Proton-transfer kinetics of photoexcited 7-hydroxy-1-naphthalenesulphonic acid in aqueous DMSO solutions.

The monoanion of 7-hydroxy-1-naphthalenesulphonic acid (HNS) undergoes pseudo-first-order dissociation and its conjugate base undergoes second-order protonation in the lowest excited singlet state. The proton transfer kinetics in water containing dimethylsulphoxide (DMSO), up to a mole fraction of about 0.4, have been evaluated as a function of DMSO concentration. At mole fractions above 0.5 of DMSO, proton-transfer does not measurably occur. At mole fractions below 0.5, steady-state and pulsed-source fluorimetries show the rate constant for dissociation to decrease exponentially with increasing mole fraction of DMSO. This is believed to be due to penetration and disruption of the aqueous solvent cage of HNS by DMSO, resulting in impairment of the Grotthus proton-transfer mechanism. The rate of neutralization of the conjugate base by hydrogen ion is found to vary only slightly with solvent composition and depends on the bulk dielectric properties of the solvent.     

The mechanism of the stabilization of the hexagonal II (HII) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO), a water-miscible organic solvent, has been used as a cryoprotectant for cells. It is known that DMSO stabilizes the HII phase of phosphatidylethanolamine (PE) membranes rather than the Lalpha phase, while most other water-miscible organic solvents such as acetone and ethanol destabilize the HII phase. To elucidate the mechanism for this stabilizing effect of DMSO on the HII phase, we have investigated its effects on the structures and physical properties of PE membranes. X-ray diffraction data indicated that dipalmitoleoylphosphatidylethanolamine (DPOPE) membranes in H2O at 20 degrees C were in the Lalpha phase and that an Lalpha to HII phase transition occurred at X=0.060 (mole fraction of DMSO) in water/DMSO mixtures. As the DMSO concentration increased, the basis vector length of the dioleoylphosphatidylethanolamine (DOPE)/ 16 wt% tetradecane membrane and also of the DPOPE/ 16 wt% tetradecane membrane in the HII phase decreased, suggesting that the spontaneous curvature of these membranes increased. We have also investigated the effects of DMSO on the physical properties of the PE membranes, and compared them with those of acetone. As the DMSO concentration increased, the excimer to monomer fluorescence intensities of pyrene-phosphatidylcholine in the PE membranes decreased, indicating that the membrane fluidity decreased, and also the generalized polarization value of the Laurdan fluorescent probe in the DPOPE membrane increased, indicating that the polarity of the membrane interface decreased. On the other hand, acetone had the opposite effects to DMSO. The interaction free energy between the membrane surface segments and solvent increased with an increase in DMSO concentration. It decreased the amount of solvent in the membrane interface, inducing an increase in the spontaneous curvature. This can reasonably explain the effects of DMSO on the phase stability and the physical properties of the membranes.     

Cosolvent-buffer mixtures as models for the cytoplasmic milieu: The enzymology of adenosine aminohydrolase.

Summary Adenosine aminohydrolase from calf intestinal mucosa is sensitive to changes in its environment produced by small mole fractions of dimethylsulfoxide (DMSO). At a mole fraction of 0.1 where the dielectric constant is lowered from that of 78 of neat water to about 76.5,V _max was reduced by 65% and affinity for substrate (adenosine) and the two competitive inhibitors, inosine and N⁶-benzyladenosine, was decreased markedly. However, this decreased affinity was such that K_i/K_m remained virtually constant for both inhibitors. DMSO itself showed the kinetics of a mixed inhibitor with K_i decreasing with increasing mole fraction. This cosolvent also decreased the heat stability of the enzyme which suggests that enzyme conformation is altered by DMSO.Comparison of data in the presence of DMSO with previously obtained data with dioxane shows that heat stability as well asV _max, at a given value of dielectric constant, is independent of the amount or nature of cosolvent used to achieve that dielectric constant. However, cosolvent induced changes in Ki indicate that colligative as well as dielectric constant effects contribute to the observed changes in kinetic behavior.These experiments may be considered as models for the behavior of enzymes in the medium of lowered dielectric constant expected in the vicinity of cytoplasmic membranes. The results indicate that in such an environment, adenosine aminohydrolase would be expected to be less efficient a catalyst, but equally susceptible to product inhibition, as compared to media of dielectric constant approaching that of water.Supported in part by Grant RR-262 from the General Clinical Research Centers Progam of the Division of Research Resources, National Institutes of Health.     

