New technique for the determination of interconversion processes based on capillary zone electrophoresis: Studies with axially chiral biphenyls

A new technique is presented for the investigation of the rotational energy barrier of axially chiral biphenyls, based on capillary zone electrophoresis using cyclodextrin derivatives as chiral selectors. Only minute amounts are required for the investigation of dynamic processes with energy barriers of 100–130 kJ/mol. The influence of the chiral selector on the conformational stability of atropisomers can be determined separately for each enantiomer. Additionally, segmentation of the capillary into different buffer zones allows us to exclude any influence of the chiral selector on the rotational energy barrier. © 1996 Wiley-Liss, Inc.     

Novel direct access to enantiomerization barriers from peak profiles in enantioselective dynamic chromatography: enantiomerization of dialkyl-1,3-allenedicarboxylates

The axially chiral allenes dimethyl-1,3-allenedicarboxylate 1 and diethyl-1,3-allenedicarboxylate 2 show characteristic plateau formation during enantioselective GC separation on the chiral stationary liquid phase Chirasil-beta-Dex. The elution profiles, obtained from temperature-dependent dynamic GC (DGC) experiments (1: 100-140 degrees C; 2: 110-150 degrees C) were evaluated with the recently derived approximation function (AF) k1(approx) = f(t(R)(A),t(R)(B),w(h)(A),h(plateau), N) to yield the enantiomerization rate constant directly k(1). These values were compared with those obtained by computer-aided simulation with ChromWin. The Eyring activation parameters of the experimental interconversion profiles were determined to be: DeltaG(#)(298.15 K) = 103.6 +/- 0.9 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.4 kJ mol(-1), DeltaS(#) = -198 +/- 7 J K(1) mol(-1) for dimethyl-1,3-allenedicarboxylate 1, and DeltaG(#)(298.15 K) = 103.5 +/- 1.1 kJ mol(-1), DeltaH(#) = 44.7 +/- 0.5 kJ mol(-1), DeltaS(#) = -197 +/- 9 J K(-1) mol(-1) for diethyl-1,3-allenedicarboxylate 2. The approximation function (AF) presented here allows the fast determination of rate constants k(1) and activation barriers of enantiomerization DeltaG(#) from chromatographic parameters without extensive computer simulation.     

Determination of enantiomerization barriers of hypericin and pseudohypericin by dynamic high‐performance liquid chromatography on immobilized polysaccharide‐type chiral stationary phases and off‐column racemization experiments

Direct enantiomer separation of hypericin, pseudohypericin, and protohypericin was accomplished by high‐performance liquid chromatography (HPLC) using immobilized polysaccharide‐type chiral stationary phases (CSPs). Enantioselectivities up to 1.30 were obtained in the polar‐organic elution mode whereby for hypericin and pseudohypericin Chiralpak IC [chiral selector being cellulose tris(3,5‐dichlorophenylcarbamate)] and for protohypericin Chiralpak IA (chiral selector being the 3,5‐dimethylphenylcarbamate of amylose) gave favorable results. Enantiomers were distinguished by on‐line electronic circular dichroism detection. Optimized enantioselective chromatographic conditions were the basis for determining stereodynamic parameters of the enantiomer interconversion process of hypericin and pseudohypericin. Rate constants delivered by computational simulation of dynamic HPLC elution profiles (stochastic model, consideration of peak tailing) were used to calculate averaged enantiomerization barriers (ΔG) of 97.6–99.6 kJ/mol for both compounds (investigated temperature range 25–45°C). Complementary variable temperature off‐column (i.e., in solution) racemization experiments delivered ΔG = 97.1–98.0 kJ/mol (27–45°C) for hypericin and ΔG = 98.9–101.4 kJ/mol (25–55°C) for pseudohypericin. An activation enthalpy of ΔH^# = 86.0 kJ/mol and an activation entropy of ΔS^# = ?37.7 J/(K mol) were calculated from hypericin racemization kinetics in solution, whereas for pseudohypericin these figures amounted to 74.1 kJ/mol and ?82.6 J/(K mol), respectively. Although the natural phenanthroperylene quinone pigments hypericin and pseudohypericin as well as their biological precursor protohypericin are chiral and can be separated by enantioselective HPLC low enantiomerization barriers seem to prevent the occurrence of an excess of one enantiomer under typical physiological conditions—at least as long as stereoselective intermolecular interactions with other chiral entities are absent. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Determination of the rotational barriers of atropisomeric polychlorinated biphenyls (PCBs) by a novel stopped-flow multidimensional gas chromatographic technique

