Do organic solvents affect the catalytic properties of lipase? Intrinsic kinetic parameters of lipases in ester hydrolysis and formation in various organic solvents

When it is assumed that organic solvents do not interfere with the binding process nor with the catalytic mechanism, the contribution of substrate-solvent interactions to enzyme kinetics can be accounted for by just replacing substrate concentrations in the equations by thermodynamic activities. It appears from the transformation that only the affinity parameters (K(m), K(sp)) are affected by this. Thus, in theory, the values of these corrected, intrinsic parameters (K(m) (int), k(sp) (int)) and the maximal rate (V(1)) should be equal for all media. This was tested for hydrolysis, transesterification, and esterification reactions catalyzed by pig pancreas lipase and Pseudomonas cepacia lipase in various organic solvents. Correction was carried out via experimentally determined activity coefficients for the substrates in these solvents or, if not feasible, from values in data bases. However, although the kinetic performances of each enzyme in the solvents became much more similar after correction, differences still remained. Analysis of the enzyme suspensions revealed massive particles, which explains the low activity of enzymes in organic solvents. However, no correlation was found between estimates of the amount of catalytically available enzyme (present at the surface of suspended particles or immobilized on beads) and the maximal rates observed. Moreover, the solvents had similar effects on the intrinsic parameters of suspended and immobilized enzyme. The possible causes for the effects of the solvents on the catalytic performance of the enzymes, remaining after correction for solvent-substrate interactions and the amount of participating enzyme, are discussed with respect to the premises on which the correction method is based. (c) 1995 John Wiley & Sons, Inc.     

The solid state chemistry of uranium. Part I: The thermal decomposition of U(NO3)4·2tdpo and U(NO3)4·2tppo

The thermal decomposition of U(NO₃)₄·2tdpo (tdpo=tris-(dimethylamino)phosphine oxide) and U(NO₃)₄·2tppo (tppo=triphenyl phosphine oxide have been examined using thermogravimetry and differential scanning calorimetry. Decomposition of the crystals approximates to the general reaction U(NO₃)₄·2L(s) → UO₂(NO₃)₂·2L(s) + gases The general thermal behaviour of the two compounds were found to be different.     

Oxides of Nitrogen (NO· and NO2 –) as Cofactors of the Myeloperoxidase System

Myeloperoxidase is the main peroxisomal protein of neutrophils, monocytes, and a subpopulation of tissue macrophages; it plays the key role in protective and inflammatory responses of the organism. This role is mediated by various diffusible radicals formed during oxidative reactions catalyzed by the enzyme heme. Myeloperoxidase and nitric oxide synthase are stored in peroxisomes. Nitric oxide reacts with the heme of myeloperoxidase. Low nitric oxide concentrations increase peroxidase activity through reduction of Compound II to native myeloperoxidase. Conversely, high nitric oxide concentrations inhibit the catalytic activity of myeloperoxidase through formation of inactive nitrosyl–heme complexes. Such effect of nitric oxide on catalytic activity of myeloperoxidase has various consequences for infectious and local inflammatory processes. Another oxide of nitrogen, nitrite, is a good substrate for myeloperoxidase Compound I but slowly reacts with Compound II. Nitrogen dioxide is formed after nitrite oxidation by myeloperoxidase. Formation of nitrogen dioxide is another protective mechanism and nitration of microbial proteins by myeloperoxidase can represent an additional protective response of peroxisomes.     

The formation of amyloid-like fibrils of α-chymotrypsin in different aqueous organic solvents