Amylose optical rotation in some mixed solvent systems

This paper extends a previous study in which a discontinuity in the specific rotation of open chain α-l,4-linked glucopyranosides in the water–dimethyl sulfoxide (H₂O–DMSO) system was attributed to a symmetry change about a polymer chain segment. Optical rotation of amylose, cyclohexamylose, methyl β-maltoside, and dextran was measured in the following mixed solvent systems: formamide–dimethyl sulfoxide (F-DMSO), ethylenediamine–dimethyl sulfoxide (E–DMSO), and hexamethylphosphoramide–dimethyl sulfoxide (HMPA–DMSO). Refractive index measurements were used in an attempt to detect hydrogen bonding between solvent components. The specific rotation of amylose corrected for variation in refractive index (CSR), as a function of solvent composition, showed a discontinuity at solvent compositions corresponding to about 1 mole F to 2 moles DMSO and to 1 mole E to at least 8 moles DMSO. A discontinuity in the CSR function of amylose in the H₂O-DMSO mixed solvent that occurs at 25°C is not observed at 70°C. The CSR function of methyl-β-maltoside exhibits a discontinuity in solvent composition corresponding to mole ratios between 2F–DMSO and 3F–DMSO. Present results indicate that an amylose chain segment may undergo a symmetry change in solvent compositions corresponding to mole ratios between F–DMSO and F–2DMSO. Our CSR measurements of amylose and model compounds in E–DMSO and HMPA–DMSO do not permit us to distinguish between possible changes in amylose chain segment symmetry and solvent interactions that could affect symmetry properties of the glucopyranose ring.     

Influence of water-miscible organic solvents on kinetics and enantioselectivity of the (R)-specific alcohol dehydrogenase from Lactobacillus brevis

Using the organic solvents acetonitrile and 1,4-dioxane as water-miscible additives for the alcohol dehydrogenase (ADH)-catalyzed reduction of butan-2-one, we investigated the influence of the solvents on enzyme reaction behavior and enantioselectivity. The NADP(+)-dependent (R)-selective ADH from Lactobacillus brevis (ADH-LB) was chosen as biocatalyst. For cofactor regeneration, the substrate-coupled approach using propan-2-ol as co-substrate was applied. Acetonitrile and 1,4-dioxane were tested from mole fraction 0.015 up to 0.1. Initial rate experiments revealed a complex kinetic behavior with enzyme activation caused by the substrate butan-2-one, and increasing K(M) values with increasing solvent concentration. Furthermore, these experiments showed an enhancement of the enantioselectivity for (R)-butan-2-ol from 37% enantiomeric excess (ee) in pure phosphate buffer up to 43% ee in the presence of 0.1 mol fraction acetonitrile. Finally, the influence of the co-solvents on water activity of the reaction mixture and on enzyme stability was investigated.     

Collapse and association of denatured lentinan in water/dimethlysulfoxide solutions

