
the reaction mixture was fractionated on a column
(2.5 × 90 cm) with Sephadex G-75. The conjugates
were eluted with 0.15 M NaCl and detected at 280 nm.
The eluates containing (Ia) and (Ib) were dialyzed
against water for 24 h and lyophilized. The enzymatic
activity of the HRP polymeric conjugates was deter-
mined by the method [35]. The content of protein com-
ponent and the molecular mass of the conjugates were
determined spectrophotometrically by the Lowry
method [37]. The molar content of VTHIQ in the poly-
meric part of the conjugate was determined spectropho-
tometrically at 280 nm as a difference between the molar
absorption coefficients of the protein component of the
conjugate and the polymeric conjugate with hapten.
1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoqui-
noline (salsolinol) [33]. A solution of dopamine hydro-
chloride (1.53 g, 8.1 mmol) and acetaldehyde (0.7 g,
15.9 mmol) in distilled water was stirred for 72 h at
room temperature. Water was removed by distillation in
a vacuum, and the residue was treated with anhydrous
ether; mp 196–197°ë (literature mp 197–198°ë [33]).
1-Vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquino-
line (1-vinyl-salsolinol) was synthesized from dopam-
ine and acrolein as described above for salsolinol; mp
94–96°ë; the elemental analysis (C, H, and N content)
coincided with the calculated data.
Starburst polymer–protein conjugates on the
basis of suc-BSA (II), (III), and (IV) (method B).
Mixtures of DEAA with MAA, VP, and VMI were
polymerized using MI-2S under the conditions
Macroinitiators on the basis of HRP (MI-1) and described above for conjugates (Ia) and (Ib). The ratio
BSA (MI-2) were prepared by the reaction of 2,2'-azo- of the macroinitiator to the monomer mixture was
bisisobutiric acid dimethylimidate dihydrochloride 1 : 2000–4000 in dependence of the number of residues
[34] with the proteins at 0°C and pH 10–11 under stir- of 2,2'-azobisisobutiric acid (m). The molar ratio of
ring for 1 h. The protein concentration was 1.0 mg/ml, monomers was 30 : 70. When the polymerization was
and the molar ratio of 2,2'-azobisisobutiric acid dimeth- completed, the reaction mixture was fractionated on a
ylimidate dihydrochloride to the protein was 100 : 1. column (2.5 × 90 cm) with Sephadex G-75 eluted with
The reaction mixture was dialyzed for 24 h against dilute hydrochloric acid (pH 3.5). The molar contents
water and fractionated on a column (2.5 × 90 cm) with of acrolein and acrolein diethylacetal were determined
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003

STARBURST CONJUGATES OF PROTEINS
41
by NMR. Within the range of studied concentrations
Conjugates of salsolinol (XIIa and XIIb) and BK
and the ratios of the protein and the polymer, the reso- (XI) with Suc-BSA were prepared as described in [32].
nances from BSA were significantly broadened, and the The water-soluble 1-ethyl-3-(3'-dimethylaminopro-
contribution of the protein protons into the integral pyl)carbodiimide (1 mol per each carboxyl group of
intensity was not taken into account to calculate the Suc-BSA) was added to a 0.5% solution of the protein
content of diethylacetal groups and aldehyde groups. in 0.1 M phosphate buffer (pH 4.5). The reaction mix-
The correctness of this assumption was checked in the ture was stirred at room temperature for 1 h, the pH
1H NMR spectra of BSA according to the intensities of
the resonances in the field near 1 ppm, which was
uncharacteristic of these polymers. The aldehyde molar
content was calculated from the resonances of aldehyde
protons at 9.12 and 9.14 ppm, methyl group of MAA at
1.25 ppm, VP protons at 3.30, 3.35, and 3.4 ppm, and
imidazole protons at 6.43 and 7.35 ppm.
value was adjusted to 7, and a 0.5% solution of BK or
THIG (30–40 mol per mol of protein) in 0.1 M borate
buffer (pH 9.6) was added. The reaction mixture was
stirred for 3 h at room temperature, dialyzed against
water for 40 h, and lyophilized. The molar content of
THIQ in the conjugate was determined spectrophoto-
metrically at 280 nm as a difference between the molar
absorption coefficients of the polymeric conjugate with
the hapten and the polymeric component in the conju-
gate. The molar content of BK was determined by
amino acid analysis after the acidic hydrolysis of the
conjugate.
Preparation of antisera. All the immunogens—
SPPC (V)–(VII), polymeric conjugates (VIII)–(X)
containing BK (Table 4), SPPC (Ia) and (Ib) containing
salsolinol (Table 3), salsolinol conjugates (XIIa) and
(XIIb) (Table 3), and conjugate of BK with BSA (XI)
(Table 4)—were dissolved in a isotonic saline solution,
and complete Freund’s adjuvant (Difco, United States)
was added. Rabbits were subcutaneously injected with
these mixtures in several points of back. The antigen
was injected with incomplete Freund’s adjuvant after a
week and with complete Freund’s adjuvant after two
weeks. The last injection was carried out after a week
without adjuvant. The total antigen amount of 4 mg was
used for the immunization. The antibody titer was
determined one week after the intravenous injection.
