Electrophoretic behavior of microgel-immobilized polyions.

Electrophoretic behavior was studied for N-isopropylacrylamide (NIPA) microgels, into which different amounts of poly(acrylic acid) (PAAc) were physically entrapped. Copolymer microgels of NIPA with acrylic acid (AAc) were also studied as a control. Electrophoretic mobility was measured in 0.1 M NaCl solution at 25 °C as a function of pH, using an electrophoretic light scattering technique. The mobility of the copolymer microgel whose COOH groups are fully ionized agreed with that of PAAc when its ionization degree (α(n)) is close to the mole fraction (f(AAc)) of the AAc unit in the copolymer gel. There was good agreement between the mobility values of the copolymer microgel and the linear NIPA/AAc copolymer when their AAc contents are very close to each other. However, the mobility of the microgel with immobilized PAAc was higher than that of the copolymer microgel, even when there was no difference in the AAc content for both microgels. Moreover, the immobilized PAAc showed a higher mobility than the free PAAc when its α(n) is equal to f(AAc) in the immobilized system. No correlation was observed between the mobility and the hydrodynamic radius. These results were discussed in terms of the free draining model (FDM) for the electrophoresis of polyelectrolytes. It became apparent that the mobility difference depending upon whether (i) the PAAc ions are in the cage of the NIPA network or (ii) the AAc units are copolymerized with the network chain is due to the structural difference of the segments considered in the FDM.

Swelling-shrinking behavior of chemically cross-linked polypeptide gels from poly(α-L-lysine), poly(α-DL-lysine), poly(ɛ-L-lysine) and thermally prepared poly(lysine): effects of pH, temperature and additives in the solution.

We synthesized the glutaraldehyde cross-linked hydrogels using four kinds of poly(lysine)s (PLs) and measured the equilibrium swelling ratio (Q) as a function of pH. Also measured was the temperature change of Q at a fixed pH (11.6) in the absence and presence of additives (LiBr, methanol and urea) that affect the secondary structure of PLs. The swelling data were examined using a force balance approach in which the repulsive and attractive interactions among the cross-linked PL chains were considered based on the conformational properties of PLs in aqueous solutions. It was found that the formation of the helical segments in the cross-linked chain has little effect in the gel collapse, but their association acts as the attractive interaction causing the gel to shrink. The formation of the beta-sheet structure within the network also acts as the attractive interaction. These attractive interactions are mainly due to the hydrogen bonding, but hydrophobic interactions between the lysine side chains should be considered. In addition, in the swelling behavior of all the PL gels the polyampholyte nature appears due to electrostatic interactions of the basic groups with the C-terminal carboxyl group.

Conductometric and light scattering studies on the complexation between cationic polyelectrolyte nanogel and anionic polyion.

This work aims to provide a basic understanding of the water dispersibility of a 1:1 stoichiometric polyelectrolyte complex (SPEC) in water in the absence of low-molecular-weight salts. We studied the complexation of a linear polyanion, potassium poly(vinyl alcohol sulfate) (KPVS), with a cationic polyelectrolyte nanogel (CPENG) composed of a lightly cross-linked copolymer of N-isopropylacrylamide and 1-vinylimidazole, in an aqueous salt-free solution (pH 3 and 25 °C), as a function of the molar mixing ratio (Mmr) of anionic to cationic groups. Also studied for comparison was the complexation of KPVS with poly(diallyldimethylammonium chloride) (PDDA), which is a standard reaction in colloid titration. Turbidimetric and conductometric measurements were used in combination of dynamic light scattering (DLS). An abrupt increase of turbidity curve and a break of conductivity curve were observed at Mmr =1 when KPVS was added to the CPENG or PDDA solution, indicating the formation of SPEC. All the complexes formed at Mmr ≤ 1 were water-dispersible and hence characterized by DLS. The CONTIN analysis of DLS data showed that (i) an increase of Mmr causes a decrease of the hydrodynamic radius (R(h)) of the nanogel complex particle but (ii) the R(h) of the PDDA complex remains unchanged at Mmr < 0.8. Taking these into account, we discussed the conductometric results in terms of the random model (RM) and all-or-none model (AONM) in polyelectrolyte complex formations. It was found that KPVS and PDDA yield a water-dispersible SPEC particle at each Mmr, accompanying the uptake of counterions (K(+) and Cl(-)) by the complex. This uptake amount was about 7% of the stoichiometric release of the counterions. In the nanogel system, a complete release of the counterions was observed at Mmr < 0.2 at which one or two KPVS chains were bound to a CPENG particle, but further KPVS binding led to about 20% of the counterion uptake to maintain electroneutrality. Thus, we suggest that the counterion uptake becomes a key factor to understand the water dispersibility of SPEC particles.

