Incapsulation of enzymes into polyelectrolyte nano- and microcapsules and the problem of the development of enzymatic microdiagnostics

The incapsulation of proteins into polyelectrolyte microcapsules (PE-microcapsules) has been studied with the aim to develop microdiagnostica for the presence of low-molecular-weight compounds in native biological fluids. The problem was solved using two enzymes: lactate dehydrogenase and urease. Polyelectrolyte microcapsules were prepared using two polyanions: polystyrene sulfonate (PSS) and dextran sulfate (DS), and two polycations: polyallylamine (PAA) and polydiallylmethylammonium (PDADMA). CaCO3 microspherulites with the incapsulated enzyme served as a "core" in the formation of polyelectrolyte microcapsules. It was shown that the main problem in the preparation of a polyelectrolyte microdiagnosticum is the selection of an oppositely charged pair of polyelectrolytes optimal for the active functioning of the enzyme. It follows from the results obtained that the best polyelectrolyte pairs for the formation of the envelope of a PE-microcapsule are PAA/DS and PAA/PSS for lactate dehydrogenase and PSS/PDADMA for urease. Taking into account these data, we designed enzyme-containing microcapsules with different polyelectrolyte compositions and different numbers of layers and studied their properties.     

Interaction of protein with charged colloidal particles

The functional state of three proteins of different molecular weight (urease, lactate dehydrogenase, and hemoglobin) in the presence of the linear polyelectrolytes poly(allylamine hydrochloride) (PAA) and sodium poly(styrenesulfonate) (PSS) in the dissolved state and of the same polyelectrolytes bound to the surface of microspheres has been investigated. Microspheres were prepared by consecutive absorption of oppositely charged polyelectrolytes so that the outer layer of the shell was PAA for the acidic protein urease, and PSS for the alkaline proteins LDH and hemoglobin. It was shown that the dissolved polyelectrolyte completely inactivates all three proteins within one minute with a slight difference in the time constant. (By Hb inactivation are conventionally meant changes in the heme environment observed from the spectrum in the Soret band.) In the presence of microspheres, the proteins were adsorbed on their surface; in this case, more than 95% of the activity was retained within two hours. The proportion of the protein adsorbed on microspheres accounted for about 98% for urease, 72% for Hb, and 35% for LDH, as determined from the tryptophan fluorescence data. The interaction of hemoglobin with another type of charged colloidal particles, phospholipid vesicles, leads to the destruction of the tertiary structure of the protein, which made itself evident in the optical absorption spectra in the Soret band, as well as the spectra of tryptophan fluorescence and circular dichroism. In this case, according to circular dichroism, the percentage of α-helical structure of Hb was maintained. The differences in the physical and chemical mechanisms of interaction of proteins with these two types of charged colloidal particles that leads to differences in the degree of denaturing effects are discussed.     

Effect of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase

Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.     

Stable stealth function for hollow polyelectrolyte microcapsules through a poly(ethylene glycol) grafted polyelectrolyte adlayer

Prospective biomedical applications of hollow polyelectrolyte microcapsules, for example, as drug delivery systems, require surface modifications that help to escape clearance by the mononuclear phagocytic system (MPS). Layer-by-layer assembled microcapsules that were alternatingly composed of polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH) were coated with adlayers of poly(ethylene glycol) (PEG)-grafted poly-L-lysine (PLL-g-PEG) and poly-L-glutamic acid (PGA-g-PEG). Their effects on MPS recognition were studied in primary cell cultures of human monocyte derived dendritic cells and macrophages. PGA-g-PEG coatings had no significant effect on cellular recognition, which may be explained by insufficient PEG density of the adlayer. Contrary, PLL-g-PEG effectively blocked phagocytosis of coated microcapsules. In addition, PLL-g-PEG coatings showed efficient adlayer stability for at least 3 weeks, and PAH/PSS microcapsules did not impair phagocyte viability. Our results demonstrate that layer-by-layer assembled polyelectrolyte microcapsules coated with a PEG-grafted polyelectrolyte, PLL-g-PEG, represent a promising platform for a drug delivery system that escapes fast clearance by the MPS.     

