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Noncovalent columnar polymers (NCPs) based on cyclodextrins (CD) are polymeric assemblies of molecules that have continuous hollow channels, the width of which is determined by the diameter of the cavity of the initial CDs. The repeating fragment in an NCP is the CD molecule. For NCPs that were obtained by the exclusion of polymer backbone macromolecule from the corresponding inclusion complexes (ICs) based on β-cyclodextrin (NCPexcl), the polymer length, expressed as the number of macrocycles in a single chain (n) is determined by the size of the included ligand, polypropylene glycol (PPG), and is the PPG polymerization degree divided by two. The determination of the molecular weight of an NCP obtained by the precipitation method (NCPprec) is rather difficult, since they are present in the aggregated state rather than in the form of individual molecules in solution. To estimate the molecular weight of NCPprec, an indirect method is used, which is based on the determination of the aggregation rate of the ICs formed as a result of the interaction between an NCP and polypropylene glycol with a fixed molecular weight (MW), in this case PPG 1000. The comparison of the aggregation rates of the inclusion between NCPexcb (which were synthesized using PPGs with different molecular weights) and PPG 1000 with the aggregation rate of the inclusion complex on the basis of NCPprec provided the estimation for the MWs of single polymer chains. The fact that the samples of NCPprec contain ∼30% of the monomeric β-CD was taken into account when constructing the calibration curve. It was demonstrated that the MW of the polypropylene glycol corresponding to NCPprec is 1320 Da. Consequently, ∼11–12 molecules of β-CD are included in the single chains of NCPprec.

Kinetic analysis of the aggregation of complexes formed by columnar types of α- and γ-cyclodextrins (α-CDcol and γ-CDcol) and poly(alkylene glycols) is performed by the dynamic light scattering method. For comparison, analogous studies were conducted for systems containing initial α- and γ-cyclodextrins (α-CD and γ-CD). Upon the aggregation of systems containing α-CD, the number of nuclei with critical sizes slowly increases at the initial part of kinetic curve throughout the solution bulk; when some limiting concentration and sizes of formed aggregates are achieved, the system is transformed into the gel-like state. The aggregation of γ-CDcol-poly(ethylene glycol) system proceeds into two stages. At the first fast stage, aggregates are formed by particles representing single-strand inclusion complexes composed of one γ-CDcol molecule and two units of ethylene oxide. At the second, much slower stage, aggregates are formed by two-strand complexes composed of one γ-CDcol molecule and four units of ethylene oxide. It follows from the comparison of aggregative properties of γ-CDcol-poly(ethylene glycol) and γ-CDcol-poly(propylene glycol) systems that the rate of aggregation is much higher in the second case.

A new method for the synthesis of associates of cyclodextrins (CDs) of the columnar type consisting of the precipitation of CDs from aqueous solutions into acetone at lowered temperatures is developed. It is shown that columnar structures exist in both a crystalline state and in aqueous solutions. Hydrodynamic radii and molecular masses of noncovalent columnar structures (NCSs) in aqueous solutions are determined by the dynamic and static light scattering methods. The degree of association of noncovalent columnar polymers is ∼40. It is revealed the NCS associates based on β-CD are stable and their hydrodynamic radius Rh is equal to 100 ± 10 nm. The kinetics of interactions of initial β-CD and NCS with poly(propylene oxide) (PPO) is studied. The pattern of kinetic curves of Rh growth upon interaction between NCS and PPO indicates that the aggregation of the particles of polymer inclusion complex proceeds in the regime of reaction-limited cluster-cluster aggregation. Kinetic curves describing the interaction processes between β-cyclodextrin and PPO are characterized by the presence of induction period t0. At t > t0, Rh ∞ t0.56 which is typical for the diffusion-limited cluster-cluster aggregation. Schemes of the formation of polymer inclusion complexes between initial β-CD or NCS and poly(propylene oxide) are proposed. Comparison of kinetic data on the complexation of β-CD in solution in the form of associates of two types with PPO demonstrates that columnar forms of associates are reactive species acting as macroreceptors.

A simple procedure is proposed for the detection of association of cyclodextrins (CDs) and their derivatives in aqueous solutions by analyzing the curves of titration of these substances with dyes. Dissociation constants of cyclodextrin-dye complexes and concentrations of sites involved in the complexation are determined by the nonlinear regression analysis. The processes of CD association were characterized using deviations of the function of CD saturation with ligands from a simple scheme CD + ligand ⇆ complex. Two association mechanisms were disclosed: cooperative association (for α-CD and dihydroxypropyl-β-CD) and step association (for β-CD, dimethyl-β-CD, hydroxypropyl-β-CD, and γ-CD). In the case of stepwise-associating compounds, association constants for head groups of cyclodextrins are found to vary from 3 × 103 to 8 × 103 M−1. The titration data on CD association are confirmed by studying solutions of the above compounds using dynamic light scattering.