Triple helical lentinan, beta-(1-->3)-D-glucan from Lentinus edodes, was denatured in dimethlysulfoxide (DMSO) into single random coils. The DMSO solutions of randomly coiled lentinan were diluted with pure water to different wH (the weight fraction of water in the mixed solvent), and their specific optical rotation [alpha]D, reduced viscosity (lnetar)/c, and hydrodynamic radius Rh were investigated as a function of wH and storage time t. With an increase of wH from 0.1 to 0.2, [alpha]D increased sharply, suggesting that transition of conformation of the macromolecules has occurred. When wH was lower than 0.1, (lnetar)/c of lentinan in water-diluted DMSO exhibited the almost same value as that in pure DMSO and changed hardly with increasing t. Interestingly, (lnetar)/c decreased to reach a minimum with a further increase of wH from 0.1 to 0.25 and then increased with a continuous increase of wH from 0.25 to 0.5. Both (lnetar)/c and Rh of the denatured lentinan in water-diluted DMSO with wH of approximately 0.25 both exhibited a minimum, indicating that collapsed coil chains have occurred. All of the experimental findings revealed that the behaviors of lentinan in water-diluted DMSO solution with wH < 0.1 were consistent with that in good solvent, DMSO. When wH = 0.25, the quality of the mixed solvents became worse, and the dominant intramacromolecular hydrogen-bond interaction enhanced, leading to minimum of viscosity and size of the chains as a result of the collapsed coils. When wH > 0.25, the quality of the mixture weakens further, and the intermolecular hydrogen-bond interaction enhanced and was dominant, leading to aggregation of the collapsed chains.     

Log P effect of organic solvents on a thermophilic alcohol dehydrogenase

An alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix was activated by water-miscible organic solvents. This activation was influenced by the kind and the concentration of the added organic solvents. The k(cat) was increased by a factor of over ten when the mole fraction of acetonitrile was 0.1. This effect was large when organic solvents with large log P values were added. In fact, the k(cat) showed a strong positive correlation with the log P value of the mixed solvent at a constant mole fraction of water, while it was not affected by the kind of organic solvents added. Both the activation enthalpy and the entropy decreased with an increase in log P. The contribution of the activation enthalpy to the free energy of activation was larger than that of the activation entropy, and the free energy of activation decreased with an increase in log P.     

Models for biological ion exchangers. II. Solvent selectivity in DMSO-water-Dowex 50W.

Dimethylsulfoxide-water-Dowex 50W-X8 systems are characterized by measurements of solvent selectively and proton magnetic resonance spectra. The H+-, Li+-, NH4+-, NHe4+-, NBu4+-, Mg2+-, Zn2+-, and La3+- forms are studied over a wide range of binary-solvent mole fractions. The relative selectivities for water by the ion exchanger, based on an integrated Kipling parameter, are Zn2+-form (reference) --1.00, Mg2+ --0.43, La3+ --0.38, Li+ +0.17, NMe4+ 0.20, NH4+ 0.31, NBU4+ 0.33, and H+ 0.68, the polyvalent counterions preferring DMSO. All of the ionic forms except the NH4+-form exhibit over much of the mole fraction range separations between the external water and the internal water peaks exceeding 50 Hz, the magnitude of the separation varying with the counter-ion. Comparison of results is facilitated by maintaining a constant ratio between the total number of moles of solvent and the number of equivalents of ions exchanger.     

Solvation properties of raft-like model membranes

Dimethyl sulfoxide (DMSO) is a universal water-soluble solvent widely used in many biotechnological and medical applications, such as cells cryopreservation, and for the treatment of different human diseases (e.g. amyloidosis). Despite the great number of reported studies, the effects of DMSO on the physico-chemical properties of biological membranes are poorly understood. Often, these studies are limited to model membranes composed of phosphatidylcholines (PCs) and cholesterol (Chol). In this work, we explored the effect of DMSO on liposomes composed of the natural egg sphingomyelin (ESM) and Chol as raft-like model membranes.With a multi-technique approach we probe the structure and the thermal stability of ESM/Chol bilayer at different Chol mole fractions. In particular, we investigate the ESM-solvent interactions to clarify the role of DMSO in perturbing the solvating conditions of lipid vesicles and show that the addition of DMSO increases the thermal stability of vesicles. An increase of transition temperature, a decrease of both enthalpy and entropy as well as a decrease of the cooperativity of the gel to liquid phase transition are observed at 0.1 DMSO mole fraction. Fluorescence experiments with the probe Laurdan and FTIR spectra strongly indicate that DMSO exerts a dehydration effect on the membrane. Besides, FTIR measurements with tungsten hexacarbonyl, in combination with fluorescence data of the probe NBD-PE, indicate that DMSO promotes the formation of a highly packed membrane by reducing the thickness of the membrane.     