The formation of amyloid-like fibrils of α-chymotrypsin was studied in aqueous ethanol, methanol, tertbutanol, dimethylformamide and acetonitrile. Thioflavin T (ThT), Congo red (CR) and 1-anilino-8-naphthalenesulfonic acid (ANS) binding, turbidity, intrinsic fluorescence and far-UV circular dichroism measurements were employed to characterize the amyloid fibril formation. The greatest extent of fibril formation after incubation for 24 h at pH 7.0 and at 24 °C was in ethanol at 55%, in methanol and dimethylformamide (DMF) at 60-70% and in tert-butanol at 60-80%. The ANS binding and intrinsic fluorescence results showed that the hydrophobic residues are more solvent-exposed in the aggregated form of α-chymotrypsin. The ThT, CR binding and far-UV CD measurements indicated that the formation of the cross-β structure of α-chymotrypsin depends on the polarity of the organic solvent. To determine the role of surface charges in the aggregation, chemically modified forms of α-chymotrypsin were prepared. The citraconylated and succinylated enzymes exhibited a higher and the enzyme forms modified with aliphatic aldehydes a lower propensity for aggregation. These results suggest the important role of surface charges in the aggregation of α-chymotrypsin.     

Synthesis and characterization of [Pt(COD)Cl(NO3)] and [Pt(COD)(NO3)2] and the use of [Pt(COD)Clx(NO3)2−x] in the preparation of cis[Pt(PPh3)2Clx(NO3)2−x] (x=0, 1, 2) and [Pt(PPh3)3L](NO3) (L=Cl,NO3)

[Pt(COD)Cl₂] (COD=1,5-cyclooctadiene) is a versatile starting material for the synthesis of Pt(II) compounds. The preparations of the new compounds [Pt(COD)Cl(NO₃)], [Pt(COD)(NO₃)₂] and [Pt(PPh₃)₃(NO₃)](NO₃) and also of the known compounds cis[Pt(PPh₃)₂Cl₂], cis [Pt(PPh₃)₂Cl(NO₃)], cis[Pt(PPh₃)₂(NO₃)₂] and [Pt(PPh₃)₃Cl](NO₃)are reported. The compounds are characterized by elemental analysis, ³¹P{¹H} NMR spectroscopy and IR spectroscopy.     

The effect of counterion,water concentration,and stirring on the stability of subtilisin BPN′ in organic solvents

The stability of subtilisin BPN′ in organic solvents or cosolvent/water mixtures was studied as a function of the type and concentration of counterion at the time of freeze-drying, water concentration, and stirring speed/method. It was found that the enzyme is stabilized by high concentrations of counterion, at least at very high cosolvent concentrations. The type of counterion also has a remarkable impact on the enzyme stability; at high concentrations of DMF (dimethylformamide), multivalent counterions with low solubility in organic solvents are far superior to monovalent, soluble ones. Sodium citrate is the best salt tested in terms of enzyme stability, increasing the half life of the enzyme better than a millionfold over Tris in 99% DMF. The stability of the enzyme was found to have a complex dependence on the amount of water in the DMF. Enzyme lyophilized from the sodium phosphate displays a stability minimum at about 90% DMF, while enzyme lyophilized from Tris becomes increasingly unstable from 30% to 99% DMF, without inflection. Vigorous stirring with a magnetic stir bar, which broke apart the enzyme particles, was found to be extremely deleterious to enzyme stability, while swirling the enzyme with a wrist-action stirrer, which did not grind the enzyme particles, had no effect. Explanations for this are discussed.     

The luminescence of europium nitrate hexahydrate,Eu(NO3)3·6H2O

Some luminescent properties of single crystals of Eu(NO₃)₃·6H₂O are reported at temperatures down to liquid helium. The water molecules are responsible for a considerable amount of quenching of the emission. The Eu³⁺ coordination changes upon lowering the temperature. The number of ⁵D₀-⁷F₂ lines observed is higher than expected.     

Comparison of the ability of β-glucosidases from almonds and Fusarium oxysporum to produce n-alkyl-β-glucosides in organic solvents

Summary The effect of water activity on the synthesis of n-alkyl-D-glucosides through condensation of glucose and n-alcohols has been studied using the commercially available almond -glucosidase and -glucosidase isolated from Fusarium oxysporum. The two enzymes exhibited a different water activity optimum. The specificity and alcohol reactivity of the two enzymes have also been investigated. Both enzymes prefer primary alcohols. -Glucosidase from F. oxysporum presents a higher affinity for primary alcohols with alkyl chain length of 4–6, whereas in the case of almond -glucosidase both initial velocity and yield decrease when the carbon chain length increases.     

