High-affinity binding of water by proteins is similar in air and in organic solvents

Published data for water adsorption by proteins suspended in organic solvents (of interest as enzyme reaction mixtures) have been converted to a basis of thermodynamic water activity (aw). The resulting adsorption isotherms have been compared with those known for proteins equilibrated with water from a gas phase. This comparison can show any effects of the solvent on the interaction between the protein and water at the molecular level. At lower water contents (aw less than about 0.4), similar adsorption isotherms are found in each solvent and in the gas phase; differences are probably less than the likely errors. Hence, it may be concluded that the presence of an organic solvent has little effect on the interaction between proteins and tightly bound water; on a molecular scale there is probably little penetration of the primary hydration layer by solvent molecules, even fairly polar ones such as EtOH. At higher aw values, there are differences between the isotherms which probably are significant. Nonpolar solvents increase the amount of water bound by the enzyme (at fixed aw), while polar solvents (mainly EtOH) may reduce the amount of water bound by the enzyme, presumably by occupying part of the secondary hydration layers in place of water.     

Denaturation of bacteriorhodopsin by organic solvents

The denaturation of bacteriorhodopsin by various organic solvents was studied using absorption, circular dichroism (CD) and fluorescence measurements. Organic solvents with a hydrogen-bonding group caused the release of retinal. The CD measurements showed that the helical structure was maintained even in the denatured state, whereas its tertiary structure was destroyed. The change in fluorescence intensity of tryptophan and fluorescent retinal also confirmed that the tertiary structure was destroyed. Comparison of the denaturation efficiency of various organic solvents showed that the concentration at denaturation was inversely proportional to the partition coefficient of the denaturant. This inverse proportionality clearly indicated that denaturation was determined by the concentration of denaturants which partitioned into the hydrophobic region of the membrane. It was discussed from the experimental results that the tertiary structure of bacteriorhodopsin was stabilized by the hydrogen-bonding networks between side chains of the helices. The results obtained from analysis of the amino acid sequence were also consistent with the hydrogen-bonding mechanism for the formation of the tertiary structure.     

Predicting helical segments in proteins by a helix-coil transition theory with parameters derived from a structural database of proteins

A novel helix-coil transition theory has been developed. This new theory contains more types of interactions than similar theories developed earlier. The parameters of the models were obtained from a database of 351 nonhomologous proteins. No manual adjustment of the parameters was performed. The interaction parameters obtained in this manner were found to be physically meaningful, consistent with current understanding of helix stabilizing/destabilizing interactions. Novel insights into helix stabilizing/destabilizing interactions have also emerged from this analysis. The theory developed here worked well in sorting out helical residues from amino acid sequences. If the theory was forced to make prediction on every residue of a given amino acid sequence, its performance was the best among ten other secondary structural prediction algorithms in distinguishing helical residues from nonhelical ones. The theory worked even better if one only required it to make prediction on residues that were “predictable” (identifiable by the theory); >90% predictive reliability could be achieved. The helical residues or segments identified by the helix-coil transition theory can be used as secondary structural contraints to speed up the prediction of the three-dimensional structure of a protein by reducing the dimension of a computational protein folding problem. Possible further improvements of this helix-coil transition theory are also discussed. Proteins 28:344–359, 1997. © 1997 Wiley-Liss, Inc.     

The use of p-toluene sulfonate to dissolve synaptosomal membrane proteins into organic solvents

A method is described by which a large proportion of membrane proteins may be dissolved in organic solvents by the use of p-toluene sulfonate. Synaptosomal membrane fractions from rat cerebral cortex were used as test material. Chloroform-methanol extraction dissolved 7% of proteins, 89% of lipid phosphorus, and 35% of reducing sugars. Further extraction of the residue with 2.5 mm p-toluene sulfonate in chloroform-methanol allowed the dissolution of 45% of the proteins. The final residue contained the rest of the protein and 60% of reducing sugars. The polypeptides in the three fractions showed marked differences in molecular weight and several glycoprotein bands were found in the final residue. The amino acid content of these three protein fractions was also different. It is concluded that p-toluene sulfonate, by lowering the pH and binding to the positive charges in the protein, is able to transfer about half of the synaptosomal membrane proteins into a hydrophobic medium.     