Insights into the dynamics of DMSO in phosphatidylcholine bilayers.

The solvation effects of dimethyl sulfoxide (DMSO) on the phase stability of dimyristoylphosphatidylcholine (DMPC) have been fully characterized using differential scanning calorimetry (DSC) and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of the sub-, pre-, and main transitions of DMPC were found to increase linearly with increasing mole fraction of DMSO up to mole fraction X=0.13 DMSO/H(2)O. Beyond X=0.13, the pre-transition peak started to merge with the peak representing the main transition. Simultaneously, the subtransition peak began to disappear as its transition temperature also decreased. At X=0.18, with both the subtransition and pre-transition absent, the main transition between the planar gel and the liquid-crystalline phase was observed at 30.3 degrees C. Transition enthalpy values indicated that the subgel, planar gel and rippled gel phases are most stable at X=0.11, 0.16 and 0.20 DMSO/H(2)O, respectively. This demonstrates that DMSO exerts distinct effects on each respective phase and corresponding transition. Temperature-dependent fluorescence emission scans show an increase in hydration as the system proceeds from the subgel phase all the way to the liquid-crystalline phase and correlated well with the effects of DMSO on the transition temperatures of DMPC observed in our calorimetry data. Initial observations for the sub- and main transition are further confirmed by fluorescence anisotropy using DPH as a probe. The results illustrate the differences in the microviscosity of each phase and how DMSO affects the phase transitions. Ultimately, our results suggest the most likely mechanism governing the biological actions of DMSO may involve the regulation of the solvation effects of water on the phospholipid bilayer.     

A differential scanning calorimetric study of the conformational transitions of schizophyllan in mixtures of water and dimethylsulfoxide

The thermal conformational transitions of two sonicated samples of schizophyllan were studied in water-dimethylsulfoxide (DMSO) mixtures by high-sensitivity differential scanning calorimetry (DSC). Two transitions were observed over most of the range of solvent compositions. These were assigned to an internal change of the triple helix [T. Itou et al. (1986) Macromolecules 19, 1234-1240] and a triple-helix-single-coil transition [T. Sato et al. (1981) Carbohydr. Res. 95, 195-204], respectively. In water, the former transition observed at lower temperature for a low molecular weight sample, U-1, is centered at 3 degrees C and characterized by the specific enthalpy, delta hcal = 3.29 J g-1. A higher molecular weight sample, M-2, showed this transition at 7 degrees C with delta hcal = 4.39 J g-1. The transition temperature for both samples increased with increasing DMSO concentration up to about 50 degrees C at 70 weight % DMSO, and then rapidly decreased with increasing DMSO concentration, with about 3 degrees C higher for M-2 than for U-1 over the DMSO concentration. The transition was not observed when the concentration of DMSO exceeded 87%. It was found that delta hcal for both samples was a linear function of t 1/2, the temperature of half-completion in degrees C, delta hcal = 0.177t + 2.96. The triple helix-coil transition was observed at around 127 degrees C for U-1 and above 130 degrees C for M-2 in the range of DMSO composition below about 70%. The transition temperature decreased with increasing DMSO concentration at above 70%, and the transition finally disappeared when the DMSO concentration exceeded 90%. The plot of delta hcal vs. t 1/2 for the transition of both samples gave a linear relation, delta hcal = 0.253t - 10.58. The reversibility of the transition at lower temperature was demonstrated by the reversibility of the curves when the first heating was stopped before the second transition. Once the heating was performed over the second transition, the reheating DSC curves showed several endothermic peaks, indicating the irreversibility of the transition and heterogeneity in the conformation of the heated schizophyllan.     

DMSO Induces Dehydration near Lipid Membrane Surfaces

Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (X_DMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at X_DMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At X_DMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw.     