Blood was taken from the marginal ear vein one time a
week during four weeks.
An immunoenzyme analysis (ELISA) of antisera.
The antibodies were isolated from the antisera by the
precipitation with ammonium sulfate, and the solutions
with the starting concentration of 10 mg/ml were pre-
pared. The titers of the antibodies were determined by
the solid-phase ELISA in the 96-well plates (Biohit OY,
Finland). A 0.01 M phosphate buffer (pH 7.2–7.4) con-
taining 1% NaCl (PBS) and 0.05% Tween-20 (PBST)
was used for washing the wells and the dilution of the
protein A conjugate with horseradish peroxidase. The
antibodies were diluted in 0.1 M PBS with 0.05%
Tween-20. The solutions (100 µl) of BSA, BK–Ova,
THIQ–Ova, and Ova (control) with concentrations of
100 µg/ml were added to the wells in 0.1 M sodium
bicarbonate buffer (pH 9.6). The mixtures were incu-
bated for 20 h at 4°ë. After the antigen solutions were
removed, the wells were washed with 0.01 M PBS, and
a 0.5% solution of casein in 0.1 M PBS (150 µl) was
added into each well. The plates were incubated for 1 h
at 37°ë. The wells were washed with PBST six times at
Starburst conjugates containing BK in poly-
methacryloyl (V) poly(N-vinylpyrrolidone) (VI), or
poly(N-vinyl-2-methylimidazole) (VII) fragments
prepared on the basis of suc-BSA. BK was attached
to the aldehyde groups of polymer–protein conjugates
as described in [9]. The solution of BK (20–40 mol per
mol of conjugate) in 0.1 M borate buffer (pH 10) was
added to a solution of a conjugate (II), (III), or (IV) in
the same buffer. The reaction mixture was kept at room
temperature for 4 h. The Schiff bases in the conjugates
were reduced by treatment with NaBH4. The reaction
mixture was dialyzed against water for 40 h and lyo-
philized. The bradykinin molar content was determined
by amino acid analysis after the acidic hydrolysis of the
conjugates.
Copolymers of DEAA with MAA, VP, and VMI
were prepared in ethanol in the argon atmosphere in
ampoules at 65°ë for 48 h in the presence of 2,2'-azo-
bisisobutyronitrile, an initiator of radical polymeriza-
tion. The total concentration of monomers was 20%,
the initiator concentration was 0.5% relative to the
monomer concentration, and the molar ratio of mono-
mers was 30 : 70. The viscous solution was diluted with
ethanol and reprecipitated by acetone. Molecular
masses of the copolymers were determined by visco-
metry. The content of methacrylic acid in the copoly-
mer was determined by the conductometric titration.
The composition of copolymer with VMI was deter-
mined by elemental analysis according to the nitrogen
content. The composition of copolymers containing VP
and DEAA was determined by NMR.
BK conjugates of copolymers (VIII), (IX), and
(X) were prepared by the BK coupling with aldehyde
groups of the copolymers similarly to the preparation of
SPPC (V), (VI), and (VII). The copolymers were
treated with hydrochloric acid (pH 3.5), lyophilized,
and dissolved in 0.1 M borate buffer (pH 10). BK was
added to the solution, and the synthesis was carried out
at room temperature for 4 h. The copolymer–peptide
molar ratio was 1 : 20–40. The Schiff bases in the con-
jugates were reduced with NaBH4. The reaction mix-
ture was dialyzed against water for 40 h and lyo- intervals of 1 min, and the solutions of antibodies
philized. The molar content of BK in conjugates (VIII), (100 µl) were added starting from the dilution of 1/20.
(IX), and (X) was determined by amino acid analysis.
The mixtures were incubated for 1 h at 37°ë. The solu-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 29 No. 1 2003

42
VLASOV et al.
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tions of antibodies were removed, the wells were six
times washed with PBST, and the solution of protein A
labeled with the horseradish peroxidase (100 µl) was
added and kept for 30 min at 37°ë. The conjugate solu-
tion was removed, the wells were six times washed with
PBST at intervals of 1 min, and the substrate (0.5%
solution of Ó-phenylenediamine in 0.1 M citrate–phos-
phate buffer with pH 5.0 containing 0.03% ç2é2) was
added. The reaction was stopped by the addition of 1 M
H2SO4 (100 µl) 30 min later. Optical absorption was
measured in the wells at 492 nm. The results were con-
sidered as positive at A/Ak > 2, where A is the absorp-
tion in the wells with the antigen and Ak, the absorption
in the control wells with Ova.
18. Ding, Z., Chen, G., and Hoffman, A.S., Bioconjug.
Chem., 1997, vol. 7, pp. 121–125.
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ACKNOWLEDGMENTS
This study was supported by MNF, projects no.
R67000 and R67300, the program New Methods of
Bioengineering, project no. 03-0003H-329, and by the
program Scientific Schools of Russian Federation,
project no. 15-97393.
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