The rotational barriers ΔG^‡ (T) of the four atropisomeric polychlorinated biphenyls (PCBs) 2,2′,3,5′,6-pentachlorobiphenyl (PCB 95), 2,2′3,3′,4,6′-hexachlorobiphenyl (PCB 132), 2,2′,3,3′,6,6′-hexachlorobiphenyl (PCB 136), and 2,2′,3,4′,5′,6-hexachlorobiphenyl (PCB 149) were determined via on-line enantiomerization kinetics by a new stopped-flow multidimensional gas chromatographic technique (stopped-flow MDGC) employing Chirasil-Dex as chiral stationary phase for enantiomer separation. The calculated rotational barriers ΔG^‡ (T) of the trichloro-ortho-substituted atropisomers are 184 ± 2 kJ/mol for PCB 95, 189 ± 4 kJ/mol for PCB 132, and 184 ± 1 kJ/mol for PCB 149 at 300°C. The rotational barrier ΔG^‡ (T) of tetrachloro-ortho-substituted PCB 136 is at least (or higher than) 210 kJ/mol at 320°C. Chirality 10:316–320, 1998. © 1998 Wiley-Liss, Inc.     

Substrate specificity,metal binding properties,and spectroscopic characterization of the DapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae

The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to approximately 20% of the Zn(II)-loaded enzyme. The order of the observed k(cat) values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) approximately equal Cu(II) approximately equal Mg(II). DapE was shown to only hydrolyze L,L-N-succinyl-diaminopimelic acid (L,L-SDAP) and was inactive toward D,L-, L,D-, and D,D-SDAP. DapE was also inactive toward several acetylated amino acids as well as D,L-succinyl aminopimelate, which differs from the natural substrate, L,L-SDAP, by the absence of the amine group on the amino acid side chain. These data imply that the carboxylate of the succinyl moiety and the amine form important interactions with the active site of DapE. The affinity of DapE for one versus two Zn(II) ions differs by nearly 2.2 x 10(3) times (K(d1) = 0.14 microM vs K(d2) = 300 microM). In addition, an Arrhenius plot was constructed from k(cat) values measured between 16 and 35 degrees C and was linear over this temperature range. The activation energy for [ZnZn(DapE)] was found to be 31 kJ/mol with the remaining thermodynamic parameters calculated at 25 degrees C being DeltaG(++) = 64 kJ/mol, DeltaH(++) = 28.5 kJ/mol, and DeltaS(++) = -119 J mol(-1) K(-1). Electronic absorption and EPR spectra of [Co_(DapE)] and [CoCo(DapE)] indicate that the first Co(II) binding site is five-coordinate, while the second site is octahedral. In addition, any spin-spin interaction between the two Co(II) ions in [CoCo(DapE)] is very weak. The kinetic and spectroscopic data presented herein suggest that the DapE from H. influenzae has similar divalent metal binding properties to the aminopeptidase from Aeromonas proteolytica (AAP), and the observed divalent metal ion binding properties are discussed with respect to their catalytic roles in SDAP hydrolysis.     

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.     

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of barriers to rotation of axially chiral 5-methyl-2-(o-aryl)imino-3-(o-aryl)thiazolidine-4-ones

Thermally interconvertible axially chiral 5-methyl-2-(o-aryl)imino-3-(o-aryl)-thiazolidine-4-ones have been synthesized diastereoselectively, and conformations of the major and minor enantiomeric pairs have been determined by (1)H nuclear magnetic resonance. Chromatographic resolutions of each compound have been performed by enantioselective high-performance liquid chromatography, and the barriers to rotation about the N(3)-C(aryl) bond have been determined by following the thermal interconversion process of the major to minor isomers until equilibrium. The rotational barriers range from 96.2 to 115.2 kJ/mol, depending on the size of ortho substituent on N(3)-aryl ring.     

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.     

On-column deracemization of an atropisomeric biphenyl by quinine-based stationary phase and determination of rotational energy barrier by enantioselective stopped-flow HPLC and CEC.