*Institute of Microbiology, Russian Academy of Sciences, pr. 60-1etiya Oktyabrya 7, k. 2, Moscow, 117811 Russia **Kazan State University, ul. Kremlevskaya 18, Kazan, 420008 Russia ***Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia Received July 12, 1999 Abstract--Alkyl-substituted hydroxybenzenes (AHBs), autoinducers of microbial dormancy (or d I factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with the Bacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renatur- ation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made that d I factors may play the role of natural chemical chaper- ons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable com- plexes with enzymes. Key words: dormancy, dormancy autoinducers, metabolic block, thermostability of enzymes, dl factors of microorganisms, chemical chaperons, aikyl-substituted hydroxybenzenes It has recently been shown that autoregulatory d~ factors, accumulating in the medium to a threshold level, induce the transition of vegetative microbial cells to a dormant state [1, 2]. In their chemical nature, the known dl factors represent mixtures of the isomers and homologues of alkyl-substituted hydroxybenzenes (AHBs) 13, 4]. The mechanism of action of dl factors during the development of a metabolically quiescent state is twofold. First, they interact with membrane lipid, causing the crystallization of the membrane lipid stroma, inhibition of the functional activity of mem- branes (including membrane-associated energy-produc- ing processes), an increase in membrane permeability to monovalent ions, and dehydration of the cell protoplast [5, 6]. Second, AHBs function as low-molecular-weight protein modifiers inhibiting the catalytic activity of enzymes (including nucleic acid depolymerases) and providing for the survival of dormant cells in the absence of functionally active energy-producing pro- cesses. The inactivation of enzymes by AHBs has been demonstrated in experiments with chymotrypsin, trypsin, RNase, invertase, and glucose oxidase [7]. The inhibition of metabolism by AHBs is associated with IE-mail for correspondence: andlm@mail.ru an enhanced resistance of resting cells to various stress factors; therefore, it can be anticipated that AHBs may enhance the stability of protein molecules. In view of the foregoing, the aim of the present work was to study the stabilizing effect of d I factors on enzymes as one of the possible mechanisms of resis- tance of dormant cells to stresses. MATERIALS AND METHODS Experiments on the structural modification of enzymes were carried out using C6-AHB, an amphiphilic compound with pK, = 9. C6-AHB (herein- after, also referred to as the dl factor) was added to reaction mixtures in the form of an ethanol solution to give the final ethanol concentration of no more than 5 vol % (in control experiments, equivalent amounts of ethanol were added). Taking into account that RNases play an important role in the functioning of the hereditary apparatus of cells, are involved in trophic chains, and serve as the extracellular autoregulators of physiological activity, experiments were primarily performed with the RNase (EC 3.1.27.5) of Bacillus intermedius purified to an apparent homogeneity (Mr = 12 300 Da) [8]. Some 0026-2617/00/6902-0180525.00 9 2000 MA1K "Nauka/Interperiodica"

New approaches to the analysis of differential scanning calorimetry (DSC) data relating to proteins undergoing irreversible thermal denaturation have been demonstrated. The experimental approaches include obtaining a set of DSC curves at various scanning rates and protein concentrations, and also reheating experiments. The mathematical methods of analysis include construction of a linear anamorphosis and simultaneous fitting of a theoretical expression for the dependence of the excess heat capacity on temperature to a set of experimental DSC curves. Different kinetic models are discussed: the one-step irreversible model, the model including two consecutive irreversible steps, the Lumry and Eyring model with a fast equilibrating first step, and the whole kinetic Lumry and Eyring model.