On the water dispersibility of a 1:1 stoichiometric complex between a cationic nanogel and linear polyanion.

This work aimed to obtain information on the water dispersibility of a 1:1 stoichiometric polyelectrolyte nanogel complex (SPENC). We synthesized a cationic polyelectrolyte nanogel (CPENG) composed of a cross-linked copolymer of 1-vinylimidazole and N-isopropylacrylamide. SPENC was then prepared at 25 degrees C from the mixing of equimolar amounts (based on fixed charges) of CPENG and potassium poly(vinyl alcohol) sulfate, which were dissolved in an aqueous solution without adding salt and at pH 3.0. We carefully observed at 25 degrees C the reduction of the imidazole-based cationic charge in the CPENG component of SPENC as a function of pH. Dynamic and static light scattering techniques were employed in combination with electrophoretic light scattering experiments. The amount of cationic charge in the SPENC was estimated from the potentiometric titration data of CPENG. It was found that, during the charge reduction process, the complex underwent aggregation, followed by a phase separation. The aggregation started at about 25% of the charge reduction (i.e., at pH approximately = 4.9), and the phase separation took place when almost half of the charge was eliminated (at pH approximately = 5.5). However, the phase-separated complexes became water-soluble again when about 90% of the charge was eliminated (pH approximately = 6.6). By colloid titration, the dissociated free polyanions were not detected in the aqueous SPENC solution before the phase separation but were detected in the complex-redispersed solution. When the pH (9.0) of the redispersion was slowly decreased to the original level (pH 3.0) by the gradual addition of HCl so as to cause again the phase separation, an intraparticle complex was reformed, the physical quantities of which were close to those of the initial SPENC. These findings clearly indicate that the whole and a part (segment) of the complexed polyanions undergoes dissociation-association reactions on the surface of a SPENC particle, depending on the ionization state of the cationic gel component. As a result, these reactions seem to be a key factor for the water dispersibility of the SPENC.

Drying dissipative structures of lycopodium spore particles in aqueous dispersion.

Drying dissipative structures of aqueous dispersions of lycopodium particles (31 microm in average diameter) from the spores of Lycopodium clavatum were studied as a function of the particle concentrations in the presence and the absence of sodium chloride. The drying patterns formed on a cover glass, a watch glass and a Petri glass dish were observed macroscopically and microscopically. Lycopodium particles were the combination of hemisphere and tetrahedron in their shape and possessed the weakly acidic groups on their surfaces. The vague broad ring structure was observed even for the very large colloidal particles, and their size on a cover glass decreased as particle concentration decreased. Microscopic drying patterns almost disappeared except the short chain-like patterns. These observations support that the convectional contribution on the dissipative drying pattern formation is still effective in the lycopodium suspensions, though the convectional forces in the suspension are very weak compared with the sedimentation forces.

On the temperature-responsive polymers and gels based on N-propylacrylamides and N-propylmethacrylamides.