Partition behavior of a nonionic detergent, octyl glucoside, between membrane and water phases, and its effect on membrane permeability

The partition equilibrium of an nonionic detergent, octyl glucoside, between the membrane phase and water and the effect of the detergent on the barrier efficiency of the vesicle membrane were studied. When the detergent concentration was lower than 4 mM in the water phase, or a mole fraction of 0.3 in the membrane phase, the partition coefficient of the detergent was independent of the detergent concentration and was 75 M-1. This value was about twice the value predicted from the critical micelle concentration. In this concentration region, the permeability of Cl- was relatively low [(2-5) x 10(-10) cm/s]. When the detergent in the membrane phase exceeded a mole fraction of 0.3, the apparent partition coefficient decreased, and the permeability of Cl- abruptly increased. These observations are explained by the following model: If the effective cross-sectional areas of phospholipid molecules and detergent molecules are similar to each other, a detergent molecule in the membrane phase will be surrounded only by phospholipid molecules as long as the mole fraction of the detergent in the membrane phase is below 0.3, and in this condition, the membrane barrier efficiency is high. At a mole fraction higher than 0.3, the detergent molecules come into contact with each other, and the membrane barrier efficiency decreases.     

Lateral diffusion rates of lipid,water, and a hydrophobic drug in a multilamellar liposome

The lateral diffusion constants of 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC), water, and ibuprofen were measured in multilamellar liposomes using pulsed field gradient magic-angle spinning (PFG-MAS) (1)H NMR. The analysis of diffusion data obtained in powder samples and a method for liposome curvature correction are presented. At 322 K POPC has a diffusion constant of (8.6 +/- 0.2) x 10(-12) m(2)/s when dehydrated (8.2 waters/lipid) and (1.9 +/- 0.1) x 10(-11) m(2)/s in excess water. The diffusion constant of water in dehydrated POPC was found to be (4.7 +/- 0.1) x 10(-10) m(2)/s. The radius of curvature is 21 +/- 2 microm for the dehydrated sample and 4.5 +/- 0.5 microm for POPC sample containing excess water. The activation energies of diffusion are 40.6 +/- 0.4 kJ/mole for dehydrated POPC, 30.7 +/- 0.9 kJ/mole for POPC with excess water, and 28.6 +/- 1.5 kJ/mole for water in dehydrated POPC. The diffusion constants and activation energies for a sample of POPC/ibuprofen/water (1:0.56:15) were also measured. The ibuprofen, which locates in the lipid-water interface, diffuses faster than POPC but has a slightly higher activation energy of lateral diffusion. Within certain restrictions, PFG-MAS NMR provides a useful method for characterizing membrane organization and mobility.     

Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change

Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.     

Electrostatic interaction in the reaction between porphyrin and DNA

The association of tetrakis(4-N-methylpyridyl)porphine with salmon testes DNA in solution has been investigated in the presence of ethanol at 25 degrees C. It was found that the association constant was 1.14 x 10(6) M-1 and it increased with increasing the mole fraction of added ethanol. The behavior was very different from that in the case of usual intercalaters.     

Low concentration of DMSO stabilizes the bilayer gel phase rather than the interdigitated gel phase in dihexadecylphosphatidylcholine membrane

We have investigated effects of dimethylsulfoxide (DMSO) on the phase stability of multilamellar vesicles of the ether-linked 1,2-dihexadecyl-sn-glycero-3-phosphatidylcholine (DHPC-MLV), which is known to be in the interdigitated gel (LbetaI) phase in excess water at 20 degrees C. The results of X-ray diffraction experiments indicate that the DHPC membrane was in the Lbeta, phase at X> or =0.12 (X=mole fraction of DMSO in DMSO/water mixture). The result of differential scanning calorimetry indicate that the gel to liquid-crystalline phase transition temperature increased, but the LbetaI to Pbeta, phase transition temperature decreased with an increase in DMSO concentration. These results show that DMSO stabilizes the bilayer gel phase rather than the LbetaI phase at its low concentration. The solubility of phosphorylcholine, which is the same structure as the headgroup of DHPC, decreased with an increase in DMSO concentration, indicating that the interaction free energy of the hydrophilic segments of the membrane with solvents increases with an increase in DMSO concentration. On the basis of the thermodynamic analysis, the mechanism of the stabilization of the bilayer gel phase of DHPC-MLV by DMSO is discussed. The decrease in the repulsive interaction between the headgroups of the phospholipid induced by the low concentrations of DMSO in water plays an important role in this stabilization.     

