N. S. Melik-Nubarov

Strategy for Stabilizing Enzymes Part One: Increasing Stability of Enzymes via their Multi-Point Interaction with a Support

This review states that the covalent multi-point attachment of enzymes to a support is the most general approach to stabilize them against different denaturing conditions, namely against their inactivation caused by protein unfolding. It is suggested that the change in the wavelength of the maximum emission in fluorescence spectra of a protein, resulting from its denaturation, can be used to evaluate a priori the effectiveness of stabilization. The copolymerization method of enzyme immobilization, as the most promising approach to stabilizing enzymes, is discussed in detail.

Interaction of tumor and normal blood cells with ethylene oxide and propylene oxide block copolymers

Ethylene oxide and propylene oxide block copolymers (pluronics) are widely known as agents that promote drug penetration across biological barriers. We have studied the interaction of normal and malignant blood cells with pluronics L61 and P85 that have different hydrophobicity. SP2/0 myeloma cells accumulated pluronics while normal cells adsorb most of the polymer on the surface. Interaction of pluronics with cells resulted in drastic changes of membrane microviscosity. Tumor cell membrane microviscosity decreased after pluronics adsorption, in contrast to normal cells, whose membrane microviscosity was enhanced. We suppose that sensitivity of tumor cell membrane microviscosity to the pluronics action correlates with its permeability for molecular substances.

Effect of polylysine on transformations and permeability of negative vesicular membranes

Small (40-60 nm in diameter) and large (300-350 nm) negative vesicles were complexed with a cationic polypeptide, poly-L-lysine (PL). Laser microelectrophoresis experiments showed that in small vesicles rendered anionic with the addition of cardiolipin (CL(2-)), only the CL(2-) in the outer leaflet is involved in the complexation with PL. Calorimetric and other data demonstrate that the binding of PL to the membrane surface causes domains (rafts) of CL(2-) to form in the outer leaflet, and it is these domains that electrostatically bind the polymer. The kinetics of transmembrane permeation of doxorubicin (Dox, a fluorescent anti-tumor drug) was monitored with and without PL binding to the outer surface of the vesicles. It was found that PL mediates the permeation of Dox into the vesicle interior. In the absence of PL, the Dox molecule (possessing an amino group of pK(a)=8.6) binds to the anionic vesicles in the protonated form and, consequently, suffers an impaired mobility through the membrane. On the other hand, when the PL covers the vesicle surface, Dox passes though the membrane with greater ease. The effects of salt and polyanion on the stability of PL-vesicle complexes and the PL-mediated Dox permeation are also discussed.

Strategy for Stabilizing Enzymes Part Two: Increasing Enzyme Stability by Selective Chemical Modification

The molecular mechanisms of change in the thermal stability of proteins modified with low molecular weight reagents are discussed. The choice of stabilization mechanisms to be used as a general strategy for increasing enzyme stability by chemical modification is addressed. Hydrophilization of nonpolar surface areas is the most simple and reliable approach to artificial stabilization of enzymes for use in applied biochemistry and biotechnology.

Protein stabilization via hydrophilization: stabilization of α-chymotrypsin by reductive alkylation with glyoxylic acid

SummaryReductive alkylation of amino groups in α-chymotrypsin by glyoxylic acid in the presence of sodium cyanoborohydride results in essential stabilization of the enzyme against irreversible thermoinactivation. At 60°C, the stabilizing effect is higher than one thousand, and it increases even more with rising the temperature.

Interaction of a cationic polymer with negatively charged proteoliposomes.

Proteoliposomes were prepared by making bilayer vesicles from neutral egg yolk lecithin and negatively charged alpha-chymotrypsin that had been previously stearoylated. Interaction of these proteoliposomes with a cationic polymer, poly-(N-ethyl-4-vinylpryidinium bromide) (PEVP) was examined. For comparison purposes, interaction of PEVP with egg lecithin vesicles containing an anionic phospholipid, cardiolipin, was also examined. Binding of PEVP to both types of vesicles was electrostatic in nature with the polymer manifesting a higher affinity to the cardiolipin relative to the enzyme. PEVP had no effect on the permeability of the bilayer membranes to sodium chloride. On the other hand, PEVP increased the transmembrane permeability of the nonionic anti-tumor drug, doxorubicin. The greater the negatively charged component in the membrane, the greater the PEVP effect. Polycation binding to the vesicles was accompanied by clustering of the stearoylated chymotrypsin (sCT) molecules within the membrane. This protein clustering is most likely responsible for the increase in the doxorubicin permeation. Enzymatic activity of the membrane-associated sCT remained unchanged upon PEVP binding. These findings seem relevant to the effects of polyelectrolytes on cellular membranes.

