Studies on horseradish peroxidase in dimethyl sulphoxide/water mixtures. The activation of hydrogen peroxide and the binding of fluoride.

We studied the variation in spectra and in reactivity towards H2O2 of solutions of horseradish peroxidase in dimethyl sulphoxide/water mixtures, obtained by diluting stock solutions of the enzyme in either water or dimethyl sulphoxide, and assayed the enzyme activity and studied the binding of F- by the peroxidase in 65% (v/v) dimethyl sulphoxide. A broadly similar pattern of changes is observed whether one starts from water or from dimethyl sulphoxide; the changes are essentially reversible, though hysteresis is observed. When the dimethyl sulphoxide content of the solvent mixture is increased, the peroxidase retains its ability to activate H2O2 up to 74% (v/v) dimethyl sulphoxide. The peroxidase in 65% (v/v) dimethyl sulphoxide binds F- together with a proton (or the equivalent loss of HO-), as already established for aqueous solutions. We point out that the occurrence in such solutions of both the ability to activate H2O2 and the inability to bind F- without taking up H+ or losing HO- supports the proposed mechanism for activating H202, whereby the protein binds the substrate in the form of the much more reactive HO2-.     

Kinetics and mechanism of the interaction between human serum albumin and monomeric haemin.

The interaction of human serum albumin with monomeric haemin has been investigated by detailed kinetic analysis in dimethyl sulphoxide/water (3:5, v/v). The results obtained under conditions of albumin saturation of haemin and under pseudo-single turnover conditions indicate that methaemalbumin is formed in a two-stage, single-intermediate process. The initial association between the haemin and human serum albumin is a chemically controlled process (k1 = 1.7 X 10(5) mol-1 . s-1 . dm3 at 24 degrees C); the variation of K1 with pH exhibited a well defined pK of 5.9. The overall equilibrium constant, calculated by using microscopic rate constants, is 1.1 (+/- 0.5) X 10(8) mol-1 at 24 degrees C. The data and conclusions are consistent with a general binding mechanism for albumin in which intermediate formation is followed by an entropy-controlled internalization of the ligand.     

Low concentrations of dimethyl sulphoxide accelerate conjugation and incorporation of glycine and glucosamine intetrahymena

Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-¹⁴C-glycine and 6-³H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide.     

Preparative isoelectric focusing in immobilized pH gradients. II. A case report

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.     

The uptake and stability of simian-virus-40 DNA after calcium phosphate transfection of CV-1 cells.

The uptake and fate of purified SV40 (Simian virus 40) DNA, transfected into permissive CV-1 cells by calcium phosphate precipitates, was examined. By using a viral plaque assay, optimum conditions for transfection were established and transfection efficiencies of up to 10(6) plaque-forming units/micrograms of SV40 DNA were obtained. After a 2h exposure to 3H-labelled SV40 DNA-calcium phosphate co-precipitates under basal conditions, up to 7% of the input DNA became cell-associated, with approx. 4% reaching the nuclear fraction. This value was never exceeded, even under conditions known to enhance significantly the ultimate transfection efficiency, such as increased exposure time, addition of carrier DNA or treatment with DMSO (dimethyl sulphoxide) or glycerol. Substantial degradation of this SV40 DNA occurred within a further 4h, apparently in both nucleus and cytoplasm. Degradation of form-II and form-III SV40 DNA, which have lower transfection efficiencies than form-I DNA, was no more rapid than degradation of form-I DNA. The results indicate that less than 0.5% of the transfected DNA which reached the nucleus is protected from nuclease attack. The mechanism of action of agents such as glycerol, DMSO or carrier DNA remains obscure, but they may be involved in conferring greater stability to the intracellular SV40 DNA rather than merely affecting its rate of entry into the cell.     

pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine.

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.     