The phenomenon of conglomerate crystallization. XV. Spontaneous resolution in coordination compounds. XIII. The crystallization behaviour and the crystal and molecular structure of Δ(λδ)-cis-Rh(en)2(NO2)2]Cl

The title compound, I, crystallizes in the monoclinic space group P2₁ with cell constants: a = 6.599(3), b = 11.121(2), c = 8.375(1) Å and β = 106.35(2)°; V = 589.74 ų and D(calc; Z = 2) = 1.974 g cm⁻³. The compound is isomorphous and isostructural with its Co analogue. A total of 2982 data were collected over the range of 4°  20  70°; of these, 2537 (independent and with I ⩾ 3σ(I)) were used in the structural analysis. Data were corrected for absorption (μ = 16.6 cm⁻¹) and the relative transmission coefficients ranged from 1.000 to 0.9504. Refinement was carried out for both enantiomeric configurations and the crystal used was found to contain cations with Δ(λδ) absolute configuration. The final R(F) and R_w(F) residuals were, respectively 0.0220 and 0.0239 for (−−−; i.e.Δ(λδ)) and 0.0231 AND 0.0317 FOR (+++; i.e.Λ(δλ)). Thus, the former was selected as correct for our specimen.In the case of I, as well as in the Co derivative [cis-Co(en)₂(NO₂)₂]Cl (II), the conformation of one of the rings is opposite that expected for the lowest energy conformation, which in the current case should be Δ(λλ)).The RhN(NO)₂ distances are 2.020(2) and 2.010(2) Å, while the RhN(amine) distances, trans to the NO₂ ligands are 2.085(2) and 2.093(1) Å, values distinctly longer than the other two RhN distances (2.064(1) and 2.068(1) Å). The latter are the RhN distances to the terminalNH₂ ligands located trans to each other. Thus, we observe a trans effect, which is more pronounced in I than in II, and which is of comparable magnitude to that observed in the case of the trien derivative, [cis-α-Rh(trien)(NO₂)₂]Cl(III).Parallel with an increase in metalN distances in going from [cis-α-Co(trien)NO₂)₂]Cl·H₂) (IV) to (III) is an increase in the torsional angles of the outer rings (NCCN) of about 10°. Comparison of these parameters in I and II reveal that this change is not so marked for this pair since in I they are −54.9° and 52.8° while in II they are 50.2° and −48.1°; i.e. a change of only 4°. This important difference between trien and en derivatives is caused by the presence of the central five-membered ring, which for compounds III and IV remains largely unchanged, except for the metalN distances.The NO bond lengths are 1.244(3), 1.220)(2), 1.237(2) and 1.211(2) Å, which are similar to those found for the analogous Co isomer. The CN bond lengths are 1.492(3), 1.474(2), 1.486(2) and 1.475(2) Å, while the CC bonds are 1.509(3) and 1.524(3) Å. These values are also comparable with those obtained for the Co isomer and, in fact, the pattern of the bonds is nearly identical in both, including the common feature of having a longer CC bond for the en ring with the conformation opposite that expected.As was the case with the Co analogue, the Cl⁻ anion is associated with the hydrogens of the secondary nitrogen (trans to the −NO₂) ligands, the Cl…H7 distance being 2.18(3) Å and the <Cl…H7N2 = 163°.     

The mechanism of antimalarial action of the ruthenium(II)–chloroquine complex [RuCl2(CQ)]2

