Noncovalent adducts of poly(ethylene glycols) with proteins.

A new method of preparation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumine) under high pressure has been developed. The involvement of polymer in the complexes was proved using (3)H-labeled PEG. The composition of the complexes (the number of polymer chains per one ChT molecule) depends on the molecular mass of PEG and decreases with the increase in molecular mass from 300 to 4000, whereas the portion of the protein (wt %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt %. The kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical with the corresponding values for the native ChT. According to the data obtained by the method of circular dichroism, the enzyme in the complexes fully retains its secondary structure. The steric availability of PEG polymer chains in the complexes was evaluated by their complexation with alpha-cyclodextrin (CyD) or polymer derivatives of beta-CyD modified with PEG (PEG-beta-CyD). In contrast to free PEG, only part of PEG polymer chains ( approximately 10%) interact with alpha-CyD. Thus, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule located far from the active site and results in the sufficient decrease in the availability of polymer chains. The complexes between PEG chains in PEG-protein adducts and PEG-beta-CyD may be considered as a novel type of dendritic structures.

[Supramolecular complexes of polyethylene glycol-alpha-Chymotrypsin covalent and noncovalent adducts with polyoxyethylene-modified cyclo dextrins]. / Supramolekuliarnte kompleksy kovalentnykh i nekovalentnykh adduktov poliétilenglikol'-alpha-khimotripsina s tsiklodekstrinami, modifitsi rovannymi poliétilenglikolem.

Complexes of covalent and noncovalent adducts of polyethylene glycol (PEG) and alpha-chymotrypsin (ChT), PEG-ChT, were generated in the presence of beta-cyclodextrin derivatives of polyoxyethylene (beta CD-PEO), and their thermal stability was studied. The covalent [PEG-ChT]c conjugates were obtained by chemical modification of the protein amino groups with the monoaldehyde derivatives of monomethoxypolyethylene glycol. The noncovalent [PEG-ChT]n complexes were obtained by the treatment of ChT-PEG mixtures with increasing pressure (1.1-400 MPa). Supramolecular structures resulting from complex formation between PEG chains of the PEG-ChT adducts (PEGad) and beta CD-PEO were studied. The decrease in the rate constant of the slow stage of ChT thermal inactivation in PEG-ChT adducts (k2) can serve as confirmation of complex formation between beta CD-PEO and PEGad. The stoichiometric composition of our supramolecular structures was determined from the k2 dependence on the molar ratio of beta CD-PEO to PEGad. It was shown that each polymeric chain in the [PEG-ChT]c conjugates forms an inclusion complex with beta CD-PEO, whereas only half of the PEGad polymeric chains participate in the formation of supramolecular structures in the case of [PEG-ChT]n complexes. Although covalent and noncovalent PEG-ChT adducts of the same composition significantly differ in their thermal stability, the maximal values of the k2 rate constants for [PEG-ChT]c and [PEG-ChT]n adducts in the triple system attainable at the (beta CD-PEO) to (PEGad) ratio corresponding to the stoichiometry of the resulting ternary systems are practically the same (k2 = 0.007 c-1 at 45 degrees C in 0.02 M Tris-HCl buffer solution, pH 8.0). Structures for the supramolecular dendrite-like ensembles formed upon the interaction of covalent and noncovalent PEG-ChT adducts with beta CD-PEO were suggested.

[Enzymic activity of polymer-protein complexes in water-miscible organic solvents]. / Fermentativnaia aktivnost' kompleksov polimer-belok v smeshivaiushch ikhsia s vodoi organicheskikh rastvoriteliakh.

The enzymic activity of noncovalent complexes of alpha-chymotrypsin with polyethylene glycol and a block-copolymer of polyethylene oxide and polypropylene oxide (proxanol) was studied in aqueous-organic media. It was shown that complex formation activated the enzyme in media with a high content of the organic solvent, whereas in systems containing more than 50% water the enzymic activity of complexes was the same as that of the native enzyme. The activation in polyethylene glycol-containing complexes was greater than in complexes with proxanol of the same molecular mass.