We studied the behavior in water of polymers, microgels, and macrogels based on the following four monomers: N-isopropylacrylamide (NiPA), N-isopropylmethacrylamide (NiPMA), N-n-propylacrylamide (NnPA), and N-n-propylmethacrylamide (NnPMA). The thermal phase separation of polymers in water as well as of microgels in the aqueous dispersion was examined by a combination of turbidity measurements and differential scanning calorimetry (DSC). The hydrodynamic radius of microgels and the swelling degree of macrogels (fine cylindrical bulk gels) were also examined as a function of temperature using the dynamic light scattering and the microscopic method, respectively. It was found that all the polymers prepared are water-soluble and clearly exhibit the phase separation on heating. The phase separation temperature varies depending on the constituent monomers and becomes higher in the order of NiPMA > NiPA > NnPMA > NnPA. The endothermic enthalpy from the heating DSC curves increases in the order of NnPMA > NnPA approximately NiPMA > NiPA. The same trends were observed in the microgels based on NiPA, NiPMA, and NnPA, which were synthesized via chemical cross-linking with N,N'-methylenebis(acrylamide) (Bis). Although we were unable to synthesize the microgel of NnPMA due to a low water solubility of the monomer, its bulk gel was obtained by gamma-ray irradiation to an aqueous poly(NnPMA) solution at a dose of 10 kGy. An irradiation-cross-linked NiPMA gel was also prepared as a counterpart to the Bis-cross-linked gel. We then studied the gel collapses upon heating by use of the chemically cross-linked gels based on NiPA, NiPMA, and NnPA as well as of the irradiation-cross-linked NnPMA and NiPMA gels. All the gels underwent the collapse transition at a certain temperature which is close to or slightly higher than the phase separation temperature of the corresponding polymer solutions or microgel dispersions. These results indicate that in both the linear and cross-linked polymers there is no difference in the thermally induced interactions between the segments as well as between the segment and the solvent, but these interactions are dependent on the structure of the constituent monomers, i.e., whether the alpha-carbon bears a hydrogen atom or a methyl group and whether the N-propyl group is branched or straight chain. The structure dependence was discussed in terms of amide-amide and amide-water hydrogen bondings as well as of a possible hydrogen bonding of solvent water with the H-C bond of the alkyl groups. Then, water clustering around both the alkyl and the amide groups was considered.

Geometrical characteristics of polyelectrolyte nanogel particles and their polyelectrolyte complexes studied by dynamic and static light scattering.

The geometric characteristics of nanogel particles in aqueous solutions were studied by determining their ratios of radius of gyration (mean-square radius; Rg) to hydrodynamic radius (Rh), Rg/Rh, derived from static light scattering and dynamic light scattering experiments, respectively. The various nanogel samples studied included ones composed of lightly cross-linked N-isopropylacrylamide (NIPA) polymer, NIPA-based anionic or cationic copolymers, and amphoteric terpolymers. Polyelectrolyte complexes between anionic or cationic nanogels and oppositely charged polyions or nanogels having opposite charges were also studied. Most NIPA and NIPA-based polyelectrolyte nanogels in a swollen state had Rg/Rh values >0.775, which is the theoretically predicted value for a solid sphere. In a collapsed state, one may expect nanogel particles to be spherical in shape; however, this was not the case for a variety of nanogel samples, either with or without charges. These data were consistent with the idea that the surfaces of these nanogel particles were decorated with attached dangling chains. The Rg/Rh data from polyelectrolyte-nanogel complexes, however, indicated different structures from this. It was found that most of the polyelectrolyte-nanogel complex particles had Rg/Rh approximately 0.775. This suggested that the complexed nanogel particles were spherical in shape and that there were no dangling surface chains.

On an intraparticle complex of cationic nanogel with a stoichiometric amount of bound polyanions.