Extraction of mitochondrial membrane proteins into organic solvents in a functional state

Protein-lipid complexes in organic solvents can be used as the starting material in the reassembly of functional planar and spherical bilayers (Montal, M., Darszon, A. and Schindler, H. (1981) Q. Rev. Biophys. 14, 1-79). The transfer of three enzymes of the inner mitochondrial membrane into organic solvents as protein-lipid complexes has been studied to understand better the extraction process. The enzymes studied were cytochrome c oxidase, ATPase and succinate dehydrogenase. These enzymes were transferred into hexane and diethyl ether in an active state, however, the activities extracted varied quantitatively, depending on the amount of protein of the starting preparation, the concentration of phospholipids and the cation employed. In all conditions cytochrome c oxidase was extracted with the highest yield and specific activity, and it was actually enriched in the organic extract. The values for succinate dehydrogenase and ATPase were lower, but their specific activities were similar to those of the starting material. This indicates that some membrane proteins are preferentially extracted into organic solvents in a functional state. The enzymes, as protein-lipid complexes, are fairly stable in organic solvents; in a month of storage at 4 degrees C in hexane some enzymes loose less than 50% of their activity.     

The oxidation of chiral alcohols catalyzed by catalase in organic solvents

The catalytic properties of bovine liver catalase have been investigated in organic solvents. In tetrahydrofuran, dioxane, and acetone (all containing 1% to 3% of water), the enzyme breaks down tert-butyl hydroperoxide several fold faster than in pure water. Furthermore, the rate of catalase-catalyzed production of tert-butanol from tert-butyl hydroperoxide increases more than 400-fold upon transition from aqueous buffer to ethanol as the reaction medium. The mechanistic rationale for this striking effect is that in aqueous buffer the rate-limiting step of the enzymatic process involves the reduction of catalase's compound I by tert-butyl hydroperoxide. In ethanol, and additional step in the reaction scheme becomes available in which ethanol, greatly outcompeting the hydroperoxide, is oxidized by compound I regenerating the free enzyme. In solvents, such as acetonitrile or tetrahydrofuran, which themselves are not oxidizable by compound I, catalase catalyzes the oxidation of numerous primary and secondary alcohols with tert-butyl hydroperoxide to the corresponding aldehydes or ketones. The enzymatic oxidation of some chiral alcohols (2,3-butanediol, citronellol, and menthol) under these conditions occurs enantioselectively. Examination of the enantioselectivity for the oxidation of 2,3-butanediol in a series of organic solvents reveals a considerable solvent dependence. (c) 1995 John Wiley & Sons, Inc.     

Glucose condensation by glucoamylase in organic solvents

Summary Oligosaccharides were synthesized through the enzymatic condensation of D-glucose by glucoamylase in water-organic mixtures with high concentrations of two of diethylene glycol diethyl ether or triethylene glycol dimethyl ether. The effect of water content on the yield of reaction was studied; maximum yield was obtained with 10% (v/v) of water in the two systems. Kinetics of synthesis and products composition were different with the two solvents. 37% of glucose were condensed by action of glucoamylase from a reaction medium containing 20 g/L of glucose and 90% (v/v) of diethylene glycol diethyl ether.     

Kinetic analysis of the mechanism for subtilisin in essentially anhydrous organic solvents

Steady-state kinetic analysis has been used to confirm the catalytic mechanism of lyophilized subtilisin suspended in a variety of organic solvents. Specifically, this article demonstrates that partial reactions can occur between subtilisin and ester substrates in organic solvents. Partitioning of common intermediates between competing acceptors at a constant ratio of products has also been described. The decomposition of a common intermediate formed from different substrates at the same rate is also further evidence of an acyl-enzyme mechanism for subtilisin suspended in anhydrous solvents. Partitioning of a common intermediate to give two products at a constant total rate, and saturation kinetics at varying substrate concentrations, complete a kinetic investigation of the enzyme mechanism. All the data generated support the formation of a stable acyl enzyme during the transesterification reaction catalzyed by subtilisin in the solvents used.     