The effect of Pluronics on the photocatalytic activity of water-soluble porphyrins

Solubilization of hydrophilic porphyrins, 2,7,12,18-tetramethyl-3,8-di(1-methoxyethyl)-13,17-di(2-oxycarbonylethyl) porphyrin disodium salt (dimegin) and N-methyl-di-D-glucose amine salt of chlorine e6 (photoditazine), as well water-insoluble meso-tetraphenylorphyrin in the micelles of Pluronics (triblock copolymers and propylene oxide and ethylene oxide), increases the photocatalytic activity of porphyrins in the course of the oxidation of L-tryptophan in aqueous salt-containing solutions. The maximum photocatalytic activity is attained for the photocatalysts based on the Pluronics P85 and F127 containing 50–70% ethylene oxide units. As a result of solubilization, the activity of tetraphenylporphyrin increases by a factor of 50, while the activities of hydrophilic dimegin and photoditazine increase by factors of 1.5 and 6, respectively. The increased activity of tetraphenylporphyrin is due to the dissolution and dissociation of aggregates in the presence of Pluronics. The increased activity of dimegin, which is known to aggregate in water, is primarily provided by disaggregation. In the case of photoditazine, which does not form aggregates in aqueous solutions and is likely to be localized in the polar micellar “crown,” the effect of a polymer is due to the local concentration of both a substrate and a catalyst in the micellar pseudophase.

Polyanions Decelerate the Kinetics of Positively Charged Gramicidin Channels as Shown by Sensitized Photoinactivation

The effects of different anionic polymers on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its positively charged analogs gramicidin-tris(2-aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine (gram-EDA) in a bilayer lipid membrane were studied using a method of sensitized photoinactivation. The addition of Konigs polyanion caused substantial deceleration of the photoinactivation kinetics of gram-TAEA channels, which expose three positive charges to the aqueous phase at both sides of the membrane. In contrast, channels formed of gram-EDA, which exposes one positive charge, and neutral gA channels were insensitive to Konigs polyanion. The effect strongly depended on the nature of the polyanion added, namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive, whereas modified polyacrylic acid induced deceleration of the channel kinetics at high concentrations. In addition, DNA was able to prevent the action of Konigs polyanion. In single-channel experiments, the addition of Konigs polyanion resulted in the appearance of long-lived gram-TAEA channels. The deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic interaction of the polyanion with gram-TAEA that reduces the mobility of gram-TAEA monomers and dimers in the membrane via clustering of channels.

Electrostatically Driven Complexation of Liposomes with a Star-Shaped Polyelectrolyte to Low-Toxicity Multi-Liposomal Assemblies†

Anionic liposomes are electrostatically complexed to a star-shaped cationic polyelectrolyte. Upon complexation, the liposomes retain their integrity and the resulting liposome-star complexes do not dissociate in a physiological solution with 0.15u2009M NaCl. This provides a multi-liposomal container for possible use as a high-capacity carrier.

Cytotoxicity of nonionic amphiphilic copolymers

The purpose of this study is to ascertain the relationship between the structure of an amphiphilic nonionic polymer and its toxicity for cells (cytotoxicity) growing in a culture. To this end, 16 polymers of different architectures and chemical structures are tested, namely, linear triblock copolymers of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronics); diblock copolymers of propylene oxide, ethylene oxide, and hyperbranched polyglycerol; alternating and diblock copolymers of ethylene oxide and dimethylsiloxane; and two surfactants containing linear (Brij-35) or branched (Triton X-100) aliphatic chains. Polymer-cell interaction is assayed in a culture medium in the absence of serum. Effective concentrations of the polymers causing 50% cell death, EC50, vary within three orders of magnitude. Toxic concentrations of the alternating copolymer, Triton X-100, and Brij-35 are lower than their CMC values. In contrast, all block copolymers, regardless of their chemical structures, become toxic at concentrations above the CMC; that is, they acquire cytotoxicity only in the micellar form. The EC50 values of the copolymers depend on their hydrophilic-liphophilic balance (HLB) through the following empirical formula: EC50 × 106 = 8.71 × HLB2.1. This relationship makes it possible to predict the cytotoxic concentration region of a block copolymer of a known structure.