A polyelectrolyte nanogel (PENG) particle consisting of lightly cross-linked terpolymer chains of N-isopropylacrylamide, acrylic acid, and 1-vinylimidazole has positive charges in an aqueous medium at pH 3 due to protonation of the imidazole groups, and thereby forms a polyelectrolyte complex with the linear polyanion, potassium poly(vinyl alcohol) sulfate (KPVS). It has been demonstrated that the hydrodynamic radius (Rh), by dynamic light scattering (DLS), and the radius of gyration (Rg), by static light scattering (SLS), of the complex particles are smallest at approximately 1:1 mixing ratio (rm) of anions to cations, in the absence of simple salts such as KCl (Langmuir 2005, 21, 4830). Here, we aimed to study the nature of the complex formed at rm=1 and examined the complex formation process by electrophoretic light scattering (ELS). It was found that the mobility of the cationic PENG with a stoichiometric amount of bound KPVS anions (i.e., the complex formed at rm=1) is positive but not zero at 25 degrees C. This was also the case when the complex was examined by ELS at 45 degrees C, where DLS and SLS show a temperature-driven collapse of the complex. We thus assumed that (a) electroneutrality is maintained in the complex particle with the aid of counterions, but (b) the complex is highly polarizable, and hence (c) during ELS the KPVS anions would dissociate in part from the complex. This hypothesis was supported by the following results: (i) Mixing complexed and uncomplexed PENG particles at different ratios brings about an increase in Rh and a decrease in the light scattering intensity of the complex at the same time, suggesting a polyelectrolyte exchange reaction. (ii) The same phenomenon is seen when poly(diallyldimethylammonium chloride) (PDDA as a polysalt) is added to the complex dispersion, meaning that the PDDA takes out the KPVS from the complex to form a stable PDDA-KPVS complex. (iii) Upon addition of KCl, the complex undergoes little change in Rh (62-67 nm) at a salt concentration (Cs)0.2 M. (iv) The Rh (78 nm) of the soluble complex at Cs from 0.3 to 0.5 M is larger than that at Cs<0.02 M, suggesting dissociation of the KPVS ions. (v) Complexation between KPVS and PDDA as mentioned in (ii) is facilitated in the presence of 0.01 M KCl.

A mycelium with polyelectrolyte complex-bunched hyphae: several important factors affecting on the fermentation performance at a very high cell density.

We have reported in the previous paper (Colloids Surf. B (2006) in press) a marked increase in the rate of gluconic acid production at a very high cell concentration (40 g/l) of filamentous fungus (Aspergillus niger IFO 31012) which was immobilized with polyelectrolyte complex consisting of potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide [6-O-(2-hydroxyethyl)-2-(trimethylammonio)-chitosan iodide]. The present study was carried out to look at what factors play a crucial role in this enhancement. We measured viscosity of broth, mass-transfer coefficient (k(L)a) for oxygen and diffusion coefficient of glucose (substrate). It has become apparent that there is only a difference in the diffusion coefficient of glucose between the free and immobilized cells. Therefore, we believe that the diffusion limitation by substrates as a problem in submerged mycelial processes is improved by immobilization based on polyelectrolyte complexes.

A mycelium with polyelectrolyte complex-bunched hyphae: preparation and fermentation performance.

We studied the immobilization of a mycelium (Aspergillus niger) using the working hypothesis as follows: (a) when polycation was added to the cell suspension, a few parts of it would bind on the surface of a hypha, allowing to gather the hyphae in part but not all; (b) upon further addition of polyanion, such a gathering of the hyphae is tightly bunched by the polyelectrolyte complex (PEC) which is resulted from the remaining polycation; (c) as a result, a mycelium with partially bunched hyphae can be obtained. Potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide [6-O-(2-hydroxyethyl-2-(trimethylamonio)-chitosan iodide) were used as the polyanion and the polycation, respectively. The optical and electron microscopic analyses showed that our immobilized cell contains many of PEC-bunched hyphae. The sedimentation rate increased with the weight ratio of PEC to dry cells and leveled off at the weight ratio larger than 0.5. The gluconic acid production from glucose was studied by a semi-large scale (1l) cultivation of the imobilized and free cells using a jar fermentor. It was found that an apparent specific activity of the immobilized cells for glucose oxidation becomes 1.44 times that of the free cells even at a high cell density of 40 g/l.

Static light scattering study of complex formation between protein and neutral water-soluble polymer.