The reversible enantiomerization of axially chiral 2'-dodecyloxy-6-nitrobiphenyl-2-carboxylic acid was studied in the presence of a brush type chiral stationary phase based on O-(tert-butylcarbamoyl) quinine as chiral selector unit by stopped-flow high-performance liquid chromatography (sfHPLC) and capillary electrochromatography (sfCEC). After initial separation of the enantiomers in the first section of the column, the flow was stopped and the resolved species allowed to enantiomerize on-column. From this conversion, which could be determined from the enantiomeric ratios at different enantiomerization times, kinetic rate constants were calculated. By sfHPLC at a constant temperature of 15 degrees C, kinetic rate constants in the presence of the CSP were found to be 4.1 x 10(-5) s(-1) and 2.2 x 10(-5) s(-1) for the (-) and (+)-enantiomers, respectively, corresponding to half-lives of 279 and 530 min. Thus, apparent activation energies of enantiomerization were calculated to be 93.0 and 94.6 kJ mol(-1) for the (-) and (+)-enantiomers. On the macroscopic level, the apparent difference of rotational energy barriers and kinetic rate constants for both enantiomers is reflected as deracemization. For example, starting from a racemic mixture, an enantiomeric excess (ee) of 14% was seen in the stopped-flow HPLC experiment described after an enantiomerization time of 220 min at 15 degrees C, and a maximal ee of 17% can be approximated after infinite enantiomerization time. There is good agreement between HPLC and CEC results as well as their experimental errors, confirming that the new sfCEC technique may be a valuable and convenient tool to study interconversion processes.     

Dynamics of interaction of vitamin C with some potent nitrovasodilators, S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and S-nitrosocaptopril (SNOCap), in aqueous solution

The reductive decomposition of both SNAP and SNOCap by ascorbate in aqueous solution (in the presence of EDTA) was thoroughly investigated. Nitric oxide (NO) release from the reaction occurs in an ascorbate concentration and pH dependent manner. Rates and hence NO release increased drastically with increasing pH, signifying that the most highly ionized form of ascorbate is the more reactive species. The experiments were monitored spectrophotometrically, and second-order rate constants calculated at 37 degrees C for the reduction of SNAP are k(b)=9.81+/-1.39 x 10(-3) M(-1) s(-1) and k(c)=662+/-38 M(-1) s(-1) and for SNOCap are k(b)=2.57+/-1.29 x 10(-2) M(-1) s(-1) and k(c)=49.7+/-1.3 M(-1) s(-1). k(b) and k(c) are the second-order rate constants via the ascorbate monoanion (HA-) and dianion (A2-) pathways, respectively. Activation parameters were also calculated and are DeltaHb++ =93+/-7 kJ mol(-1), DeltaSb++ =15+/-2 J K(-1) mol(-1) and DeltaHc++ =51+/-5 kJ mol(-1), DeltaSc++ =-28+/-3 J K(-1) mol(-1) with respect to the reactions involving SNAP. Those for the reaction between SNOCap and ascorbate were calculated to be DeltaHb++ =63+/-11 kJ mol(-1), DeltaSb++ =-71+/-20 J K(-1) mol(-1) and DeltaHc++ =103+/-7 kJ mol(-1), DeltaSc++ =118+/-8 J K(-1) mol(-1). The effect of Cu2+/Cu+ ions on the reductive decompositions of these S-nitrosothiols was also investigated in absence of EDTA. SNOCap exhibits relatively high stability at near physiological conditions (37 degrees C and pH 7.55) even in the presence of micromolar concentrations of Cu2+, with decomposition rate constant being 0.011 M(-1) s(-1) in comparison to SNAP which is known to be more susceptible to catalytic decomposition by Cu2+ (second-order rate constant of 20 M(-1) s(-1) at pH 7.4 and 25 degrees C). It was also observed that the reductive decomposition of SNAP is not catalyzed by alkali metal ions, however, there was an increase in rate as the ionic strength increases from 0.2 to 0.5 mol dm(-3) NaCl.     

Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding.

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (k(U)) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea-induced denaturation where spectroscopic methods do not allow direct measurement of k(U). The results show that the approach can be applied successfully to denaturing (free energy of unfolding Delta G < 10 kJ.mol(-1)) and to marginally native conditions (Delta G = 10-25 kJ.mol(-1)). Under moderately (Delta G = 25-30 kJ.mol(-1)) and strongly native conditions (Delta G > 30 kJ.mol(-1)), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.     