Lipid membrane structure and interactions in dimethyl sulfoxide/water mixtures.

In this paper we have investigated via x-ray diffraction the influence of dimethyl sulfoxide (DMSO), known for its biological and therapeutic properties, on the structure of lipid membranes of dipalmitoylphosphatidylcholine (DPPC) in excess of the solvent (DMSO/water) at mole DMSO fractions XDMSO in (0.1) and under equilibrium conditions. At small XDMSO </= 0.133 the repeat distance d is reduced remarkably, whereas wide-angle x-ray diffraction pattern remains almost unchanged with the increase in XDMSO. It agrees well with previous study (Yu and Quinn, 1995). At 0.133 < XDMSO < 0.3 the repeat period d reduces slowly; however, an orthorombic in-plane lattice of hydrocarbon chains transfers to a disordered quasihexagonal lattice. The increase in XDMSO from 0.3 up to approximately 0.9 leaves d almost unchanged, whereas it leads to less disordered packing of hydrocarbon chains. At XDMSO approximately 0.9, Lbeta' phase transfers into interdigitated phase. The chain-melting phase transition temperature of DPPC membranes increases by several degrees with the increase of DMSO concentration. It points to a strong concentration-dependent solvation of membrane surface by DMSO. Thus DMSO strongly interacts with the membrane surface, probably displacing water and modifying the structure of the lipid bilayer. It appears to determine some of the properties of DMSO as a biologically and therapeutically active substance.     

Calcium ion binding between lipid bilayers: the four-component system of phosphatidylserine, phosphatidylcholine, calcium chloride, and water

Ca2+ binding between lamellae of phosphatidylserine (PS) and phosphatidylcholine (PC) gives rise to a rigid phase of Ca(PS)2. When aqueous Ca2+, hydrated PS/PC, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant and is characteristic of the PS/PC ratio. This characteristic Ca2+ concentration is 0.040 microM for palmitoyloleoylphosphatidylserine without PC and increases as the inverse square of the PS mole fraction at high PS concentration (Raoult's law) and as the inverse square of the PS mole fraction multiplied by a constant at low PS concentration (Henry's law). For example, for palmitoyloleoylphosphatidylserine/palmitoyloleoylphosphatidylcholi ne = 0.6/0.4 or 0.2/0.8, this characteristic Ca2+ concentration is about 0.1 or about 6 microM, respectively. These observations at constant temperature are summarized in a quaternary phase diagram for the four-component system CaCl2/PS/PC/water.     

Inhibition of lactate and malate dehydrogenase by dyes containing vinylsulfone and chlorotriazine groups

The difference spectra of lactate and malate dehydrogenase complexes with four native dyes containing vinylsulfonic and triazinic groups (light-resistant yellow 2KT, red-violet 2KT, etc.) were monitored in 0.1 M phosphate buffer pH 8.2 at 20 degrees C. The dissociation constants were calculated from the spectral data. The most stable complexes were lactate dehydrogenase--light-resistant yellow 2KT and malate dehydrogenase--light-resistant yellow 2KT ones. The values of delta H degree = 5.75 kcal/mole and standard thermodynamic parameters, delta G degree = -6.5 kcal/mole and delta S degree = 41.2 e. u., were calculated from the values of association constants for temperature dependence. The thermodynamic characteristics confirmed the key role of hydrophobic interactions in lactate dehydrogenase--reactive dye complex formation. All the dyes under study competitively inhibit lactate and malate oxidation by the corresponding dehydrogenases. The inhibition constants of both enzymes by the four dyes were determined at 20 degrees C in 0.1 M phosphate buffer pH 8.2. Light-resistant yellow 2KT appeared to be the most effective inhibitor of the enzymes.