Using hydrated reversed micelles to evaluate the dimensions of polymer-protein adducts.

Hydrolysis of N-trans-cynnamoylimidazole catalyzed by conjugates and complexes of alpha-chymotrypsin (ChT) with poly(ethylene glycol) (PEG) of different molecular mass (from 300 to 5000 daltons) was studied in the system of the hydrated reversed micelles of aerosol OT (AOT) in octane at 25 degrees C. The plot of the deacylation constant k3 for PEG--ChT conjugates and complexes versus the degree of hydration of reversed micelles (w0 = [H2O]/[AOT]) was studied. These plots are bell-shaped with maxima shifted to higher degrees of micelle hydration compared to the corresponding value of the shift for ChT. As for PEG--ChT conjugates, the value of the shift of w0 increases with increasing of molecular mass of the attached PEG and/or with the number of polymer chains per ChT molecule. Another picture was observed for PEG--ChT complexes for which the position of the maximum on k3 versusw0 curves was practically the same for all compounds. The values of the thickness of the polymer layer for PEG--ChT conjugates and complexes were calculated. Thus, polymer chains in conjugates placed in hydrated micelles are highly packed, whereas in the case of complexes they form a flat layer on the surface of the protein.

Selective assembly of cyclodextrins on poly(ethylene oxide)-poly(propylene oxide) block copolymers.

This paper presents a computational study on the formation of a molecular necklace formed by specific threading of cyclodextrins (CDs) on block copolymers. Structural as well as energetic principles for the selective complexation of alpha- and beta-cyclodextrin with poly(ethylene oxide)-poly(propylene oxide) block copolymers (PEO-PPO) are elucidated considering a diblock copolymer of equimolecular composition (PEO)4-(PPO)4 as guest. A non-statistical distribution of CDs, i.e. alpha-CDs primarily located on the PEO chain and beta-CDs on PPO blocks of the polymer, is based on a variety of structural features and energetic preferences considering both potential as well as solvation energies. This selectivity becomes already obvious considering 1:1 complexes between PEO and PPO monomers and the two CDs, but is increasingly evident when calculating higher order ensembles. Besides the host-guest interaction, docking between CDs themselves is an important, also non-statistical, prerequisite for the self-assembly of highly ordered tubes. The formation of intermolecular hydrogen bonds between adjacent CDs in a tubular aggregate gives an important contribution to the overall stability of the molecular necklace. The net effect, based on the preferential interaction between host and guest as well as between the host molecules themselves, results in the formation of a stable, highly ordered macromolecular, multicomponent aggregate.

Noncovalent complexes between poly(ethylene glycol) and proteins.

A new method of formation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumin) under high pressure has been developed. The existence of polymer in complexes was proved using 3H-labeled PEG. Complexes between PEG and ChT were studied in detail. It was shown that the composition of complexes (the number of polymer chains per ChT molecule) depends on the molecular mass of PEG and decreases with the increase of molecular mass from 300 to 4000. At the same time, the portion of the protein (wt. %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt. %. It was shown that kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical to the corresponding values for the native ChT. The conformational properties of ChT in complexes were studied by circular dichroism. It was shown that the enzyme in complexes fully retains its secondary structure. The estimation of steric availability of PEG polymer chains in complexes was evaluated by the complexation with alpha-cyclodextrin (CyD). It was shown that in contrast to free PEG, only part (approximately 10%) of PEG polymer chains in PEG--ChT complexes participate in the complexation with CyD. Hence, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule in a region far from the active site of the enzyme and results in the significant decrease in the mobility of polymer chains.

Novel derivatives of cyclodextrins, modified with poly(ethylene oxide) and their complexation properties.

