Interconnection of physico-chemical characteristics of organic solvents and their denaturing ability in relation to proteins]

Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effectiveness of acylation of protein amino groups with fatty acid chlorides in a system of reversed micelles of aerosol OT in octane

The characteristics of water-soluble enzyme (alpha-chymotrypsin) modification with [3H] palmitoyl chloride in the reversed Aerosol OT micelles in octane were determined. The degree of enzyme modification depends on the molar ratio [palmitoyl chloride]/[protein]. The modification reaction is characterized by the wide pH-optimum range and proceeds with high speed.     

A new strategy for the study of oligomeric enzymes: gamma-glutamyltransferase in reversed micelles of surfactants in organic solvents

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.     

The inconsistencies of present-day mathematical-physical methods pertaining to current generators in volume conductors used in electrocardiography

The present-day practices of electrocardiography and vectorardiography are based upon the theory that the surface potential differences can be assumed to be due to a single dipole inside the body. It is shown in this paper that a dipole cannot account for all the surface potentials due to realistic current generators, and hence the determination of the current generator from surface potential measurements based upon such a theory will lead to inconsistent representations of the heart for one and the same subject. To demonstrate this point two eccentric dipoles of different strengths and locations representing two muscle fibers are taken to be the current generator in a homogeneous spherical conductor. The exact surface potentials are then expressed by means of the “interior sphere theorem” of the authors. With these expressions the magnitude, direction, and location of the resultant dipole are determined by the method of D. Gabor and C. V. Nelson (J. App. Physics,25, 413–16, 1954). The surface potentials due to this resultant dipole are again exactly expressed by means of the “interior sphere theorem” and compared with those due to the eccentric dipoles assumed. It can be seen that the differences can be considerable. It is suggested that the multipole model of the authors (Bull. Math. Biophysics,20, 203–16, 1958) be used as a more accurate and the only unique representation of the heart. This investigation was supported by the National Heart Institute under a research grant H-2263(c).     

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.     

Reversed micelles of polymeric surfactants in nonpolar organic solvents. A new microheterogeneous medium for enzymatic reactions.

A new microheterogeneous non-aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n-butanol mixtures. The hydrodynamic radius of polymeric-reversed micelles was estimated to be in the range 22-51 nm, depending on the water content of the system, as determined by means of the quasi-elastic laser-light scattering. Polymeric reversed micelles were capable of solubilizing enzymes (alpha-chymotrypsin and laccase) in nonpolar solvents with retention of catalytic activity. Due to the strong buffering properties of CEPEI over a wide pH range, it could maintain any adjusted pH inside hydrated reversed micelles. It was found that catalytic behavior of enzymes entrapped in polymeric reversed micelles was rather insensitive to the pH of the buffer solution introduced into the system as an aqueous component, but determined mostly by acid-base properties of the polymeric surfactant itself. Both catalytic activity and stability of entrapped alpha-chymotrypsin and laccase were found to increase with increasing water content of the system. Under certain conditions, the entrapment of alpha-chymotrypsin into CEPEI reversed micelles resulted in a considerable increase in catalytic activity and stability as compared to aqueous solution. CEPEI reversed micelles were demonstrated to be promising enzyme carriers for use in membrane reactors. Owing to the large dimensions of CEPEI reversed micelles, they are effectively kept back by a semipermeable membrane, thus allowing an easy separation of the reaction product and convenient recovery of the enzyme.     

Catalysis by alpha-chymotrypsin entrapped into surface-modified polymeric nanogranules in organic solvent.

Physicochemical characteristics of previously suggested surface-modified polymeric nanogranules (SMPN) and catalytic and stability properties of alpha-chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N,N'-methylene-bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2-ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F-108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i.e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi-elastic light scattering to be 84 +/- 1 nm. Catalytic and stability properties of alpha-chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and wo = 15, respectively. alpha-Chymotrypsin-containing SMPN were used as a catalyst in the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and ethanol, performed in a membrane reactor.     

Effects of antibodies to choriogonadotropin in malignant growth

Summary We have studied the effects of preimmunization with a conjugate of the -subunit of choriogonadotropin and tetanus toxoid (CG-tt) on the growth of the implanted R 3230 AC mammary adenocarcinoma (in Fischer 344 rats) and the implanted 5123 1-1 hepatoma (in Buffalo rats) after 20 days, in order to determine if the in vivo production of antibodies against CG could modify the relationship between host and malignant growth. The results obtained demonstrated that active immunization against CG retarded significantly (P<0.01) the growth of the two transplantable tumors. Anti-CG antibodies were also determined and constantly found in the sera of all the preimmunized rats of both strains while no antibodies to CG were found in the control animals.