Thermal stability of lactate dehydrogenase in the complex with the anion polyelectrolyte poly(styrene sulfonate)

The temperature stability of the cytoplasmic enzyme of the glycolysis of lactate dehydrogenase from a pig muscle (isoenzyme M4) in a complex with the anion polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, the own protein fluorescence, and circular dichroism. Calorimetric investigations of complex of lactate dehydrogenase with poly(styrenesulphonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulphonate)/lactate dehydrogenase, though at 20 degrees C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulphonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

Temperature stability of lactate dehydrogenase in complex with anionic polyelectrolyte poly(styrenesulfonate)

The temperature stability of the cytoplasmic enzyme of glycolysis, lactate dehydrogenase from pig muscle (isoenzyme M4) in complex with anionic polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, own protein fluorescence, and circular dichroism. Calorimetric investigations of the complex of lactate dehydrogenase with poly(styrenesulfonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulfonate)/lactate dehydrogenase, though at 20°C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulfonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

Spontaneous loading of positively charged macromolecules into alginate-templated polyelectrolyte multilayer microcapsules

A simple and high-efficiency approach to loading macromolecules into microscale carriers is presented. Calcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique and then used as negatively charged templates to form polyelectrolyte multilayer coatings. A calcium ion chelator, EDTA, was used to free the Ca(2+)-cross-linked alginate hydrogel within {poly(allylamine hydrochloride)/poly (styrene sulfonate)}(4) ({PAH/PSS}(4)) coating, allowing partial release of alginate. The retention of alginate in {PAH/PSS}(4) microcapsule was confirmed by FTIR spectroscopy and confocal microscopy. Real-time confocal microscopy was used to investigate the loading process of positively charged macromolecules (dextran-amino, and peroxidase) into alginate-templated microcapsules, which showed the loading occurred in <2 min for dextran-amino and <10 min for peroxidase, respectively. A high loading efficiency of 25 mug peroxidase in approximately 1.0 x 10(7) microcapsules (2.5 pg POx/capsule) was achieved with a low concentration of peroxidase loading solution (10 mug/mL). This spontaneous loading technique for encapsulating positively charged molecules in alginate-templated polyelectrolyte microcapsules shows strong potential for biosensor and drug delivery applications.     

Designing anti‐diabetic β‐cells microcapsules using polystyrenic sulfonate,polyallylamine, and a tertiary bile acid: Morphology,bioenergetics, and cytokine analysis

Purpose: Recently sodium alginate (SA)‐poly‐l‐ornithine (PLO) microcapsules containing pancreatic β‐cells that showed good morphology but low cell viability (<27%) was designed. In this study, two new polyelectrolytes, polystyrenic sulfonate (PSS; at 1%) and polyallylamine (PAA; at 2%) were incorporated into a microencapsulated‐formulation, with the aim of enhancing the physical properties of the microcapsules. Following incorporation, the structural characteristics and cell viability were investigated. The effects of the anti‐inflammatory bile acid, ursodeoxycholic acid (UDCA), on microcapsule morphology, size, and stability as well as β‐cell biological functionality was also examined. Methods: Microcapsules were prepared using PLO‐PSS‐PAA‐SA mixture and two types of microcapsules were produced: without UDCA (control) and with UDCA (test). Microcapsule morphology, stability, and size were examined. Cell count, microencapsulation efficiency, cell bioenergetics, and activity were also examined. Results: The new microcapsules showed good morphology but cell viability remained low (29% ± 3%). UDCA addition improved cell viability post‐microencapsulation (42 ± 5, P < 0.01), reduced swelling (P < 0.01), improved mechanical strength (P < 0.01), increased Zeta‐potential (P < 0.01), and improved stability. UDCA addition also increased insulin production (P < 0.01), bioenergetics (P < 0.01), and decreased β‐cell TNF‐α (P < 0.01), IFN‐gamma (P < 0.01), and IL‐6 (P < 0.01) secretions. Conclusions: Addition of 4% UDCA to a formulation system consisting of 1.8% SA, 1% PLO, 1% PSS, and 2% PAA enhanced cell viability post‐microencapsulation and resulted in a more stable formulation with enhanced encapsulated β‐cell metabolism, bioenergetics, and biological activity with reduced inflammation. This suggests potential application of UDCA, when combined with SA, PLO, PSS, and PAA, in β‐cell microencapsulation and diabetes treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:501–509, 2016     

