A kinetic model for production of glucose by hydrolysis of levoglucosan and cellobiosan from pyrolysis oil

Anhydro sugars, produced during wood pyrolysis, can by hydrolyzed to sugars under acidic conditions. The acid hydrolysis of two common anhydro sugars in wood pyrolysis oils, levoglucosan (1,6-anhydro-beta-D-glucopyranose) and cellobiosan (beta-D-glucopyranosyl-(1-->4)-1,6-anhydro-D-glucopyranose), was investigated. Levoglucosan hydrolysis to glucose follows a first-order reaction, with an activation energy of 114 kJ mol(-1). For cellobiosan hydrolysis, 44% of the cellobiosan is hydrolyzed initially via the beta-(1-->4) glycosidic bond to form levoglucosan and glucose. The remaining cellobiosan is hydrolyzed initially at the 1,6 anhydro bond to form cellobiose. Both reactions are first order with respect to cellobiosan, with an activation energy of 99 kJ mol(-1). The intermediate levoglucosan and cellobiose are hydrolyzed to glucose.     

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.     

Estimation of cytoplasmic nitrate and its electrochemical potential in barley roots using 13NO3 and compartmental analysis

13NO3 was used to determine the intracellular compartmentation of NO3 in barley roots (Hordeum vulgare cv. Klondike), followed by a thermodynamic analysis of nitrate transport.Plants were grown in one-tenth Johnson's medium with 1 mol m3 NO3 (NO3-grown plants) or 1 mol m3 NH4NO3 (NH4NO3-grown plants).The cytoplasmic concentrations of NO3 in roots were only approx. 3-6 mol m3 (half-time for exchange approx. 21 s) in both NO3 and NH4NO3 plants. These pool sizes are consistent with published nitrate microelectrode data, but not with previous compartmental analyses.The electrochemical potential gradient for nitrate across the plasmalemma was +26 +/-1 kJ mol1 in both NO3- and NH4NO3-grown plants, indicating active uptake of nitrate. At an external pH of 6, the plasmalemma electrochemical potential for protons would be approx. -29 +/- 4 kJ mol1. If the cytoplasmic pH was 7.3 +/- 0.2, then 2H+/1NO3 cotransport, or a primary ATP-driven pump (2NO3/1ATP), are both thermodynamically possible. NO3 is also actively transported across the tonoplast (approx. +6 to 7 kJ mol1).     

Role of 2-deoxy-D-glucose in the inhibition of phagocytosis by mouse peritoneal macrophage

2-Deoxy-D-glucose inhibits Fc and complement receptor-mediated phagocytosis of mouse peritoneal macrophages. To understand the mechanism of this inhibition, we analyzed the 2-deoxy-D-glucose metabolites in macrophages under phagocytosis inhibition conditions and conditions of phagocytosis reversal caused by glucose, mannose and 5-thio-D-glucose, and compared their accumulations under these conditions. Macrophages metabolized 2-deoxy-D-glucose to form 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucose 1-phosphate, UDP-2-deoxy-D-glucose, 2-deoxy-D-glucose 1, 6-diphosphate, 2-deoxy-D-gluconic acid and 2-deoxy-6-phospho-D-gluconic acid. The level of bulk accumulation as well as the accumulation of any of these 2-deoxy-D-glucose metabolites did not correlate with changes in macrophage phagocytosis capacities caused by the reversing sugars. 2-Deoxy-D-glucose inhibited glycosylation of thioglycolate-elicited macrophage by 70-80%. This inhibition did not cause phagocytosis inhibition, since (1) the reversal of phagocytosis by 5-thio-D-glucose was not followed by increases in the incorporation of radiolabelled galactose, glucosamine, N-acetylgalactosamine or fucose; (2) cycloheximide at a concentration that inhibited glycosylation by 70-80% did not affect macrophage phagocytosis. The inhibition of protein synthesis by 2-deoxy-D-glucose similarly could not account for phagocytosis inhibition, since cycloheximide, when used at a concentration that inhibited protein synthesis by 95%, did not affect phagocytosis. 2-Deoxy-D-glucose lowered cellular nucleoside triphosphates by 70-99%, but their intracellular levels in the presence of different reversing sugars did not correlate with the magnitude of phagocytosis reversal caused by these sugars. The results show that 2-deoxy-D-glucose inhibits phagocytosis by a mechanism distinct from its usual action of inhibiting glycosylation, protein synthesis and depleting energy supplies, mechanisms by which 2-deoxy-D-glucose inhibits other cellular processes.     