Complex formation of poly(N-isopropylacrylamide) (PNIPA) having a weight-average molecular weight of 1,720,000g/mol with human serum albumin (HSA), ovalbumin (OVA) and lysozyme (LYZ) was studied in an aqueous medium containing 0.01 M NaCl and adjusted to pH 3. The polymer-protein mixtures at different molar ratios (r(m)) were examined by static light scattering (SLS). The analysis of SLS data using our own approach [Kokufuta et al., Langmuir 15 (1999) 940; Biomacromolecules 4 (2003) 728] showed that the molecular weight of each resulting complex is smaller than that of the interpolymer complex composed of two polymer chains plus one protein. This indicates the formation of an intrapolymer complex in all the polymer-protein systems studied. Thus, at each r(m) we calculated the number of bound proteins per polymer, the value of which was OVA>HSA>LYZ in order. These results were compared with the hydropathy profiles of each protein which are a good tool for obtaining an information about distribution of hydrophobic and hydrophilic segments in a protein. It has become apparent that the hydrophobic interaction between polymer and protein plays an important role in the intrapolymer complex formation.

Light scattering study of complex formation between protein and polyelectrolyte at various ionic strengths.

Formation of protein-polyelectrolyte complexes (PPCs) between bovine serum albumin (BSA) and potassium poly (vinyl alcohol) sulfate (KPVS) was studied at pH 3 as a function of ionic strength. Turbidimetric titration was employed by a combination of dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The formal charge (Z(PPC)) of the resulting PPCs at different ionic strengths were estimated from ELS data by assuming the free draining and the non-free draining model. The radius of a BSA molecule in the complex was used in the former model for calculation of Z(PPC) with the Henry's equation, while in the latter case the hydrodynamic radius of a PPC particle determined from DLS was employed. The results obtained were compared with the Z(PPC) values calculated using a relation of Z(PPC)=n(b)Z(BSA)+alphaZ(KPVS), where Z(BSA) (> or =0) and Z(KPVS) (< or =0) denote the formal charge of BSA and KPVS, respectively. Moreover, n(b) is the number of bound proteins per complex composed of alpha polymer chains. It was suggested that the PPC between BSA and KPVS behaves as a free draining molecule during the electrophoresis, at least at a high ionic strength. Also suggested is that the PPC formation at low ionic strength follows a 1:1 stoichiometry in the charge neutralization.

Light-scattering study of polyelectrolyte complex formation between anionic and cationic nanogels in an aqueous salt-free system.

We studied complex formation in an aqueous salt-free system (pH approximately 3 and at 25 degrees C) between nanogel particles having opposite charges. Anionic gel (AG) and cationic gel (CG) particles consist of lightly cross-linked N-isopropylacrylamide (NIPA) copolymers with 2-acrylamido-2-methylpropane sulfonic acid and with 1-vinylimidazole, respectively. The number of charges per particle was -4490 for AG and +20 300 for CG, as estimated from their molar masses (3.33 MD for AG and 11.7 MD for CG) by static light scattering (SLS) and their charge densities (1.35 mmol/g for AG and 1.74 mmol/g for CG) by potentiometric titration. The complexes were formed through the addition of AG to CG and vice versa using a turbidimetric titration technique. At the endpoint of the titration, the aggregate formed was a complex based upon stoichiometric charge neutralization: CG(n)()(+) + xAG(m)()(-) --> CG(n)()(+) (AG(m)()(-))(x)() where x = (n)()/(m)(). At different stages of the titration before the endpoint, the resulting complexes were examined in detail using dynamic light scattering, SLS, and electrophoretic light scattering (ELS). The main results are summarized as follows: (i) When AG with a hydrodynamic radius (R(h)) of 119 nm is added to CG (R(h) approximately 156 nm), the (R(h)) of the complex size decreases from 156 to 80 nm. (ii) In contrast to this (R(h)) change, the molar mass increases from 11.7 MD to 24 MD with increasing amounts of added AG. (iii) Upon addition of CG to AG, the complex formed has the same size ((R(h)) approximately 80 nm) and the same molar mass (55 +/- 2.5 MD) until 55 +/- 5% of AG has been consumed in the complexation. To understand these results, we used the following two models: the random model (RM), in which the added AG particles uniformly bind to all of the CG particles in the system via a strong electrostatic attraction, and the all-or-none model (AONM), in which part of the AG particles in the system preferably bind to the added CG particles to neutralize their electric charges but the other AG particles are uncomplexed and remain in the system. The complex formations upon addition of AG to CG and CG to AG were elucidated in terms of RM and AONM, respectively.