The synthesis and analysis of nanostructures in the cavities of protein molecules is a promising research field in the industry of nanosystems. In this study, atomic force microscopy (AFM) has been used to evaluate the properties of CdS quantum dots synthesized in the tunnel cavities of R-phycoerythrin, a 290 kDa water-soluble pigment protein responsible for light harvesting in red algae. It has been shown that R-phycoerythrin dissolved in deionized water to a concentration of 50 μg/ml is prone to self-organization into regular spatial structures upon adsorption on the surface of mica, but no such structuring takes place in films prepared from R-phycoerythrin solutions diluted tenfold. In the latter case, protein molecules are deformed, as judged from the analysis of the surface profile. R-phycoerythrin with CdS quantum dots in protein cavities (the concentration of the preparation was (48 μg/ml) loses the self-organization ability and is not deformed upon adsorption on the mica surface. Analysis of AFM images by flicker-noise spectroscopy has shown that incorporation of CdS quantum dots into R-phycoerythrin molecules provides for "smoothing" of the protein surface, with various irregularities being leveled off. Conversely, the irregularity of the protein surface increases when R-phycoerythrin molecules are arranged into three-dimensional branching structures. It is concluded that CdS quantum dots interfere with protein-protein interactions and restrain the conformational mobility of the protein. The anomalously rigid structure of R-phycoerythrin in the presence of CdS is due to its conformational rearrangements during the synthesis of quantum dot.

This review summarizes data on structure of muscle glycogen phosphorylase b and the role of the cofactor pyridoxal 5'-phosphate in catalysis and stabilizing the native conformation of the enzyme. Specific attention is paid to the stabilizing role of pyridoxal 5'-phosphate upon denaturation of phosphorylase b. Stability of holoenzyme, apoenzyme, and enzyme reduced by sodium borohydride is compared.

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52 degrees C in a wide range of protein concentrations, [P]0. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52 degrees C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]0 (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42 degrees C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]0 and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates [A/A(lim); t] (t is time and A(lim) is the limiting value of A at t --infinity). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.

The experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A(lim) and kI (A(lim) is the limiting value of A at t --infinity and kI is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant kI calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A(lim) and kI may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.

Kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of AMP concentration under the saturation of the enzyme by glycogen. Applicability of some variants of the kinetic model involving the interaction of AMP- and glucose 1-phosphate-binding sites in the dimeric enzyme molecule is considered. A kinetic model of the enzymatic reaction describing adequately the activation of the enzyme by AMP and inhibition at sufficiently high concentrations of AMP is proposed.

The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity, an intermediate plateau, and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min(-1); 1 M GuHCl, 25 degrees C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscle was studied in the system of aerosol OT (AOT) reversed micelles in octane. The sedimentation experiments have shown that the enzyme oligomeric state depends on the degree of micelle hydration. The enzyme monomer, dimer, trimer, tetramer, hexamer, and octamer were observed, depending on the degree of hydration.

An empirical equation that describes deviations from Michaelian kinetics is proposed. The equation allows the limiting values of the Michaelis constant at v/Vmax --0 and v/Vmax --1 to be estimated (v is the rate of the enzymatic reaction and Vmax is the limiting value of v at saturating concentrations of substrate). The applicability of the equation is demonstrated for kinetic data obtained for glutamate dehydrogenases from various sources (negative kinetic cooperativity for coenzyme) and for biosynthetic threonine deaminase from pea seedlings (sharper approaching the limiting value of the enzymatic reaction rate with increasing substrate concentration in comparison with the hyperbolic law). The negative cooperativity for the function of saturation of protein by ligand is also analyzed (data on binding of spin-labeled NAD, NADH, and NADPH by beef liver glutamate dehydrogenase and binding of cupric ions by BSA are used as examples).

Three-parameter Hill's equation, which is used in enzyme kinetics, was shown to applicable to calibration curves of both potentiometric (glucose, pesticides, urea, etc.) and amperometric (surfactants, biphenyl, etc.) biosensors. Possible causes of errors of analyte concentration measurements are discussed.

Alkyl-substituted hydroxybenzenes (AHBs), which are auto-inducers of microbial dormancy (d1 factors), were found to stabilize the structure of protein macromolecules and modify the catalytic activity of enzymes. In vitro experiments showed that C6-AHB at concentrations from 10(-4) to 10(-2) M, at which it occurs in the medium as a true solution and a micellar colloid, respectively, nonspecifically inhibited the activity of chymotrypsin, RNase, invertase, and glucose oxidase. C6-AHB-induced conformational alterations in protein macromolecules were due to the formation of complexes, as evidenced by differences in the fluorescence spectra of individual RNase and C6-AHB and their mixtures and in the surface tension isotherms of C6-AHB and trypsin solutions. Data on the involvement of dormancy auto-inducers in the post-translational modification of enzymes and their inhibition will provide further insight into the mechanisms of development and maintenance of dormant microbial forms.