The first example of a Hoogsteen base-paired DNA duplex in dynamic equilibrium with a Watson-Crick base-paired duplex--a structural (NMR), kinetic and thermodynamic study.

A single-point substitution of the O4' oxygen by a CH2 group at the sugar residue of A6 (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C1G2C3G4A5A6T7T8C9G10C11G12)2(-3), has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)<==>(1B): Keq = k1/k(-1) = 0.56+/-0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9 0.8 sec(-1); deltaHdegrees++ = 164+/-14 kJ/mol; -TdeltaS degrees++ (298K) = -92 kJ/mol giving a deltaG degrees++ 298 of 72 kJ/mol. Ea (k1) = 167 14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 0.6 sec-1, deltaH degrees++ = 153 13 kJ/mol; -TdeltaSdegrees++ (298K) = -82 kJ/mol giving a deltaGdegrees++(298) of 71 kJ/mol. Ea (k-1) = 155 13 kJ/mol]. Acomparison of deltaGdegrees++(298) of the forward (k1) and backward (k-1) conversions, (1A)<==>(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2'-deoxyaristeromycin moiety, A6, as a result of substitution of the endocyclic 4'-oxygen in the natural sugar with a methylene group in A6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in alpha, sigma and gamma torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.     

Carboxylate binding modes in zinc proteins: A theoretical study

The relative energies of different coordination modes (bidentate, monodentate, syn, and anti) of a carboxylate group bound to a zinc ion have been studied by the density functional method B3LYP with large basis sets on realistic models of the active site of several zinc proteins. In positively charged four-coordinate complexes, the mono- and bidentate coordination modes have almost the same energy (within 10 kJ/mol). However, if there are negatively charged ligands other than the carboxylate group, the monodentate binding mode is favored. In general, the energy difference between monodentate and bidentate coordination is small, 4-24 kJ/mol, and it is determined more by hydrogen-bond interactions with other ligands or second-sphere groups than by the zinc-carboxylate interaction. Similarly, the activation energy for the conversion between the two coordination modes is small, approximately 6 kJ/mol, indicating a very flat Zn-O potential surface. The energy difference between syn and anti binding modes of the monodentate carboxylate group is larger, 70-100 kJ/mol, but this figure again strongly depends on interactions with second-sphere molecules. Our results also indicate that the pK(a) of the zinc-bound water ligand in carboxypeptidase and thermolysin is 8-9.     

On the water and proton permeabilities across membranes from erythrocyte ghosts

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.     

Anion-induced increases in the affinity of colcemid binding to tubulin

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.     

Thermodynamics of hexokinase catalyzed reactions. II. Measurement and calculation of enthalpies of reaction as a function of magnesium ion concentration.

Enthalpies of phosphorylation of glucose by adenosine 5'-triphosphate have been measured as a function of concentrations of magnesium chloride in TRIS/TRIS-HCl buffer in the pH range 8.64 to 8.98. These measurements are compared with the results of calculations of these enthalpies that use a coupled equilibrium formalism with equilibrium data and enthalpy values selected from the literature. The experimental results span the range of magnesium ion concentrations 1 X 10(-6) to 0.3 mol alpha-1 and show a total variation in the enthalpy of reaction of almost 10 kJ mol-1, with the most exothermic reaction occurring at a magnesium ion concentration of 6.0 X 10(-4) mol alpha-1. The calculated enthalpies of reaction, except for the magnesium ion concentration range 4 X 10(-6) to 5 X 10(-4) mol alpha-1, are, within estimated uncertainty intervals (0.8 to 10.2 kJ mol-1), in agreement with the measured values.     