Polyelectrolyte gel transitions: experimental aspects of charge inhomogeneity in the swelling and segmental attractions in the shrinking.

This paper aims to provide a systematic discussion based on our experimental results both previously published and unpublished, to promote better understanding of volume-phase transitions in polyelectrolyte gels. Special attention was paid to the distribution of network charges as well as to the attractive interaction among polymer segments. From looking at how these effects appear in the swelling curves, an exploration of the nature of polyelectrolyte gel transitions was attempted. Two sorts of polyelectrolyte gels, temperature-responsive ionic gels based on N-isopropylacrylamide (NIPA) and cationic poly(ethyleneimine) (PEI) gels, were mainly employed with various modifications. The charge inhomogeneity within the gel phase was created by surfactant binding, immobilized enzyme reaction and physical entrapment of polyions. The attractive interactions holding the gel in a collapsed state were studied in comparison with phase separations of the corresponding linear polyelectrolyte. The main conclusions are summarized as follows: (i) The charge inhomogeneity exhibits a large influence on the volume transition in ionic gels. (ii) Hydrogen bonding and hydrophobic association, other than electrostatic attraction, can be considered to play an important role in the segmental association. (iii) Stably associated segments via one or more of these attractive interactions causes a large hysteresis in the swelling process, in which the repulsive interaction among the fixed charges on the network is dominant as shown in the Katchalsky's model. (iv) A distribution of "neutral but hydrophilic" moieties (e.g., ion pair or salt-linkage formed between the opposite charged groups) within the gel shows a marked effect on the temperature-induced volume collapse, the aspect of which is similar to that observed in the gels with a charge inhomogeneity.

Formation of intra- and interparticle polyelectrolyte complexes between cationic nanogel and strong polyanion.

Polyelectrolyte complex formation of a strong polyanion, potassium poly(vinyl alcohol) sulfate (KPVS), with positively charged nanogels was studied at 25 degrees C in aqueous solutions with different KCl concentrations (C(s)) as a function of the polyion-nanogel mixing ratio based on moles of anions versus cations. Used as the gel sample was a polyampholytic nanogel consisting of lightly cross-linked terpolymer chains of N-isopropylacrylamide, acrylic acid, and 1-vinylimidazole; thus, the complexation was performed at pH 3 at which the imidazole groups are fully protonated to generate positive charges. Turbidimetric titration was employed to vary the mixing ratio. Also employed for studies of the resulting complexes at different stages of the titration were dynamic light scattering (DLS) and static light scattering (SLS) techniques. It was found from the titration as well as DLS and SLS that there is a critical mixing ratio (cmr) at which both the size and molar mass of the complexed gel particles abruptly increase. The value of the cmr at C(s) = 0 or 0.01 M (mol/L) was observed at approximately 1:1 mixing ratio of anions versus cations but at lower mixing ratios than the 1:1 ratio under conditions of C(s) = 0.05 and 0.1 M. At the mixing ratios less than the cmr, the molar mass of the complex agrees with that of one gel particle with the calculated amount of the bound KPVS ions, indicating the formation of an "intraparticle" KPVS-nanogel complex, by the aggregation of which an "interparticle" complex is formed at the cmr. During the process of the intraparticle complex formation, both the hydrodynamic radius by DLS and the radius gyration by SLS decreased with increasing mixing ratio, demonstrating the gel collapse due to the complexation. At C(s) = 0 or 0.01 M and under conditions where the amount of KPVS bindings was less than half of the nanogel cations, however, the decrease of the hydrodynamic radius was very small, while the radius gyration fell monotonically. These results were discussed in connection with a collapse of dangling chains attached to the nanogel surface by the binding of KPVS.

New type of glucose sensor based on enzymatic conversion of gel volume into liquid column length.