The mechanism of antimalarial action of the ruthenium-chloroquine complex [RuCl(2)(CQ)](2) (1), previously shown by us to be active in vitro against CQ-resistant strains of Plasmodium falciparum and in vivo against P. berghei, has been investigated. The complex is rapidly hydrolyzed in aqueous solution to [RuCl(OH(2))(3)(CQ)](2)[Cl](2), which is probably the active species. This compound binds to hematin in solution and inhibits aggregation to beta-hematin at pH approximately 5 to a slightly lower extent than chloroquine diphosphate; more importantly, the heme aggregation inhibition activity of complex 1 is significantly higher than that of CQ when measured at the interface of n-octanol-aqueous acetate buffer mixtures under acidic conditions modeling the food vacuole of the parasite. Partition coefficient measurements confirmed that complex 1 is considerably more lipophilic than CQ in n-octanol-water mixtures at pH approximately 5. This suggests that the principal target of complex 1 is the heme aggregation process, which has recently been reported to be fast and spontaneous at or near water-lipid interfaces. The enhanced antimalarial activity of complex 1 is thus probably due to a higher effective concentration of the drug at or near the interface compared with that of CQ, which accumulates strongly in the aqueous regions of the vacuole under those conditions. Furthermore, the activity of complex 1 against CQ-resistant strains of P. falciparum is probably related to its greater lipophilicity, in line with previous reports indicating a lowered ability of the mutated transmembrane transporter PfCRT to promote the efflux of highly lipophilic drugs. The metal complex also interacts with DNA by intercalation, to a comparable extent and in a similar manner to uncomplexed CQ and therefore DNA binding does not appear to be an important part of the mechanism of antimalarial action in this case.     

The rate of the deoxygenation reaction limits myoglobin- and hemoglobin-facilitated O₂ diffusion in cells

A mathematical model describing facilitation of O(2) diffusion by the diffusion of myoglobin and hemoglobin is presented. The equations are solved numerically by a finite-difference method for the conditions as they prevail in cardiac and skeletal muscle and in red cells without major simplifications. It is demonstrated that, in the range of intracellular diffusion distances, the degree of facilitation is limited by the rate of the chemical reaction between myglobin or hemoglobin and O(2). The results are presented in the form of relationships between the degree of facilitation and the length of the diffusion path on the basis of the known kinetics of the oxygenation-deoxygenation reactions. It is concluded that the limitation by reaction kinetics reduces the maximally possible facilitated oxygen diffusion in cardiomyoctes by ～50% and in skeletal muscle fibers by ～ 20%. For human red blood cells, a reduction of facilitated O(2) diffusion by 36% is obtained in agreement with previous reports. This indicates that, especially in cardiomyocytes and red cells, chemical equilibrium between myoglobin or hemoglobin and O(2) is far from being established, an assumption that previously has often been made. Although the "O(2) transport function" of myoglobin in cardiac muscle cells thus is severely limited by the chemical reaction kinetics, and to a lesser extent also in skeletal muscle, it is noteworthy that the speed of release of O(2) from MbO(2), the "storage function," is not limited by the reaction kinetics under physiological conditions.     

Simulation of homology models for the extracellular domains (ECD) of ErbB3, ErbB4 and the ErbB2–ErbB3 complex in their active conformations

Epidermal growth factor receptors (EGFR) are associated with a number of biological processes and are becoming increasingly recognized as important therapeutic targets against cancer. In this work, we provide models based on homology for the extracellular domains (ECD) of ErbB3 and ErbB4 in their active conformations, including a Heregulin ligand, followed by further refinement of the models by molecular dynamics simulations at atomistic scale. We compare the results with a model built for ErbB2 based on crystallographic information and analyze the common features observed among members of the family, namely, the periscope movement of the dimerization arm and the hinge displacement of domain IV. Finally, we refine a model for the interaction of the ECDs corresponding to a ErbB2–ErbB3 heterodimer, which is widely recognized to have a high impact in cancer development.     

The α2(XI) collagen gene lies within 8 kb of Pb in the proximal portion of the murine major histocompatibility complex

A number of serious hereditary disorders are now known to be associated with defective expression of collagen genes, and these findings have underscored the important and varied roles that the collagen family of genes must play during normal mammalian development. Although the activities of genes encoding the quantitatively major types of collagen are fairly well characterized, functions of the many minor types of collagen remain a matter of speculation. As a first step toward a functional analysis of type XI collagen, a member of this class of poorly understand minor collagen proteins which is expressed primarily in hyaline cartilage, we have used human probes for the gene encoding the protein's 2-subunit (COL11A2) to isolate and map homologous murine DNA sequences. Our results demonstrate that Col11a-2 is embedded within the major histocompatibility complex (MHC), within 8.4 kb of the class II pseudogene locus, Pb, and confirm that human and murine 2(XI) collagen genes are located in very similar genomic environments. The conserved location of these genes raises the possibility that type XI collagen genes may contribute to one or more of the diverse hereditary disorders known to be linked to the MHC in mouse and human.     