A new family of bouquet-like molecules based on cyclodextrins is described. These compounds were obtained by polymerization of ethylene oxide. This reaction was initiated by primary and secondary hydroxyl groups of cyclodextrins, which constitute an organizing core. Analysis of structure and composition of conjugates based on alpha-CD and beta-CD was performed using the data of MALDI-MS, GC-MS, and 13C-NMR spectra. Glass transition behavior of the conjugates shows that the above compounds are amorphous. Complexation properties of the conjugates are described with respect to sodium 4-nitrophenolate and calcium acetylhomotaurinate, which are used as guest molecules. Binding interaction between cyclodextrins or their conjugates and sodium 4-nitrophenolate was studied using differential absorption spectra. Association constants Ka between CD hosts and calcium acetylhomotaurinate composed of two equal anionic moieties were studied using 1H-NMR spectroscopy. The values of binding constant for beta-CD were found to increase by more than 2 orders of magnitude than that of the corresponding system based on alpha-CD. alpha-CD was shown to form the inclusion complex with one anionic moiety, whereas beta-CD produces a ternary complex with two anionic moieties of CAHT. For PEO derivatives of CDs, Ka decreases as compared with that of parent CD for both guests. These conjugates may be used as potent drug-delivery systems.

Artificial chaperone-assisted refolding of proteins.

A new two-step procedure of protein refolding in vitro, proposed by Rozema and Gellman and named artificial chaperone-assisted refolding, is discussed. The new approach has been inspired by the two-step mechanism of the GroE system. In the first step, the protein is captured by a detergent under conditions that would normally lead to irreversible protein aggregation (heating or denaturant removal). In the second step, removal of detergent from the protein--detergent complex is triggered by addition of a cyclodextrin which is capable of forming "inclusion complexes" with detergent, allowing the protein to refold. The protein refolded with artificial chaperones (detergent and cyclodextrin) may be purified via a two-step protocol. After refolding was complete, the solution was passed through a 0. 22-micro(m) filter, to remove aggregated protein, and then through a M = 10 kD cutoff filter. The second filtration was intended to allow the low-molecular-weight artificial chaperones to pass, but to retain the refolded enzyme. The application of the above procedure for refolding of carbonic anhydrase B from human erythrocytes, hen egg white lysozyme, pig heart citrate synthase, and creatine kinase from rabbit skeletal muscles (MM isoenzyme) is discussed.

Application of the method of thermal denaturation for investigation of alpha-chymotrypsin adducts with poly.

The thermostability of conjugates, non-covalent complexes and mixtures of alpha-chymotrypsin (alpha-ChT) with poly(alkylene oxides)--poly(ethylene glycol) (PEG) with molecular mass of 1.9 kD and diblock copolymers of ethylene and propylene oxides (proxanols)--has been investigated. It was shown that the addition of PEG in concentration up to 2 wt. % to the solution of alpha-ChT did not affect the rate of the enzyme thermoinactivation. Meanwhile the addition of proxanol in the same concentration resulted in twofold decrease in the rate constant for the slow inactivation step, k2. Even more pronounced decrease in the thermoinactivation rate was observed for alpha-ChT--proxanol complexes obtained by heating or under the action of high pressure. The general tendency in the behavior of complexes of both types was the decrease in the k2 constant as the temperature or pressure used for complex preparation increased. The highest stabilizing effect was observed for complex obtained by heating up to 52 degreesC and containing maximal number of polymer chains (molar ratio proxanol/alpha-ChT was 10). For this complex fourfold decrease in the k2 value was observed. Covalent attachment of PEG or proxanol to enzyme gives maximal stabilizing effect with up to tenfold decrease in the k2 value. The investigation of the thermal denaturation kinetics of alpha-ChT and its adducts with poly(alkylene oxides) by means of fluorescence spectroscopy has shown that the presence of polymer chains practically does not affect the rate of protein denaturation registered by the decrease in the intensity of protein fluorescence. The polymer chains, probably, diminish the rate of melting of the active site-containing region of the protein molecule. At the same time, the overall denaturation rate is independent of the presence of polymer chains in the vicinity of the protein globule.