Kinetic study of hydrolysis of xylan and agricultural wastes with hot liquid water

We investigated the kinetics of hot liquid water (HLW) hydrolysis over a 60-min period using a self-designed setup. The reaction was performed within the range 160–220 °C, under reaction conditions of 4.0 MPa, a 1:20 solid:liquid ratio (g/mL), at 500 rpm stirring speed. Xylan was chosen as a model compound for hemicelluloses, and two kinds of agricultural wastes–rice straw and palm shell–were used as typical feedstocks representative of herbaceous and woody biomasses, respectively. The hydrolysis reactions for the three kinds of materials followed a first-order sequential kinetic model, and the hydrolysis activation energies were 65.58 kJ/mol for xylan, 68.76 kJ/mol for rice straw, and 95.19 kJ/mol for palm shell. The activation energies of sugar degradation were 147.21 kJ/mol for xylan, 47.08 kJ/mol for rice straw and 79.74 kJ/mol for palm shell. These differences may be due to differences in the composition and construction of the three kinds of materials. In order to reduce the decomposition of sugars, the hydrolysis time of biomasses such as rice straw and palm shell should be strictly controlled.     

Coupling of GTP hydrolysis by elongation factor G to translocation and factor recycling on the ribosome

The translocation step of elongation entails the coordinated movement of tRNA and mRNA on the ribosome. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis, which affects both translocation and turnover of EF-G. Both reactions are much slower (50-100-fold) when GTP is replaced with non-hydrolyzable GTP analogues or GDP, indicating that the reaction rates are determined by conformational transitions induced by GTP hydrolysis. Compared to the rate of uncatalyzed, spontaneous translocation, ribosome binding of EF-G with any guanine nucleotide reduces the free energy of activation by about 18 kJ/mol, whereas GTP hydrolysis contributes another 10 kJ/mol. The acceleration by GTP hydrolysis is due to large decrease in activation enthalpy by about 30 kJ/mol, compared to the reaction with GTP analogues or GDP, whereas the activation entropy becomes unfavorable and is lowered by about 20 kJ/mol (37 degrees C). The data suggest that GTP hydrolysis induces, by a conformational change of EF-G, a rapid conformational rearrangement of the ribosome ("unlocking") which determines the rates of both tRNA-mRNA translocation and recycling of the factor.     

Characterization of molecular mobility in seed tissues: an electron paramagnetic resonance spin probe study.

The relationship between molecular mobility (tauR) of the polar spin probe 3-carboxy-proxyl and water content and temperature was established in pea axes by electron paramagnetic resonance (EPR) and saturation transfer EPR. At room temperature, tauR increased during drying from 10(-11) s at 2.0 g water/g dry weight to 10(-4) s in the dry state. At water contents below 0.07 g water/g dry weight, tauR remained constant upon further drying. At the glass transition temperature, tauR was constant at approximately 10(-4) s for all water contents studied. Above Tg, isomobility lines were found that were approximately parallel to the Tg curve. The temperature dependence of tauR at all water contents studied followed Arrhenius behavior, with a break at Tg. Above Tg the activation energy for rotational motion was approximately 25 kJ/mol compared to 10 kJ/mol below Tg. The temperature dependence of tauR could also be described by the WLF equation, using constants deviating considerably from the universal constants. The temperature effect on tauR above Tg was much smaller in pea axes, as found previously for sugar and polymer glasses. Thus, although glasses are present in seeds, the melting of the glass by raising the temperature will cause only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.     

Effects of structure on alpha C-H bond enthalpies of amino acid residues: relevance to H transfers in enzyme mechanisms and in protein oxidation.