A way to convert the volume change of a biochemo-mechanical gel into the change in liquid column length was developed. Our trial sensor device consisted of a small compartment for incorporating the gel, a flow channel with a filled dye solution, and a poly(dimethylsiloxane) (PDMS) diaphragm by which the gel and the dye solution were separated. A lightly cross-linked N-isopropylacrylamide (NIPAAm)/acrylic acid (AA) copolymer gel with immobilized glucose oxidase was used as a sensing element. It was found that a change in the gel volume caused by the immobilized enzyme reaction was accurately converted into a change of the column length (Deltal) with the help of the PDMS diaphragm. By use of a cylindrical gel (diameter approximately 2 and thickness approximately 1 mm), the time curve of Deltal varied depending upon glucose concentration over a range of 0.2-50 mM; in particular, it is of importance that semilogarithmic plots of Deltal (in mm) against glucose concentration (in mM) can be used as a calibration curve. For glucose solutions of mM order, 1 min was enough to determine the concentrations, whereas 10 min was required for concentrations of microM order. When the measurement time was limited within 10 min, the lower detection limit was 200 microM. The response was affected by buffering capacity of the samples, but this was controllable through reduction of the sample volume. These results indicate that the present way can be used for the determination of glucose concentration.

Swelling-shrinking behavior of a polyampholyte gel composed of positively charged networks with immobilized polyanions.

Polyampholyte gels were prepared by free radical polymerization of aqueous monomer solutions with the following composition: 69% N-isopropylacrylamide (thermosensitive neutral monomer), 1% N,N'-methylenebisacrylamide (cross-linker), 15% 1-vinylimidazole (cationic monomer), and either 15% acrylic acid (AAc, anionic monomer) or poly(acrylic acid) (PAAc, polyanion). We thus obtained two sorts of polyampholyte gels; that is, G1 with immobilized PAAc and G2 with randomly copolymerized AAc. The equilibrium swelling ratio (Qe) was studied as a function of the pH, NaCl concentration, and temperature. Also studied was the kinetics of swelling and shrinking in response to a sudden pH change. The significant results obtained were as follows: (i) A fully collapsed state was observed at pH 4.5-9.0 for G1 and at pH 4.5-7.0 for G2. (ii) Below and above these pH ranges, both gels were in a swollen state; therefore, an isoelectric point (pI) appeared in a wide pH range. (iii) At alkaline pH regions where a hysteresis was observed in the Qe versus pH curves of G1 and G2 as the pH was first increased then decreased, G1 exhibited very slow swelling-shrinking kinetics. (iv) An increase in the NaCl concentration allowed the gel to swell at pH approximately pI (antipolyelectrolyte behavior) but to shrink at pHs below and above the pI range (polyelectrolyte behavior). (v) The magnitude of the salt-induced shrinking of G1 is smaller than that of G2 at pH 10 and at NaCl concentrations > 0.01 M. (vi) At pH 10, an increase in the temperature from 35 to 50 degrees C led to a shrinking change of G1 but not of G2. These results were found to be explicable in terms of a different distribution of negative charges within the polyampholyte gel network.

Complex formation of protein with different water-soluble synthetic polymers.

The formation of protein-polymer complexes was studied in an aqueous system using dynamic light scattering (DLS) and static light scattering (SLS) as the main experimental tools. Human serum albumin (HSA) was used as a protein and complexed with four representative water-soluble polymers: poly(N-isopropylacrylamide) (PNIPA), poly(ethylene glycol) (PEG), poly(vinyl pyrrolidone) (PVP), and poly(vinyl alcohol) (PVA). The first three molecular weights were within 420,000-540,000 and the last one was 270,000. The complexation was performed at 25 degrees C in 0.01 M NaCl solution adjusted to pH 3 with HCl as a function of mixing ratio (rm; molar ratio of polymer to HSA). From SLS experiments, we determined the molecular weight of the resulting complexes, from the value of which the number (nb) of bound proteins per polymer was estimated. It was found that each polymer forms an intrapolymer complex over a wide range of rm (1.2 > or = rm > or = 0.01). Then, a marked decrease in nb with increasing rm was found. Over the whole rm range, the HSA-PNIPA complex exhibited a large nb value, as compared with the other three complexes whose nb values at the same rm were close to one another. Both the hydrodynamic radius (Rh) by DLS and the radius of gyration (Rg) by SLS for the complexes of PNIPA, PVP, and PVA decreased and then reached a constant value as nb decreased with increasing rm. In the PEG system, however, there were a few changes in Rh and Rg with nb. The Rg/Rh ratio, as an indication of chain expansion, was found to increase with decreasing nb in the PNIPA system. The complexes of PVA and PVP displayed a similar tendency, although the magnitude of the increasing trend was smaller than that of the PNIPA complex. In contrast, the Rg/Rh ratio of the PEG complex hardly varied depending on nb. These results were discussed in connection with the differences of physicochemical properties among four water-soluble polymers.