Charge equilibria and pK(a) of malvidin-3-glucoside by electrophoresis

Paper electrophoresis has been used over the pH range 1.2 to 10.4 to measure apparent pK(a) values for malvidin-3-O-glucoside of pK(a(1)) 1.76+/-0.07, pK(a(2)) 5.36+/-0.04, and pK(a(3)) 8.39+/-0.07. Using solvent partitioning between buffered aqueous solutions and n-octanol, several micro-pK(a) constants for malvidin-3-O-glucoside were also identified, highlighting the complex nature of malvidin-3-glucoside equilibria. As a nonspectrophotometric procedure, the charge-dependent electrophoretic mobility method provided independent information on the net charge and color of anthocyanin species at wine pH (ca. 3.6). At this pH, the color of malvidin-3-glucoside in red wines is consistent only with the uncharged quinonoidal base as a major colored component of the equilibria.     

Osmotically inactive volume, hydraulic conductivity, and permeability to dimethyl sulphoxide of human mature oocytes

Controlled ovarian stimulation during an in vitro fertilization cycle usually produces large numbers of oocytes and, consequently, it is likely that more embryos will be generated than can be transferred in a given cycle. It is desirable to freeze-bank surplus oocytes before insemination to avoid the ethical and legal complications of disposing of or storing embryos. Although many attempts have been made to cryopreserve human oocytes, to date, post-thaw survival has been poor, and viable pregnancies after in vitro fertilization have been rare. A possible explanation for the lack of success is that the freezing methods have been adapted from animal studies but have not been optimized for the human oocyte. In this study, video microscopy was used to determine the volumetric responses of mature human oocytes to changes in osmolarity during preparation for freezing. A Boyle van't Hoff plot of data collected in static experiments with fresh human oocytes gave a value of 0.19 +/- 0.01 (mean +/- SEM) for the osmotically inactive volume. Dynamic measurements during exposure to dimethyl sulphoxide at room temperature (22 degrees C) were analysed by a two-parameter transport model and produced values of 1.30 x 10(-6) cm atm-1 s-1 for the hydraulic conductivity of the plasma membrane and 3.15 x 10(-5) cm s-1 for dimethyl sulphoxide permeability (chi-squared = 0.43, df = 20) of fresh human oocytes. Oocytes that had failed to fertilize had a slightly lower hydraulic conductivity and dimethyl sulphoxide permeability and, after exposure to 1.5 mol dimethyl sulphoxide l-1, these cells appeared to become permeable to normally impermeable solutes. These permeability properties have been used to design a protocol for the addition and removal of dimethyl sulphoxide to control the magnitude of volumetric changes.     

Factorization of the Michaelis functions.

Each Michaelis function that expresses the concentration of one of the species AL2, AL and A in terms of the concentration of free ligand (or its logarithm) is the product of two functions each of which represents the degree of ligation or de-ligation of a single site. These hypothetical sites have pK values of pK (SEE ARTICLE) where pK and alpha are defined by writing the two molecular pK values as pK +/- log2alpha. The factors are thus real if alpha larger than or equal to 1, i.e. if the binding of L by A is not positively co-operative. The dependence of [AL] on 1n[L] is compared with relations that represent other ligand-dependent equilibria.     

Treatment of Zoster with Idoxuridine in Dimethyl Sulphoxide. Results of Two Double-blind Controlled Trials

The antiviral effect of 5% idoxuridine in dimethyl sulphoxide intermittently applied and of 40% idoxuridine in dimethyl sulphoxide continuously applied for four days to the lesions in patients with zoster of recent onset was studied in two double-blind controlled trials. Most, but not all, of the patients receiving intermittent active treatment had pain for a short period only. The effect of continuous treatment was striking: pain had disappeared within nine days in all the patients and healing was accelerated. The results were statistically significant.     