The bond dissociation enthalpies (BDE) of all of the amino acid residues, modeled by HC(O)NHCH(R)C(O)NH(2) (PH(res)), were determined at the B3LYP/6-31G//B3LYP/6-31G level, coupled with isodesmic reactions. The results for neutral side chains with phi, psi angles approximately 180 degrees, approximately 180 degrees in ascending order, to an expected accuracy of +/-10 kJ mol(-)(1), are Asn 326; cystine 330; Asp 332; Gln 334; Trp 337; Arg 340; Lys 340; Met 343; His 344; Phe 344; Tyr 344; Leu 344; Ala 345; Cys 346; Ser 349; Gly 350; Ile 351; Val 352; Glu 354; Thr 357; Pro-cis 358; Pro-trans 369. BDEs calculated at the ROMP2/6-31G//B3LYP/6-31G level exhibit the same trends but are approximately 7 kJ mol(-)(1) higher. All BDEs are smaller than those of typical secondary or tertiary C-H bonds due to the phenomenon of captodative stabilization. The stabilization is reduced by changes in the phi,psi angles. As a result the BDEs increase by about 10 kJ mol(-)(1) in beta-sheet and 40 kJ mol(-)(1) in alpha-helical environments, respectively. In effect the alpha C-H BDEs can be "tuned" from about 345 to 400 kJ mol(-)(1) by adjusting the local environment. Some very significant effects of this are seen in the current literature on H-transfer processes in enzyme mechanisms and in oxidative damage to proteins. These observations are discussed in terms of the findings of the present study.     

The energetics of rearrangement and water elimination reactions in the radiolysis of the DNA bases in aqueous solution (eaq- and *OH attack): DFT calculations

DFT calculations on the relative stability of various nucleobase radicals induced by e(aq)(-) and (*)OH have been carried out for assessing the energetics of rearrangements and water elimination reactions, taking the solvent effect of water into account. Uracil and thymine radical anions are protonated fast at O2 and O4, whereby the O2-protonated anions are higher in energy (50 kJ mol(-1), equivalent to a 9-unit lower pK(a)). The experimentally observed pK(a)=7 is thus that of the O4-protonated species. Thermodynamically favored protonation occurs slowly at C6 (driving force, thymine: 49 kJ mol(-1), uracil: 29 kJ mol(-1)). The cytosine radical anion is rapidly protonated by water at N3. Final protonation at C6 is disfavored here. The kinetically favored pyrimidine C5 (*)OH adducts rearrange into the thermodynamically favored C6 (*)OH adducts (driving force, thymine: 42 kJ mol(-1)). Very similar in energy is a water elimination that leads to the Ura-5-methyl radical. Purine (*)OH adducts at C4 and C5 (plus C2 in guanine) eliminate water in exothermic reactions, while water elimination from the C8 (*)OH adducts is endothermic. The latter open the ring en route to the FAPY products, an H transfer from the C8(*)OH to N9 being the most likely process.     

Kinetic characterization for dilute sulfuric acid hydrolysis of timber varieties and switchgrass

Hydrolysis of four timber species (aspen, balsam fir, basswood, and red maple) and switchgrass was studied using dilute sulfuric acid at 50 g dry biomass/L under similar conditions previously described as acid pretreatment. The primary goal was to obtain detailed kinetic data of xylose formation and degradation from a match between a first order reaction model and the experimental data at various final reactor temperatures (160-190 degrees C), sulfuric acid concentrations (0.25-1.0% w/v), and particle sizes (28-10/20 mesh) in a glass-lined 1L well-mixed batch reactor. Reaction rates for the generation of xylose from hemicellulose and the generation of furfural from xylose were strongly dependent on both temperature and acid concentration. However, no effect was observed for the particle sizes studied. Oligomer sugars, representing incomplete products of hydrolysis, were observed early in the reaction period for all sugars (xylose, glucose, arabinose, mannose, and galactose), but were reduced to low concentrations at later times (higher hemicellulose conversions). Maximum yields for xylose ranged from 70% (balsam) to 94% (switchgrass), for glucose from 10.6% to 13.6%, and for other minor sugars from 8.6% to 58.9%. Xylose formation activation energies and the pre-exponential factors for the timber species and switchgrass were in a range of 49-180 kJ/mol and from 7.5 x 10(4) to 2.6 x 10(20)min(-1), respectively. In addition, for xylose degradation, the activation energies and the pre-exponential factors ranged from 130 to 170 kJ/mol and from 6.8 x 10(13) to 3.7 x 10(17)min(-1), respectively. There was a near linear dependence on acid concentration observed for xylose degradation. Our results suggest that mixtures of biomass species may be processed together and still achieve high yields for all species.     