Intramolecular complex formation of poly(N-isopropylacrylamide) with human serum albumin.

Complexation of human serum albumin (HSA) with poly(N-isopropylacrylamide) (PNIPA) ranging in molecular weight (M(PNIPA)) from 2.1 x 10(4) to 1.72 x 10(6) was studied in an aqueous system (pH 3) containing NaCl as a supporting salt. Dynamic light scattering, static light scattering, electrophoretic light scattering, and dialyzing techniques were used as the experimental tool in a suitable combination. The measurements were performed mainly at 25 degrees C and at 0.01 M NaCl as a function of mixing ratio (r(m), molar ratio of PNIPA to HSA). The results of DLS and ELS evidently demonstrated the formation of a water-soluble complex through mixing of HSA and PNIPA. A detailed analysis of SLS data with the aid of dialysis data revealed that the resulting complex is an "intramolecular" complex consisting of a PNIPA chain with several of bound HSA molecules. Both hydrodynamic radius (R(h)) and radius gyration (R(g)) of intramolecular complexes decreased as r(m) was increased. This result correlated well to the fact that the number (n) of bound proteins per polymer decreases with increasing r(m). The size and the molar mass of the complex became large depending on M(PNIPA), but the increase of M(PNIPA) led to a decrease in n at r(m) < 1. The increase in NaCl concentration from 0.01 to 0.3 M brought about the increase in the size and the molar mass of an intramolecular HSA-PNIPA complex prepared at r(m) = 1.1. This was found to be due to an increase of n. A similar trend was observed when temperature rose from 25 to 32 degrees C (close to lower critical solution temperature of PNIPA). However, the effect of temperature on the increase of was strong in comparison with that of ionic strength. On the basis of these results obtained, the complexation mechanism was discussed in detail.

Coimmobilization of gluconolactonase with glucose oxidase for improvement in kinetic property of enzymatically induced volume collapse in ionic gels.

The object of this paper is to provide an enzymatic means to attain faster swelling or shrinking kinetics of polyelectrolyte gels that undergo volume phase transition as an immobilized enzyme reaction sets in. For this, we studied the coimmobilization of gluconolactonase (GL) with glucose oxidase (GOD). A gel used was in the shape of a small cylinder (several hundred micrometers in diameter) and composed of a lightly cross-linked copolymer of N-isopropylacrylamide and acrylic acid. GL was isolated from Aspergillus niger and purified about 100-fold. It was found that in a substrate solution containing glucose, the gel with the coimmobilized GL and GOD shrinks very rapidly. The shrinking rate was identical to that of the enzyme-free gel that undergoes a shrinking transition in response to a sudden pH change of the outer medium from 7 to 5. This indicates the rate-limiting step in the shrinking process to be diffusion of the networks, but not the enzyme reaction. In the gel with singly immobilized GOD, a very slow shrinking was observed because the process is governed by the enzyme reaction. These results were discussed in full in connection with an enzymatically induced decrease in pH within and in the vicinity of the gel phase. As a result, it has become apparent that the faster shrinking kinetics in the coimmobilized enzyme system is attained by the GL-catalyzed hydrolysis of D-glucono-delta-lactone resulting from the oxidation of glucose with GOD.