Solution structures of ferrihaem in some dipolar aprotic solvents and their binary aqueous mixtures

1. Conductivity and u.v. and visible spectroscopic techniques were used to investigate the solution structure of the prosthetic group of the ferric haemoproteins (ferrihaem) in dimethyl sulphoxide, NN-dimethylacetamide, NN-dimethylformamide and sulpholane, and certain of their aqueous mixtures. 2. In neutral or acid dimethyl sulphoxide, chlorohaemin is monomeric and completely dissociated into Cl⁻ion and a ferrihaem species with dimethyl sulphoxide molecules in the fifth and sixth co-ordination positions on iron. 3. In neutral NN-dimethylacetamide and NN-dimethylformamide chlorohaemin is monomeric but is largely undissociated, giving different spectra from that of chlorohaemin in dimethyl sulphoxide. On acidification, dissociation occurs and the dimethyl sulphoxide type of spectrum results. 4. Studies in a fourth solvent, sulpholane, indicate that solvent co-ordinating power (ligand strength) rather than bulk dielectric constant is responsible for dissociation of chlorohaemin. 5. In neutral dimethyl sulphoxide–water mixtures chlorohaemin remains monomeric and completely dissociated, and spectra are independent of mixture composition, except at high water concentrations, when precipitation occurs. In alkaline dimethyl sulphoxide–water mixtures, where the complete solvent mixture range is accessible, ferrihaem is polymeric (probably dimeric) and spectra are dependent on solvent composition. A quantitative analysis indicates that the spectral changes are due to replacement by water of one molecule of co-ordinated dimethyl sulphoxide per ferrihaem aggregate, and do not involve a two-molecule replacement as has been suggested for the alkaline pyridine–water system.     

Glass-forming property of the system diethyl sulphoxide/water and its cryoprotective action on Escherichia coli survival

In this work the thermal properties of diethyl sulphoxide (Et2SO), as well as its cryoprotective ability are studied and related to other well-known cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have investigated the thermal properties of Et2SO/water systems using Differential Scanning Calorimetry at a very low heating/cooling rate (2 degrees C/min). Liquid/solid or glassy/crystalline transitions have been observed only for the solutions with content of Et2SO ranging from 5 up to 40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found a noticeable glass-forming tendency and a great stability of the amorphous state to the reheating. In samples with Et2SO content ranging from 80 to 85%, we observed a great stability of the glass forming by cooling, but a lesser stability to the subsequent reheating. The glass-forming tendency of these solutions is discussed in terms of existing competitive interactions between molecules of Et2SO, on the one hand, and Et2SO and water molecules, on the other hand. The results are well explainable on the basis of the model structure of water/Et2SO solutions, deduced by Raman and infrared studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of Et2SO on Escherichia coli survival has been also investigated, and a comparison among Et2SO and other widely used cryoprotectants, like Me2SO and glycerol has been done. Survival of E. coli, determined after freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85% viability). It should be noted that at the same concentration the survival is only about 35% in the presence of Me2SO and not more than 15% in the presence of glycerol. These features are well consisted with the glass-forming properties of Et2SO.     

Stabilization of Na+, K+-adenosine triphosphatase by dimethyl sulfoxide under inactivation by urea]

Hydrophobic agents, e.g. methanol, ethanol, isopropanol, acetone and dioxane were shown to induce irreversible inactivation of Na+, K+-adenosine triphosphatase beginning with their concentrations of 20 to 35%, whereas dimethyl sulphoxide exerted similar effect only at concentration of 50% and higher. Urea also irreversibly inactivated Na+, K+-adenosine triphosphatase, beginning with a concentration of about 20%. It was found that, dimethyl sulphoxide contrary to the other hydrophobic agents studied, protected Na+, K+-adenosine triphosphatase against the inactivating (denaturing) action of urea. The highest stabilizing effect of dimethyl sulphoxide was displayed at concentrations from 20 to 30%.     

The methionine sulphoxide reductase activity of the yeast dimethyl sulphoxide reductase system

Abstract Glycyl- l -methionine sulphoxide and N-acetyl- l -methionine sulphoxide were less effective inhibitors of the dimethyl sulphoxide (DMSO) reductase activity of Saccharomyces cerevisiae NCYC240 than was l -methionine (±)-sulphoxide. Methionine sulphoxide reductase and DMSO reductase activities from crude extracts co-purified over five purification steps and the two activities eluted from columns in exactly the same fractions. Both activities were sensitive to the same inhibitors. It was concluded that: (1) the DMSO reductase activity of S. cerevisiae is a property of a methionine sulphoxide reductase different from that of Black et al. [J. Biol. Chem. 235 (1960) 2910–2916], and (2) free methionine sulphoxide rather than peptidyl methionine sulphoxide is probably the enzyme's true substrate.     

Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: a site-directed mutagenesis approach.

The catalytic rate of wild type, two single (Lys 120-->Leu, Lys 134-->Thr), and one double (Lys 120-->Leu-Lys 134-->Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double mutants indicate that pK1 is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK2 is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.     

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.     

Rheology and microstructure of solutions of the microbial polysaccharide from Pseudomonas elodea

Rheological studies of solutions and gels of the microbial polysaccharide from the organism Pseudomonas elodea have been combined with X-ray diffraction studies of fibres and pulsed electric-birefringence studies of dilute solutions, to investigate the conformation and interaction of the polymer molecules. Rheological data are suggestive of a locally rigid conformation for the biopolymer in solution. X-Ray diffraction studies suggest that the molecules adopt a three-fold helical structure. O-Acetyl substituents have been shown to inhibit the packing of these helices into crystalline domains. Studies of pulsed electric-birefringence suggest an extended, kinetically rigid structure in solution. Dissolving the polysaccharide in dimethyl sulphoxide inhibits the gelation and shear-thinning characteristics of aqueous solutions. Comparative studies of electric birefringence of solutions in water and dimethyl sulphoxide suggest that the differences in rheological properties may result from a change in molecular conformation.     

Isolation and analysis of lignin-carbohydrate complexes from Lolium multiflorum

Lignin-carbohydrate complexes were extracted from grass cell walls by a variety of solvents. The yield of complexes was greatly enhanced if the sample was finely milled in a ball mill; dimethyl sulphoxide and N alkali extractions gave the highest yields. Hydrolysis showed that the carbohydrate fraction of the alkali-extracted complex contained mainly xylose (ca. 70%) and arabinose (ca. 20 %) whereas the dimethyl sulphoxide extracted complex contained glucose (ca. 50 %), xylose (ca. 30%), arabinose (ca. 12 %) and galactose (ca. 5 %). The UV spectrum of the dimethyl sulphoxide extracted complex showed lignin absorbance at 280 nm, but, in addition, ester bonding was also observed by the presence of a secondary absorbing region near 325 nm. This secondary absorbing region was absent from the spectrum of the alkali-extracted complexes. Fractionation of the complexes by ethanol precipitation gave a major component which appeared homogeneous by molecular sieve chromatography and had a MW of ? 150,000.     

The influence of different freezing procedures and different cryoprotective agents on the immunological capacity of frozen-stored lymphocytes

The influence of a series of factors on the frozen storage of lymphocytes was investigated. The cells were frozen using different freezing programmes, using the cryoprotectants dimethyl sulphoxide and glycerol in different concentrations in the freezing medium, and with variations in the period of exposure of cells to cryoprotectants before freezing and after thawing. Cell viability was evaluated by quantitative measurements of the cell-mediated immune response after stimulation with phytohemagglutinin, pokeweed mitogen, concanavalin A, and allogenic cells in mixed lymphocyte cultures. The factors investigated were found to have an important effect on the immune response, so that careful investigation and exact specification of the freezing system are necessary before frozen cells are used in blast-transformation tests. The best freezing programme had a duration of approximately 40 min with a smooth progression through the temperature range where phase transition takes place. The optimum dimethyl sulphoxide concentration in this programme was 8–10%. Dimethyl sulphoxide had no toxic effect on the cells, and no equilibration period was necessary prior to freezing. An equilibration period of 15 min with 10% glycerol was even better than the optimum programme with dimethyl sulphoxide.     

pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates,chromophore deprotonation by hydroxyl uptake,and protonation relaxation of the dark state

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.