Gene delivery using biodegradable polyelectrolyte microcapsules prepared through the layer-by-layer technique

Biodegradable and non‐biodegradable microcapsules were prepared via the layer‐by‐layer (LbL) technique consisting of the polyelectrolyte pairs of dextran sulphate/poly‐L ‐arginine and poly(styrene sulfonate)/poly(allylamine hydrochloride), respectively, in an attempt to encapsulate plasmid DNA (pDNA) for efficient transfection into NIH 3T3 cells. Results indicated the retention of bioactivity in the encased pDNA, as well as a correlation between the level of in vitro gene expression and biodegradability properties of polyelectrolyte. Furthermore, the incorporation of iron oxide nanoparticles within the polyelectrolyte layers significantly improved the in vitro transfection efficiency of the microcapsules. As a novel pDNA delivery system, the reported biodegradable microcapsules provide useful insight into plasmid‐based vaccination and where there is a prerequisite to deliver genes into cells capable of phagocytosis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1088–1094, 2012     

Ultrathin antibiotic walled microcapsules

Ultrathin microcapsules comprised of anionic polyelectrolytes (PE) and a polycationic aminoglycoside (AmG) antibiotic drug were prepared by depositing PE/AmG multilayers on zinc oxide (ZnO) colloid particles using the layer-by-layer self-assembly technique and subsequently dissolving the ZnO templated cores. The polyelectrolytes, dextran sulfate sodium (DxS) and poly(styrenesulfonate) (PSS), were selected owing to their different backbone structure. An aminoglycoside, tobramycin sulfate (TbS), was used for studying DxS/TbS or PSS/TbS multilayer films. The multilayer growth on ZnO cores was characterized by alternating zeta potential values that were different for the DxS/TbS and PSS/TbS multilayers due to the PE chemistry and its interaction with Zn(2+) ions. Transmission and scanning electron microscopy provide evidence of PE/TbS multilayer coating on ZnO core particles. The slow acid-decomposition of the ZnO cores using weak organic acids and the presence of sufficient quantity of Zn(2+) in the dispersion were required to produce antibiotic multilayer capsules. There was no difference in the morphological characteristics of the two types of capsules; although, the yield for [PSS/TbS](5) capsules was significantly higher than for [DxS/TbS](5) capsules which was related to the physicochemical properties of DxS/TbS/Zn(2+) and PSS/TbS/Zn(2+) complexes forming the capsule wall. The TbS quantity in the multilayer films was determined using a quartz crystal microbalance and high performance liquid chromatography techniques which showed less TbS loading in both, capsules and multilayers on planar gold substrate, than the theoretical DxS:TbS or PSS:TbS stoichiometric ratio. The decomposition of the [PE/TbS](6) multilayers was fastest in physiological buffer followed by mannitol and water. The decomposition rate of the [PSS/TbS](6) multilayers was slower than [DxS/TbS](6) monolayers. The incomplete decomposition of DxS/TbS under saline conditions suggests the major role of hydrogen bonding for stability of DxS/TbS multilayers. A combination of hydrogen bonding and hydrophobic interaction between phenyl rings in PSS was responsible for PSS/TbS multilayer stability. In vivo studies in rabbits highlight the safety and sustained drug delivery potential of the PE/AmG microcapsules. The antibiotic walled ultrathin capsules presented here are suitable for sustained ophthalmic antibiotic delivery.     