Alkyl-substituted hydroxybenzenes (AHBs), auto-inducers of microbial dormancy (or d1 factors), were found to stabilize the structure of protein macromolecules, making them metabolically less active and more resistant to stresses. In vitro experiments with the Bacillus intermedius ribonuclease and chymotrypsin showed that the degree of the physical and chemical stability of these enzymes treated with AHBs depends on their concentration and incubation time. Experiments with RNase, which is capable of refolding, i.e., renaturation after heat denaturation, revealed that AHBs efficiently interact with both intact and denatured proteins. The data obtained allow the inference to be made that d1 factors may play the role of natural chemical chaperons, blocking metabolism in dormant cells through the formation of catalytically inactive thermostable complexes with enzymes.

Large-scale functionally significant changes in the intramolecular dynamics of muscle glycogen phosphorylase b (EC 2.4.1.1) in solution upon ligand binding, transition from dimeric to tetrameric form, temperature denaturation and aggregation were registered at room temperature using the tryptophan phosphorescence technique. It was shown that binding of glucose-1-phosphate (substrate, 0.25-4 mM) and glucose (competitive inhibitor, 0.5-8 mM) to the active site and temperature-induced protein aggregation decrease large-scale structural fluctuations of the protein matrix at the level of domains and subunits; whereas the transition of glycogen phosphorylase b to tetrameric form (R-conformation) leads to a dramatic increase in the structural flexibility of the peripheral parts of the protein globule.

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.

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.

The mechanism of heat aggregation of proteins proposed by the author involves the stage of irreversible denaturation, the stage of nucleation, and the stage of growth of aggregates. It was shown that the initial parts of the kinetic curves of aggregation followed by monitoring the increase in absorbance (A) or intensity of light scattering (I) are linearized in coordinates (dA/dt; t) and (A; t2) (or, respectively, in coordinates (dI/dt; t) and (I; t2)). The slope of these linear anamorphoses is proportional to the product of the rate constant of irreversible denaturation and the rate constant of growth of aggregates. The mechanism of heat aggregation proposed is fulfilled for pig heart citrate synthase. The dI/dt versus t curves for heat aggregation of glycogen phosphorylase b from rabbit skeletal muscles display a lag period whose appearance is caused by intramolecular predenaturational changes in the enzyme molecule.

The use of biosensors for detecting aromatic compounds (aniline, hydroquinone, phenol, and N,N-dimethyl-p-phenylenediamine), heterocyclic compounds (adenosine, AMP, ATP, bilirubin, hypoxanthine, guanine, inosine, inosine 5'-phosphate, xanthine, creatinine, uric acid, and NAD), carbohydrates (glucose, galactose, lactose, maltose, and sucrose), vitamins, antibiotics, steroids, mutagens, and organophosphorous pesticides is discussed. Basic metrological characteristics of these biosensors are reviewed.

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.

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.

Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride is accompanied by the displacement of the maximum in the protein fluorescence spectrum (lambda max) from 331 to 348 nm. The half-maximal change in the lambda max position is observed at 1.18 M guanidine hydrochloride. For this concentration of denaturant, the sedimentation pattern consists of two boundaries, one of which corresponds to the motion of the hexameric enzyme form (s20,w = 8.2 S) and other represents a monomer (s20,w = 2.6 S). In the presence of 2 M guanidine hydrochloride the enzyme moves as a monomer. The kinetics of inactivation of uridine phosphorylase by guanidine hydrochloride are complex (minima and maxima are observed on the kinetic curves). The initial rate of the enzyme reactivation after dilution of the enzyme preincubated with guanidine hydrochloride is second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of low-activity monomers into dimers followed by a rapid hexamer formation. The second-order rate constant for the reassociation of the enzyme is 3.0.10(4) M-1.sec-1 (50 mM borate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20 degrees C). Thiol groups become accessible to titration by 5,5'-dithiobis-(2-nitrobenzoic acid) after treatment of uridine phosphorylase with guanidine hydrochloride. Uridine and uracil inhibit the unfolding of the protein globule by guanidine hydrochloride.

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.

It has been shown that the rate constant, k, for thermal inactivation of rabbit skeletal muscle glycogen phosphorylase b decreases as the enzyme concentration increases. This effect is interpreted within the framework of a kinetic model which includes two parallelly occurring processes, namely: phosphorylase b denaturation in solution and denaturation of the enzyme absorbed on test-tube walls. The contribution of the latter process increases with a decrease in the enzyme concentration. The protective effect of the allosteric activator (AMP), allosteric inhibitors (glucose 6-phosphate and FMN) and the competitive inhibitor (glucose) against heat denaturation of glycogen phosphorylase b has been demonstrated. Quantitative analysis of the dependence of the rate constant, k, on concentration of AMP, glucose 6-phosphate and FMN allows the calculation of microscopic constants for dissociation of phosphorylase b complexes with these ligands for the given experimental conditions as equal to 0.34, 0.50 and 0.30 mM, respectively. The S-shaped dependence of the rate constant of thermal inactivation on glucose concentration points to the existence of positive cooperative interactions between glucose-binding sites in the dimeric molecule of phosphorylase b (nH = 1.8).