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

High sensitivity analysis of oxprenolol in urine by capillary electrophoresis with C18 packed on-line preconcentrator

High sensitivity analysis of oxprenolol in spiked human urine has been performed by capillary zone electrophoresis (CZE) in ammonium formate buffer pH 2.5 using an uncoated capillary with 1cm length C18 on-capillary preconcentrator at the inlet side. The preconcentrator was fabricated in laboratory using the packing method and not encapped C18 5 microm particles as stationary phase material. The packed path was retained into the capillary by sintered stationary phase frits. Before running the CZE analysis, the oxprenolol was eluted from the preconcentrator by injecting a short plug of acetonitrile/water mixtures. With respect to classical CZE, the use of on-line preconcentrator widely increased the method sensitivity allowing the detection of the drug at 0.5 ng/mL (injected concentration). The method showed a linear response in the range of 1-150 ng/mL oxprenolol standard compound. The intra-day repeatability (n = 11) R.S.D. values for migration time, peak area and normalized peak area were 0.72%, 3.96% and 3.66%, respectively, while inter-day repeatability (n = 5 days) R.S.D. values were 2.74%, 9.41% and 9.83%, respectively. The method was successfully applied to the analysis of oxprenolol in extracted urine spiked at 250 pg/mL (oxprenolol LOQ concentration in urine).     

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.     

alpha-Amylase adsorption on starch crystallites

The goal of this work was to characterize the adsorption of Bacillus subtills alpha-amylase onto crystalline starchy materials of the B-type polymorph. Monodisperse spherulitic particles (R z6; 5.0 mum), essentially resistant to alpha-amylolysis at 25 degrees C were prepared from short amylose chains (DP(n) approximately 15). The alpha-amylase adsorbed specifically onto the spherulites, and adsorption was found to be a prerequisite step for hydrolysis. Adsorption was inhibited by the presence of maltose and maltotriose in the reaction mixture. Adsorption isotherm of the enzyme on the particles showed a well developed plateau of 1.62 mug/cm(2) at 25 degrees C corresponding to a monolayer adsorption process. The binding free energy calculated from the initial slope of the isotherm was DeltaG approximately -20.7 kJ/mol. This is smaller than published values for the binding of alpha-amylase to soluble amylosic chains (DeltaG < -30 kJ/mol).     

Spirobipyridopyrans and indolinospiropyridopyrans: Synthesis, X-ray crystal structure, separation of enantiomers, and barriers to thermal cleavage of the C(sp(3))-O bond

The novel chiral spirobipyridopyrans 1 and 2 were synthesized by the acid catalyzed aldol type condensation of 5-deoxypyridoxal with the appropriate ketone and subsequent reaction of the resulting pyrylium salt with base. The indolinospiropyridopyrans 3-5, which contain the modified B(6) unit, were prepared by aldol reaction of 5-deoxypyridoxal with 1,3,3-trimethyl-2-methylenindolines. Analytical separation of enantiomers was accomplished by low-pressure liquid chromatography (LPLC) on triacetylcellulose. The barriers to thermal racemization were determined by on-line measurements of the enriched enantiomers after LPLC. Gibbs energies of activation DeltaG superset, not equals for reversible cleavage of the C(spiro)-O bond in 1, 3, and 4 were in the range 103-108 kJ/mol. The lower limits of the barriers in 2 and 5 were estimated to be greater than 102 and 109 kJ/mol by attempted thermal racemizations. The increase of the barriers from 3 to 4 and 5 was explained by the influence of electron withdrawing groups, which reduce the stability of the ring-opened transition states to C(sp3)-O bond cleavage. Geometrical data from X-ray structure analysis showed that the angle [C3-C2-C3'] around the spiro carbon atom increases with elongation of the chain in the C3-C3' bridge. This angle widening is explained by a ring-strain effect, which is greater in the five-membered ring in the skeleton of 7 than in the six- and seven-membered rings of 1 and 2.     

Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase

Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol). Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol). At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine [delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)] to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)].     

Analysis of coals and biomass pyrolysis using the distributed activation energy model

The thermal decomposition of coals and biomass was studied using thermogravimetric analysis with the distributed activation energy model. The integral method resulted in Datong bituminous coal conversions of 3-73% at activation energies of 100-486 kJ/mol. The corresponding frequency factors were e(19.5)-e(59.0)s(-1). Jindongnan lean coal conversions were 8-52% at activation energies of 100-462 kJ/mol. Their corresponding frequency factors were e(13.0)-e(55.8)s(-1). The conversion of corn-stalk skins were 1-84% at activation energies of 62-169 kJ/mol with frequency factors of e(10.8)-e(26.5)s(-1). Datong bituminous coal, Jindongnan lean coal and corn-stalk skins had approximate Gaussian distribution functions with linear ln k(0) to E relationships.