Block polyelectrolyte networks from poly(acrylic acid) and poly(ethylene oxide): sorption and release of cytochrome C

A new family of block polyelectrolyte networks containing cross-linked poly(acrylic acid) (PAA) and poly(ethylene oxide) (PEO) was synthesized by copolymerization of acrylic acid and bisacrylated PEO (10 kDa). Two materials with different PEO/PAA ratios were compared with a weakly cross-linked PAA homopolymer network. The networks bound a cationic protein, cytochrome C, due to the polyion coupling, leading to the network contraction. After binding the protein the block polyelectrolyte networks were more porous compared to a homopolymer network, facilitating protein absorption within the gel. The protein was released by adding Ca2+ ions or a polycation. Ca2+ ions migrated within the gels and reacted with PAA chains, thus displacing the protein. The polycation transfer into hydrogels, as a result of polyion substitution reactions, was inhibited by the excess of PEO chains in the block polyelectrolyte networks. Overall, these findings advance development of functional polyelectrolyte networks for immobilization and controlled release of proteins.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.     

Lactate dehydrogenase in an interpolyelectrolyte complex. Function and stability

A new method for encapsulating enzymes by multilayer polyelectrolyte coating is proposed. The method consists in a stepwise adsorption of polyelectrolytes from solution onto protein aggregates formed by salting out the proteins in highly concentrated salt solutions. Polystyrene sulfonate and fluorescence-labeled polyalylamine were used for capsule formation. The size of lactate dehydrogenase aggregates covered by four layer pairs of electrolytes was 1-5 microns, as indicated by fluorescence microscopy. The catalytic characteristics and stability of pig muscle lactate dehydrogenase (EC 1.1.1.13) incapsulated in multilayer electrolyte complex obtained by this method were studied. It was found that the affinity of the substrate pyruvate for the enzyme in the polyelectrolyte complex (K(M)) did not essentially change as compared with the free enzyme. Incapsulated lactate dehydrogenase showed the following features that distinguish it from the free form: (1) the lifetime in diluted solutions increases from 30 min (without capsules) to 1-2 days (in capsules); (2) a higher stability to basic denaturation (up to pH 10); and (3) the absence of substrate inhibition of enzyme in the polyelectrolyte complex. The changes in the catalytic characteristics of incapsulated lactate dehydrogenase are discussed in terms of an increase in effective pK values of amino acid perturbed by polyelectrolyte coating of enzyme.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Protein-calcium carbonate coprecipitation: a tool for protein encapsulation

A new approach of encapsulation of proteins in polyelectrolyte microcapsules has been developed using porous calcium carbonate microparticles as microsupports for layer-by-layer (LbL) polyelectrolyte assembling. Two different ways were used to prepare protein-loaded CaCO3 microparticles: (i) physical adsorption--adsorption of proteins from the solutions onto preformed CaCO3 microparticles, and (ii) coprecipitation--protein capture by CaCO3 microparticles in the process of growth from the mixture of aqueous solutions of CaCl2 and Na2CO3. The latter was found to be about five times more effective than the former (approximately 100 vs approximately 20 mug of captured protein per 1 mg of CaCO3). The procedure is rather mild; the revealed enzymatic activity of alpha-chymotrypsin captured initially by CaCO3 particles during their growth and then recovered after particle dissolution in EDTA was found to be about 85% compared to the native enzyme. Core decomposition and removal after assembly of the required number of polyelectrolyte layers resulted in release of protein into the interior of polyelectrolyte microcapsules (PAH/PSS)5 thus excluding the encapsulated material from direct contact with the surrounding. The advantage of the suggested approach is the possibility to control easily the concentration of protein inside the microcapsules and to minimize the protein immobilization within the capsule walls. Moreover, it is rather universal and may be used for encapsulation of a wide range of macromolecular compounds and bioactive species.     