Quantum chemical calculations of the reorganization energy of blue-copper proteins.

The inner-sphere reorganization energy for several copper complexes related to the active site in blue-copper protein has been calculated with the density functional B3LYP method. The best model of the blue-copper proteins, Cu(Im)2(SCH3)(S(CH3)2)(0/+), has a self-exchange inner-sphere reorganization energy of 62 kJ/mol, which is at least 120 kJ/mol lower than for Cu(H2O)4(+/2+). This lowering of the reorganization energy is caused by the soft ligands in the blue-copper site, especially the cysteine thiolate and the methionine thioether groups. Soft ligands both make the potential surfaces of the complexes flatter and give rise to oxidized structures that are quite close to a tetrahedron (rather than tetragonal). Approximately half of the reorganization energy originates from changes in the copper-ligand bond lengths and half of this contribution comes from the Cu-S(Cys) bond. A tetragonal site, which is present in the rhombic type 1 blue-copper proteins, has a slightly higher (16 kJ/mol) inner-sphere reorganization energy than a trigonal site, present in the axial type 1 copper proteins. A site with the methionine ligand replaced by an amide group, as in stellacyanin, has an even higher reorganization energy, about 90 kJ/mol.     

Equilibrium unfolding of dimeric desulfoferrodoxin involves a monomeric intermediate: iron cofactors dissociate after polypeptide unfolding

Here we report the conformational stability of homodimeric desulfoferrodoxin (dfx) from Desulfovibrio desulfuricans (ATCC 27774). The dimer is formed by two dfx monomers linked through beta-strand interactions in two domains; in addition, each monomer contains two different iron centers: one Fe-(S-Cys)(4) center and one Fe-[S-Cys+(N-His)(4)] center. The dissociation constant for dfx was determined to be 1 microM (DeltaG = 34 kJ/mol of dimer) from the concentration dependence of aromatic residue emission. Upon addition of the chemical denaturant guanidine hydrochloride (GuHCl) to dfx, a reversible fluorescence change occurred at 2-3 M GuHCl. This transition was dependent upon protein concentration, in accord with a dimer to monomer reaction [DeltaG(H(2)O) = 46 kJ/mol of dimer]. The secondary structure did not disappear, according to far-UV circular dichroism (CD), until 6 M GuHCl was added; this transition was reversible (for incubation times of < 1 h) and independent of dfx concentration [DeltaG(H(2)O) = 50 kJ/mol of monomer]. Thus, dfx equilibrium unfolding is at least three-state, involving a monomeric intermediate with native-like secondary structure. Only after complete polypeptide unfolding (and incubation times of > 1 h) did the iron centers dissociate, as monitored by disappearance of ligand-to-metal charge transfer absorption, fluorescence of an iron indicator, and reactivity of cysteines to Ellman's reagent. Iron dissociation took place over several hours and resulted in an irreversibly denatured dfx. It appears as if the presence of the iron centers, the amino acid composition, and, to a lesser extent, the dimeric structure are factors that aid in facilitating dfx's unusually high thermodynamic stability for a mesophilic protein.     

Blocking nonspecific adsorption of native food-borne microorganisms by immunomagnetic beads with iota-carrageenan