Protein conjugates with water-soluble poly(alkylene oxide)s entrapped in hydrated reversed micelles.

Conjugates of alpha-chymotrypsin (ChT) with poly(ethylene glycol) (PEG) and block copolymers of ethylene and propylene oxides (proxanols) have been synthesized. The molecular mass of the polymers used was 2 kDa. The conjugates contained five to seven polymer chains per enzyme molecule. Hydrolysis of N-trans-cinnamoylimidazole catalyzed by the conjugates of ChT with poly(alkylene oxide)s was studied in 0.05 M Tris-HCl buffer at pH 8.0 and in the system of the hydrated reversed micelles of aerosol OT (AOT) in octane at 25 degrees C. The deacylation rate constant k3 for the conjugates in buffer solution was 1.5-1.8-fold higher than that for native ChT. The value of the [H2O]:[AOT] ratio corresponding to the maximum on k3 versus [H2O]:[AOT] curves for the conjugates (ca. 16) allows the dimensions of their molecules to be evaluated. The radius of the conjugate molecules was found to be about 2.8 nm. The value of k3 for the conjugate of ChT with PEG, as in the case of native ChT, remains constant when the concentration of AOT is varied. However, the deacylation rate constant for the conjugates of ChT with proxanols decreases with the increase in AOT concentration, which indicates that these conjugates are able to interact with the micellar matrix and therefore may be considered membranotropic compounds.

[Complex formation between alpha-chymotrypsin and block copolymers based on ethylene and propylene oxide, induced by high pressure]. / Kompleksoobrazovanie mezhdu alpha-khimotripsinom i blok-sopolimerami na osnove okisi étilena i okisi propilena, indutsiruemoe deistviem vysokikh davlenii.

A new method of formation of non-covalent adducts based on an amphiphilic diblock copolymer of ethylene and propylene oxides with molecular mass of 2 kDa and alpha-chymotrypsin (ChT) under high pressure, has been developed. The composition of the complexes corresponds to seven polymer molecules per one ChT molecule in the pressure range of 1.1 to 400 MPa. The complexes fully retain the catalytic activity. Kinetic constants (Km and kcat) for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester catalyzed by the complexes are identical with the corresponding values for native ChT. Analysis of kinetics of thermal inactivation of the complexes revealed that the constant of the rate of the slow inactivation step is markedly lower than for ChT.

[Conjugates of alpha-chymotrypsin with polyalkylene oxides in hydrated reverse micelles]. / Con''iugaty alpha-khimotripsina s polialkilenoksidami v gidratirovannykh obrashchennykh mitsellakh.

Conjugates of alpha-chymotrypsin (ChT) with poly(ethylene glycol) (PEG) and block copolymers of ethylene oxide and propylene oxide (proxanols) have been synthesized. The molecular mass of the polymers was 2 kDa. The conjugates contained 5 to 7 polymeric chains per enzyme molecule. Hydrolysis of N-trans-cinnamoylimidazole catalyzed by conjugates of ChT with poly(alkylene oxides) was studied in 0.05 M Tris-HCl buffer, pH 8.0, and in hydrated reversed micelles of aerosol OT (AOT) in octane at 25 degrees C. The deacylation constant, k3, for the conjugates in the buffer solution was found to be 1.5-1.8-fold higher than the corresponding value for native ChT. The value of the [H2O]/[AOT] ratio corresponding to the maximum on the k3 versus [H2O]/[AOT] curves for the conjugates (around 16) allows the dimensions of their molecules to be characterized. The radius of the conjugate molecules was found to be approximately equal to 2.8 nm. The k3 value for the conjugate of ChT with PEG, as in the case of native ChT, remains constant when the concentration of AOT is varied. However, the deacylation constant for ChT conjugates with proxanols decreases with a rise in AOT concentration. This fact indicates that ChT conjugates with proxanols are able to interact with the micellar matrix, i.e., they possess membranotropic properties.