The denaturation transition of DNA in mixed solvents

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The "melting" transition temperature was found to be 94 degrees C for 4% mass fraction DNA/d-water and 38 degrees C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 A across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains.     

Inhibition of human lymphocyte function by organic solvents

We studied the direct effect of reactive hydroxyl precursors and inhibitors on CD4+ T-cell function. We used hydrogen peroxide plus ferrous chloride as the hydroxyl radical-generating system and di-methyl sulphourea, di-methyl sulfoxide, pyrrolidine dithiocarbonate, methanol, and ethanol, at a noncytotoxic concentration, as inhibitors. The immune parameter studies were proliferation and interleukin-2 production by peripheral blood lymphocytes stimulated with anti-CD3 antibody, phytohemagglutinin and alloantigens; proliferation, interleukin-2 production and mRNA expression of interleukin-4 and interferon gamma by allogeneic CD4+ T-cell clones stimulated with alloantigens. The results show that lymphocytes produce significant amounts of reactive oxygen species as measured by malondialdehyde produced in cultures. The hydroxyl radical-generating system did not change any of the cellular responses studied although it doubled Malondialdehyde production. Hydroxyl radical scavengers signi tly inhibited all responses at doses that didn't significantly decrease malondialdehyde production. DNA analysis failed to show evidence for apoptosis. Conclusion: Hydroxyl radical scavengers inhibit lymphocyte mitogenesis by a process that is independent of scavenging hydroxyl radicals.     

Luminescence of isolated chloroplasts induced by organic solvents

Preilluminated lettuce chloroplasts emit light when injected with pure organic solvents. A positive linear correlation was found between the luminescence intensity and the dielectric constant of the solvent. It is suggested that the dissolution of the solvent in the chloroplast membrane increases its dielectric constant. This modifies the energy levels of charged trapped photoproducts and enables their recombination leading to luminescence.     

Proteins in organic solvents

Catalysis in organic solvents and the mapping of protein surfaces using multiple solvent crystal structures are two rapidly developing areas of research. Recent advances include the study of protein folding and stability in different solvents, and the demonstration that it is possible to qualitatively rank the affinities of protein binding sites for a given organic solvent using the multiple solvent crystal structures method.     

Improvement of hydrogenase enzyme activity by water-miscible organic solvents

Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.     

The biodegradation of mixtures of organic solvents by mixed and monocultures of bacteria

Much more information concerning the biodegradation kinetics of mixtures of common industrial chemicals, such as organic solvents, needs to be gathered before wastewater biotreatment process performance can become a matter of design. Here, the biooxidation of a solvent mixture comprizing methanol, acetone, isopropanol, and methylene chloride is examined. The fact that the enrichment culture obtained comprized only two solvent-utilizing strains, together with only minor percentages of nonsolvent utilizing satellite strains, was contrary to the theory of microbial competition. In addition, the complex relationship between the two solvent-utilizing strains indicates that further work is necessary on the pathways involved in isopropanol and acetone biooxidation and on the effects of operating conditions on the fluxes along such pathways.     

Some properties of erythrocuprein treated by organic solvents.

The effect of various types of organic solvents on the properties of bovine erythrocuprein was studied. Three organic solvents were found in which the protein is soluble, these were: dimethyl sulfoxide, formamide and N-methylformamide. It was shown that in formamide and dimethyl sulfoxide media the protein possees superoxide dismutase activity, but in N-methylformamide the protein has negligible activity. In organic solvents the substrate (superoxide radical) and solvated electron result in reduction of the protein copper. At high concentrations of superoxide radical or solvated electrons an inactivation of protein and stabilization of superoxide radicals was noted. The stabilization is most pronounced in N-methylformamide. The protein that is reduced by the radical or the solvated electron may be reoxidized by molecular oxygen, the latter being reduced to the superoxide radical.