We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av DeltaG0=-20 +/- 4 kJ mol(-1)) even in the presence of casein [1% (w/v): DeltaG0=-18 +/- 3 kJ mol(-1); DeltaDeltaG0 approximately -2 kJ mol(-1)]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; DeltaG0=-19 +/- 2 kJ mol(-1)) was tested for nonspecific binding in the presence of iota-carrageenan (0.03-0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency xi (DeltaG0=-11 +/- 4 kJ mol(-1); DeltaDeltaG0 approximately -8 kJ mol(-1)). Other anionic polysaccharides (0.1% kappa-carrageenan and polygalacturonic acid) had no significant effect (av DeltaG0=-19 +/- 1 kJ mol(-1); DeltaDeltaG0 approximately 0 kJ mol(-1)). Varying iota-carrageenan from 0% to 0.02% resulted in xi significantly diminishing from 0.69 (e.g., 69% of the cells captured; DeltaG0=-19 +/- 3 kJ mol(-1)) to 0.05 (DeltaG0=-11 +/- 2 kJ mol(-1); DeltaDeltaG0 approximately -9 kJ mol(-1)) at about 0.03% iota-carrageenan where xi leveled off. An optimum blocking ability was achieved with 0.04% iota-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of iota-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.     

Slow inhibition of almond beta-glucosidase by azasugars: determination of activation energies for slow binding

The thermodynamic and activation energies of the slow inhibition of almond beta-glucosidase with a series of azasugars were determined. The inhibitors studied were isofagomine ((3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 1), isogalactofagomine ((3R,4S,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 2), (-)-1-azafagomine ((3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine, 3), 3-amino-3-deoxy-1-azafagomine (4) and 1-deoxynojirimycin (5). It was found that the binding of 1 to the enzyme has an activation enthalpy of 56.1 kJ/mol and an activation entropy of 25.8 J/molK. The dissociation of the enzyme-1 complex had an activation enthalpy of -2.5 kJ/mol and an activation entropy of -297 J/molK. It is suggested that the activation enthalpy of association is due to the breaking of bonds to water, while the large negative activation entropy of dissociation is due at least in part to the resolvation of the enzyme with water molecules. For the association of 1 DeltaH(0) is 58.6 kJ/mol and DeltaS(0) is 323.8 J/molK. Inhibitor 3 has an activation enthalpy of 39.3 kJ/mol and an activation entropy of -17.9 J/molK for binding to the enzyme, and an activation enthalpy of 40.8 kJ/mol and an activation entropy of -141.0 J/molK for dissociation of the enzyme-inhibitor complex. For the association of 3 DeltaH(0) is -1.5 kJ/mol and DeltaS(0) is 123.1 J/molK. Inhibitor 5 is not a slow inhibitor, but its DeltaH(0) and DeltaS(0) of association are -30 kJ/mol and -13.1 J/molK. The large difference in DeltaS(0) of association of the different inhibitors suggests that the anomeric nitrogen atom of inhibitors 1-4 is involved in an interaction that results in a large entropy increase.     

Specificity and kinetics of hexose transport in Trypanosoma brucei

Transport of 6-deoxy-D-glucose was studied in Trypanosoma brucei in order to characterise the kinetics of hexose transport in this organism using a nonphosphorylated sugar. Kinetic parameters for efflux and entry, measured using zero-trans and equilibrium exchange protocols, indicate that the transporter is probably kinetically symmetrical. Comparison of the kinetic constants of D-glucose metabolism with those for 6-deoxy-D-glucose transport shows that transport across the plasma membrane is likely to be the rate-limiting step of glucose utilisation. The transport rate is nevertheless very fast and 6-deoxy-D-glucose, at concentrations below Km, enters the cells with a half filling time of less than 2 s at 20 degrees C. Thus the high metabolic capacity of these organisms is matched by a high transport rate. The structural requirements for the trypanosome hexose transporter were explored by measuring inhibition constants (Ki) for a range of D-glucose analogues including fluoro and deoxy sugars as well as epimeric hexoses. The relative affinities shown by these analogues indicated H-bonds from the carrier to the C-3, C-4 and C-5 hydroxyl oxygens and from the C-1 and C-3 hydroxyl hydrogens to the binding site. Hydrophobic interactions are likely at the C-2 and C-6 regions of the glucose molecule. Spatial constraints appear to occur around C-4 indicating that the transport site at this position is not freely open to the external solution as is the case with the mammalian hexose transporter. However, the trypanosome transporter appears to accept D-fructose but the common mammalian (erythrocyte type) hexose transporter does not.