Synthesis and physicochemical properties of protein conjugates with water-soluble poly(alkylene oxides).

Conjugates of proteins (bovine serum albumin (BSA) and alpha-chymotrypsin (CHT) with poly(ethylene glycol) and amphiphilic block copolymers of ethylene oxide and propylene oxide (proxanols) were synthesized, using monoaldehyde polymer derivatives as the amino group modifying reagents. Four types of conjugates varying in the placement of hydrophobic block and type of polymer chain distribution were obtained. Methods of purification and characterization of proteins conjugated with proxanols were developed. It was shown that conjugates based on CHT retain high enzymatic activity toward both substrates investigated--N-benzoyl-L-tyrosine and casein-up to high degrees of modification (11 polymer chains per protein molecule). At the same time, CHT--proxanol conjugates were characterized by higher thermostability, the stabilizing effect increasing in parallel with the degree of modification. It was shown that the alteration of sedimentation coefficients of proteins caused by modification was negligible. On the basis of data obtained by the methods of hydrophobic chromatography, sedimentation, and differential scanning calorimetry, conformational models of protein-proxanol conjugates were suggested. It was supposed that conjugates form compact structures in aqueous solutions, which resemble intramolecular micelles, stabilized by hydrophobic interactions between poly(propylene oxide) blocks of proxanols.

[Noncovalent complexes between alpha-chymotrypsin and block copolymers from ethylene and propylene oxides]. / Nekovalentnye kompleksy mezhdu alpha-khimotripsinom i blok-sopolimerom oksidov étilena i propilena.

The ability of alpha-chymotrypsin to form complexes with amphiphilic block copolymer of ethylene oxide and propylene oxide upon heating up to 44-60 degrees C has been demonstrated for the first time. Depending on temperature and the initial component ratio, some complexes were obtained which varied in both composition and enzymatic activity. With a rise in the complexation temperature, the polymer content in the complex increased, while the enzymatic activity of the complex decreases. The complexes are very stable in water, but dissociate in 8 M urea and are characterized by enhanced thermal stability as compared with the original enzyme. It is assumed that both hydrophobic interactions and hydrogen bonds between the components are involved in the complex formation.

[Membranotropic properties of conjugates of proteins with ethylene oxide and propylene oxide block copolymers]. / Membranotropnye svoistva kon''iugatov belkov s blok-sopolimerami okisiétilena i okisi propilena.

It has earlier been established that conjugates of hydrophilic proteins with block copolymers of ethylene oxide and propylene oxide (proxanols) used at low concentrations decrease the oxidation of NAD-dependent substrates in mitochondria and cells. The dependence of this property on the chemical structure and structural organization of such conjugates has been studied. The conjugates have been shown to inhibit the oxygen consumption of lymphocytes when the hydrophobic (polypropylene oxide) blocks are located on the periphery of the conjugate and the polymer chains form domains that are irregularly distributed on the protein globule surface. The minimum number of the polymer chains in the conjugate sufficient to provide this property is three. Additional evidence has been obtained in favour of the ability of conjugates to penetrate through the cellular membrane of lymphocytes.

The influence of pluronics and their conjugates with proteins on the rate of oxygen consumption by liver mitochondria and thymus lymphocytes.

