As. Dilgimen et al., Water-soluble covalent conjugates of bovine serum albumin with anionic poly(N-isopropyl-acrylamide) and their immunogenicity, BIOMATERIAL, 22(17), 2001, pp. 2383-2392
We have conjugated bovine serum albumin (BSA) to poly(N-isopropylacrylamide
-co-acrylic acid) (poly(NIPAAm-AA)) by using water-soluble carbodiimide, an
d the effects of the bulk mass ratio of protein to polymer (r) on the forma
tion of polymer-protein conjugates have been studied. HPLC, electrophoresis
, viscosimetry and fluorescence spectroscopy suggest that the mode of coval
ent binding of BSA to poly(NIPAAm-AA) depends upon the weight concentration
ratio (r) of BSA to poly(NIPAAm-AA). At r less than or equal to 1, free po
ly(NIPAAm-AA) molecules coexist with conjugate, and when r reaches I the am
ount of free polymer is too small to be observed. It is shown that dependin
g on the ratio r, two types of conjugate particles were formed: at r < 1, t
he protein molecules in the structure of conjugate particles are densely co
vered as a shell by polymer chain and practically "fenced off" from water e
nvironment; at r > I the conjugate-forming particles possess more friable s
tructures in which protein molecules are practically exposed to the solvent
. The complex formation involving electrostatic interactions between BSA an
d carbodiimide activated polymer are proposed as the driving force for the
covalent binding of BSA to polymer macromolecules. The coil-globule transit
ion of macromolecules in low and thermally induced precipitation in more co
ncentrated solutions of bioconjugates was observed. The immunogenic propert
ies of covalent conjugates of CP-BSA were investigated and the temperature-
modulated solubility-immunogenicity alterations was analyzed. A single immu
nization of mice with conjugates at the thermally precipitating concentrati
on without an adjuvant evoked increased specific immune response to BSA, wh
ich practically did not depend on the initial conjugation ratio of componen
ts. Such a modulated system is attractive for application as a novel immuno
genic system in vaccine technology. (C) 2001 Elsevier Science Ltd. All righ
ts reserved.