A model of O·2 - generation in the complex III of the electron transport chain

Oxidation of semiquinone by O₂ in the Q cycle is known to be one of the sources of superoxide anion (O·₂ ^-) in aerobic cells. In this paper, such a phenomenon was analyzed using the chemical kinetics model of electron transfer from succinate to cytochrome c, including coenzyme Q, the complex III non-heme iron protein FeS_III and cytochromes b₁, b_h and c₁. Electron transfers from QH₂ to FeS_III and cytochrome b₁ were assumed to occur according to direct transfer mechanism (dynamic channelling) involving the formation of FeS^red _III -Q·^- and Q·^--cytochrome b₁ complexes. For oxidation/reduction reactions involving cytochromes b_h and b₁, the dependence of the equilibrium and elementary rate constants on the membrane potential () was taken into consideration. The rate of O·₂ ^- generation was found to increase dramatically with increase in above the values found in State 3. On the other hand, the rate of cytochrome c reduction decreased sharply at the same values of the membrane potential. This explains experimental data that the O·2^- generation at State 4 appears to be very much faster than at State 3. A mild uncoupling in State 4 can markedly decrease the superoxide generation due to a decrease in below the above mentioned critical level. pH appears to be equally effective as in stimulation of superoxide production which depends, in fact, upon the ^- _H + level.     

Activity and HPLC measured action pattern of Bacillus amyloliquefaciens α -amylase in water soluble organic solvents

The influence of two water miscible solvents (ethanol and isopropanol) on the activity of Bacillus amyloliquefaciens -amylase was studied.In ethanol-aqueous buffer (1:4, v/v) retained about 60% of the activity shown in water alone, both after l h hydrolysis. Isopropanol - aqueous buffer (1: 4,v/v) reduced the activity at 40%. The amount and the quality of produced oligosaccharides were effected by ethanol and isopropanol presence. In the mixture of produced oligosaccharides formed in the presence of the solvents only DP2, DP3 and DP6 were found. The disappearance of DP4, DP5 and DP7 which were formed in aqueous buffer suggest that a change in substrate affinity at the active centre is induced in the ethanol or isopropanol presence in buffer.Abbreviations DP degree of polymerization     

The fluted giant clam (Tridacna squamosa) increases nitrate absorption and upregulates the expression of a homolog of SIALIN (H+:2NO3− cotransporter) in the ctenidium during light exposure

Coral Reefs - Giant clams flourish in nutrient-poor waters of tropical Indo-Pacific because they live in symbiosis with extracellular dinoflagellates (zooxanthellae) and receive photosynthates from...     

Squamous cell carcinoma of the head and neck—The role of diffusion and perfusion imaging in tumor recurrence and follow-up

Computed tomography and magnetic resonance imaging-based techniques of functional imaging are proven to be sensitive and reliable tools for detection and staging of head and neck cancer.These new techniques enable to visualize and differentiate subtle pathologic changes before they become evident on standard cross-sectional images.However, it is their role in evaluating possible recurrence and estimation of treatment response that holds the biggest promise.This article describes the role and usefulness of diffusion and perfusion in detecting recurrence and follow-up in patients with head and neck squamous cell carcinoma.     

Synthesis and structure of the complex tetra-μ-prolinatodirhodium(II)

The dinuclear Rh^IIRh^II complex with proline [Rh₂(pro₄][NEt₄]₂ was synthesized and its structure studied by means of spectroscopic (IR, EPR and ESCA) and magnetochemical methods. It was shown that two proline molecules serve as bridging ligands, while the other two are only axially coordinated through their N atoms.