The kinetics of the thermal inactivation of rabbit skeletal muscle phosphorylase b have been studied. Under the condition used (0.08 M HEPES, pH 6.8, 75 mu g/ml phosphorylase b) the enzyme quickly loses its activity at the temperature higher then 49 degrees C. All the specific ligands tested (substrate -glucose 1-phosphate, competitive inhibitor - glucose, allosteric activator - AMP, and allosteric inhibitors - glucose 6-phosphate and FMN) have the pronounced protective effect. The strongest protective action have glucose 6-phosphate and FMN. Quantitative analysis of the inactivation rate constant versus ligand concentration curves allow the enzyme affinity for ligands to be characterized.

The kinetic behaviour of rabbit skeletal muscle phosphorylase kinase at variable concentrations of the enzyme and the substrate (glycogen phosphorylase b) has been studied. The kinetic curves reveal a lag period whose duration decreases with a rise in the phosphorylase kinase concentration (when the reaction is initiated by an addition of the ATP + MgCl2 mixture to the enzyme preincubated with phosphorylase b, CaCl2, glycogen and glucose-1-phosphate or inorganic phosphate). A decrease of the phosphorylase b concentration eliminates the lag period. Under these conditions the specific activity of phosphorylase kinase decreases with a rise in the enzyme concentration. The kinetic behaviour of phosphorylase kinase is interpreted in terms of a model of a linearly associating system, such as M reversible M2 reversible M3 reversible ...Mi, where M is the dexadecameric molecule of phosphorylase kinase. Acceleration of the phosphorylase kinase-catalyzed reaction in the course of the enzymatic process seems to be due to the breakdown of inactive enzyme associates (Mi) caused by phosphorylase b. The short gamma-subunit of phosphorylase kinase devoid of the calmodulin-binding domain does not display any hysteretic properties.

The inhibition of glycogen phosphorylase b from rabbit skeletal muscles by the derivatives of riboflavin, FMN, FAD, and 2', 3', 4', 5'-tetraacetylriboflavin substituted in positions 6 and 8 of the isoalloxazine part of the flavin molecule is found to be cooperative (the Hill coefficient, h, exceeds 1.0). The modification of the flavin molecule slightly changes the value of the Hill coefficient, but results in the increase of the "half-saturation" concentration [I]0.5.

The mathematical models of amperometric biosensors, with single- or two-substrate reaction occurring in their cover, were developed. A linear two-parametric equation of steady state was suggested for the biosensor detecting the product of single-substrate reaction corresponding to the Michaelis-Menten scheme. The conditions of equation applicability to description of standard biosensor kinetics in the wide range of substrate concentration, diffusion-module values, and permeability coefficients of three-layer-cover components were studied.

The turbidimetric method for determining the phosphorylase kinase activity has been developed. The reaction mixture contained, alongside with other components of the kinase reaction, also the substrates of glycogen phosphorylase a, the final product of the kinase reaction-glycogen and glucose 1-phosphate (or inorganic phosphate). The kinetics of the cascade enzymatic system were followed by the increment (decrement) of absorbance of the glycogen solution at 360 nm (delta A). The initial rate of the phosphorylase kinase-catalyzed enzymatic reaction, nu 0, can be calculated according to the formula: nu 0 = 2tg alpha/a2, where tg alpha is the initial slope of the kinetic curve in the coordinates: delta A-(time)2 and a2 is the specific enzymatic activity of phosphorylase a. The latter was estimated from the initial rates of the phosphorylase reaction measured by the addition of glycogen to the reaction mixture after the completion of the kinase reaction. The reliability of the turbidimetric method for determining the phosphorylase kinase activity was proved by comparison with a direct method based on the measurement of the amount of incorporated 32P.

The interaction of the myosin subfragment-1 (S-1) isoforms containing different alkali light chains A1 and A2 with F-actin has been studied using the stopped-flow technique and sedimentation in an analytical ultracentrifuge. The data obtained suggest a different mode of attachment of the S-1 isoforms to F-actin filaments.