The membranotropic properties of block co-polymers and their protein conjugates were studied by their effect on the rate of oxygen consumption by isolated liver mitochondria and on thymus-derived lymphocytes. The block co-polymers consisted of poly(ethylene oxide) (PoE) [poly(ethylene glycol)] and poly(propylene oxide) (PoP) to give either PoE-PoP or PoE-PoP-PoE. Both types inhibited uncoupled respiration of liver mitochondria in a medium containing glutamate and malate and also of lymphocytes. They also uncoupled respiration in the presence of succinate in K(+)-containing medium and of lymphocytes. A method is described for linking protein to the block polymers to form conjugates. Such conjugates were formed from alpha-chymotrypsin, BSA and cytochrome c, all of which produced similar effects on the respiration of the isolated mitochondria and lymphocytes. The data suggest that both the block co-polymers and their protein conjugates inhibit the NADH dehydrogenase complex and induce a K(+)-conductivity of the mitochondrial inner membrane; the surface activity of the conjugates allows them to pass through the plasma membrane and interact with the mitochondrial inner membrane.

[Structure-functional properties of conjugates of proteins with polyalkylene oxides: study by a fluorescence method]. / Strukturno-funktsional'nye svoistva kon''iugatov belkov s polialkilenoksidami: issledovanie metodom fluorestsentsii.

A fluorescent study of some structural and functional properties of conjugates of a number of proteins (bovine serum albumin, pyruvate kinase, alpha-chymotrypsin, and the two toxic proteins of plant origin--ricin and viscumin) with polyalkylene oxides (polyethylene glycol and pluronic) has been carried out. Analysis of the intrinsic protein fluorescence showed that the structure and stability of various protein conjugates to denaturing agents change only slightly: the conformational mobility of tryptophan residues accessible to the solvent decreases, whereas that of tryptophan residues localized in the protein regions of low polarity remains unchanged. Besides, the conjugates display a higher thermal stability in comparison with their native proteins. The fluorescence of 1-anilinonaphthalene-8-sulfonic acid and water insoluble 2',3',4',5'-tetrabenzoylriboflavin bound to the native and modified proteins indicated that modification of the proteins with polyalkylene oxides decreased the polarity and increased the viscosity of the microenvironment. Hence, this modification makes it possible to change some functional characteristics of the protein without causing any significant changes in its structure.

[The effect of bovine serum albumin conjugates with polyalkylene oxides on the respiration of heart mitochondria]. / Vliianie kon''iugatov bych'ego syvorotochnogo al'bumina s polialkilenoksidami na dykhanie mitokhondrii serdtsa.

The effect of polyalkylene oxides and pluronics (block copolymers of polyethylene oxide and polypropylene oxide) and their conjugates with bovine serum albumin (BSA) on respiration and oxidative phosphorylation in rat heart mitochondria has been studied. The pluronics and their conjugates with BSA stimulate state 4 respiration in mitochondria, inhibit state 3 respiration and 2,4-dinitrophenol-uncoupled state respiration, decrease the respiratory control, ADP/O ratio values and display weak uncoupling properties. The pluronics inhibit respiration and oxidative phosphorylation more effectively than their conjugates with BSA. The effect of the conjugate on the mitochondrial membrane is reversible, in contrast with that of the pluronics. The compounds under study act preferentially on the NADH-dehydrogenase complex of the respiratory chain. Inhibition of respiration in thymocytes by these compounds confirms their ability to penetrate into the cell membrane. The dependence of membranotropic properties of polyalkylene oxides and their conjugates with the protein on their structure is discussed.

[Conjugation of classic Bowman-Birk soy inhibitor with a copolymer of ethylene oxide and propylene oxide]. / Kon''iugirovanie klassicheskogo soevogo ingibitora tipa Baumana-Birk s sopolimerom okisi étilena i okisi propilena.

A classical soybean inhibitor of the Bowman-Birk type (BBI) with a copolymer of ethylene oxide and propylene oxide (PE) has been synthesized. The BBI-PE conjugate contain five covalently bound polymeric chains per one protein molecule and retains its capacity to inhibit trypsin (Ki = 10(-10) M), alpha-chymotrypsin (Ki = 7 x 10(-8) M) and human granulocyte elastase (Ki = 3 x 10(-8) M). The preservation of the antiproteinase activity in the antichymotrypsin center creates a prerequisite for the manifestation of the anticarcinogenic effect of the inhibitor.