A mild methods of general use for covalent coupling of enzymes to chemically activated collagen films

Films of highly polymerized collagen, prepared in industrial conditions, were chosen for surface covalent binding of enzymes because of their insolubility, mechanical resistance, proteic nature, hydrophilic properties and for their abundance in chemically activable ? COOH. Untanned films, previously acid- methylated, were activated by acyl azide formation. After removal of reagents by repeated washing, the coupling of enzyme was performed by immersion of the activated film in the enzyme solutions (2 to 3 hr, 0°C). The procedure is particularly mild since the enzymes never come into contact with chemical reagents, and thus avoid all denaturing processes. All the enzymes tested were successfully bound: glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, glutamate pyruvate transaminase, creatine kinase, hexokinase, trypsin, and urease. As tested with aspartate amino transferase, enzymatic activity remained constant for months (100% after 5 months) in spite of repeated use of the film at 30°C, washing and storage in buffer at 4°C between assays.     

The electrostatic contribution to interactions of some enzymes with polyelectrolytes

To explain the inhibitory action of polyelectrolytes on enzymes and, in particular, to define potentially reactive zones for the binding of polyelectrolyte, the electric potential of enzymes lactate dehydrogenase and glutamate dehydrogenase was calculated using the solution of the Poisson-Boltzmann equation by a numerical method with the use of the Gauss-Seidel relaxation method at three pH values: 6.5, 7.0, and 8.0 and three values of ionic strength: 50, 100, and 150 mm. On the basis of these calculations and their visualization, representative sites for favorable binding of polyanions were determined as extended areas on the surface of proteins with the positive potential in the neutral pH region. It was shown that there is a correlation between the area of positive potential and the efficiency of enzyme inactivation for a number of pH values and concentrations of salt for two enzymes. The calculations performed allowed one to explain the inhibitory action of polyelectrolytes on the specified enzymes to understand the difference between the values of polyelectrolyte inactivation constants for the two enzymes and estimate the minimal areas of the positive potential on the protein surface that provide their effective inhibition.     

Aqueous two-phase systems with a liquid protein (bovine serum albumin) phase for partitioning of enzymes

New aqueous liquid-liquid two-phase systems based on bovine serum albumin and sodium thiocyanate in combination with either poly(vinyl alcohol) or poly(ethylene glycol) were investigated. Phase diagrams are presented. Lactate dehydrogenase and some mitochondrial enzymes were partitioned in the systems. All the phase components used influenced, either positively or negatively, the activity of lactate dehydrogenase. The enzymes showed a strong preference for the albumin phase. Possible scientific and biotechnological uses are discussed.     

A special role of phosphate in the stability of lactate dehydrogenase against destruction by a polyelectrolyte

It has been shown by X-ray structure analysis that proteins have specific anion-binding sites for sulfate, citrate, and phosphate ions; however, the functional role of these anions is not always clear. Thus, it is unknown which of two phosphate anions, mono- or divalent, determines the stability of cellular proteins under stress conditions. In the present work, the influence of phosphate, sulfate, and chloride on the stability of lactate dehydrogenase (LDH) in the presence of poly(styrenesulfonate) (PSS) has been investigated by the methods of steady-state kinetics and intrinsic protein fluorescence. The study is based on the analysis of differences between the influence of phosphate and sulfate ions on the process at two pH values, 6.2 and 7.0, at which the ratio of the concentrations of mono- and bivalent phosphate forms differs, whereas sulfate remains in the bivalent form. It was shown that the differences between the influence of phosphate and sulfate ions at pH 7.0 were greater; divalent phosphate ions much more effectively stabilized LDH against destruction by a polyelectrolyte compared with sulfate and monovalent phosphate. It was concluded that, of two anion-binding sites of the LDH molecule, the intersubunit center plays the most important role in its stabilization against destruction by polyelectrolyte, and, of two forms of phosphate anions, its bivalent form HPO ₄ ^?2 plays the stabilizing role.