Based on a theoretical analysis of functioning of a monocascade enzymatic system, a method for continuous monitoring of the phosphorylase kinase-catalyzed enzymatic reaction has been developed. The method is based on the ability of the kinase reaction product--the phosphorylated form of glycogen phosphorylase (form a)-to catalyze glycogen phosphorolysis (with inorganic phosphate as the low molecular weight substrate) or synthesis (with glucose 1-phosphate) in the absence of AMP. A turbidimetric method may be used for the monitoring of the reaction of glycogen degradation (or synthesis) by phosphorylase a formed in the course of the kinase reaction. A method to calculate the initial rate of the kinase reaction from an absorbance versus (time)2 plot has been theoretically substantiated.

The kinetics of glycogen chains growth catalyzed by glycogen phosphorylase b from rabbit skeletal muscles at saturating glycogen concentrations have been studied. The applicability of twelve different variants of the Monod-Wyman-Changeux model and six kinetic schemes assuming the interaction of AMP- and glucose 1-phosphate binding sites in the dimeric enzyme molecule is discussed. The kinetic model of the reaction is proposed which suggests: (1) independent binding of AMP and a molecule of glucose 1-phosphate to the dimeric enzyme molecule saturated by glycogen; (2) exclusive binding of the second glucose 1-phosphate molecule by the dimeric enzyme molecule containing two molecules of bound AMP and one molecule of bound glucose 1-phosphate; (3) exclusive ability of the enzyme complex with glycogen, two molecules of AMP and two molecules of glucose 1-phosphate in catalytic transformation. The parameters of the rate equation were calculated by nonlinear regression analysis. The proposed kinetic scheme satisfies the following criteria: (1) convergence in the regressional analysis; (2) reliability of the parameter values; (3) the minimal sum of the weighed squares of residuals. This model was found to fit adequately the kinetics of muscle phosphorylase b reaction at saturating glycogen concentrations.

The effect of specific ligands (the substrate glucose 1-phosphate, AMP, and flavins) on the initial rate of tryptic digestion of muscle glycogen phosphorylase b has been studied (0.02 M HEPES, pH 6.8; 37 degrees C). Differences were observed between the kinetic curves of the enzyme trypsinolysis initiated by the addition of a trypsin/ligand mixture and once trypsin was added to the enzyme preincubated with a ligand for 10 min. It was suggested that the specific ligands studied induce relatively slow conformational changes in the phosphorylase b molecule.

The effect of specific ligands (the substrate glucose 1-phosphate, AMP, and flavins) on the initial rate of tryptic digestion of muscle glycogen phosphorylase b has been studied (0.02 M HEPES, pH 6.8; 37°C). Differences were observed between the kinetic curves of the enzyme trypsinolysis initiated by the addition of a trypsin/ligand mixture and once trypsin was added to the enzyme preincubated with a ligand for 10 min. It was suggested that the specific ligands studied induce relatively slow conformational changes in the phosphorylase b molecule.

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 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.

Solubilization of 2',3',4',5'-tetrabenzoyl-5-acetyl-1,5-dihydroriboflavin (benzaflavin) by conjugates of BSA with poly(alkylene oxide)s [poly(ethylene glycol) and block co-polymers of ethylene oxide and propylene oxide (pluronics)] was investigated. Conjugates of BSA with pluronics were shown to have more solubilizing efficiency towards benzaflavin than BSA itself. Solubilized forms of benzaflavin are able to inhibit NADPH-dependent peroxidation of lipids in rat liver microsomes. A study of solubilized riboflavin ester transfer to mitochondria demonstrated that conjugates of BSA with pluronics may be advantageously employed for this purpose. Inhibitory properties of solubilized forms of benzaflavin were demonstrated by the study of their influence on the enzyme activity of D-amino-acid oxidase from pig kidney. The results show that solubilized forms of biologically active compounds based on conjugates of proteins with poly(alkylene oxide)s may be used for testing of chemical substances in biochemical systems.

A theoretical analysis of the functioning of membrane biosensors based on consecutive polyenzymatic transformations of the substrate and detectable by indicator electrodes with electrochemical regeneration of the enzyme active sites, has been carried out. Correlations between the substrate concentration, diffusion-catalytic characteristics of the coating components, and rate of the indicator reaction on the one hand, and the level of the steady-state response of the polyenzymatic biosensors, on the other, have been determined for relatively low values of the polyenzymatic layer thickness. Various regimens of functioning of bienzymatic electrodes have been considered.