Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.     

Interaction of ribonuclease A with aqueous 2-methyl-2,4-pentanediol at pH 5.8.

The interactions between ribonuclease A and solvent components in aqueous 2-methyl-2,4-pentanediol (MPD) have been investigated by differential refractometry and light scattering at pH 5.8, i.e., conditions similar to those used to crystallize the protein from this solvent system. Application of multicomponent thermodynamic theory shows that, at all solvent compositions up to 50% (v/v) MPD, the protein is preferentially hydrated; i.e., addition of ribonuclease to the mixed solvent leads to an increase in the chemical potential of MPD. This unfavorable thermodynamic interaction leads to phase separation, probably caused by local salting out of the MPD by the charges on the surface of the protein molecule. A parallel examination by circular dichroism (CD) has shown that the CD spectrum of ribonuclease in 50% MPD is indistinguishable from that in dilute buffer.     

A simple preparative method for the isolation of amylose and amylopectin from potato starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8,0.1 M) + urea (2 M) + 1-butanol (8.5%, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and beta -amylolysis limit.     

A Simple Preparative Method for the Isolation of Amylose and Amylopectin from Potato Starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8, 0.1 M) ± urea (2 M) ± 1-butanol (8.5 %, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and β -amylolysis limit.     

An improved histochemical method for the demonstration of disaccharidases with natural substrates

Summary A simple histochemical method for the demonstration of disaccharidases is described. The medium consists of natural disaccharides (as substrates) and glucose oxidaseperoxidase-3,3-diaminobenzidine tetrahydrochloride (as the detecting agent) buffered to pH 6.0 with 0.05 M citric acid phosphate buffer in 1/2% agar gel. This method which enables the localization of disaccharidases on the histological level is very suitable for the routine assessment of activities of disaccharidases in enterobiopsies.     

Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.     

Determination of acyclovir in maternal plasma,amniotic fluid,fetal and placental tissues by high-performance liquid chromatography

Acyclovir [9-[(2-hydroxyethoxy)-methyl]-guanosine, Zovirax, ACV] is a synthetic purine nucleoside analog active against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella zoster virus. Acyclovir has frequently been used in HSV-2 seropositive mothers to prevent prenatal transmission of herpes virus to their unborn children. A fast and reproducible HPLC method for the determination of the highly polar acyclvoir in maternal rat plasma, amniotic fluid, placental tissue, and fetal tissue has been developed and validated. Plasma and amniotic fluid samples were prepared by protein precipitation using 2 M perchloric acid and syringe filtering. Tissue samples were homogenized in distilled water, centrifuged, and extracted using a C(18) solid-phase extraction method prior to analysis. Baseline resolution was achieved for acyclovir and the internal standard gancyclovir, an anti-viral of similar structure to acyclovir, using an Agilent Eclipse XDB C(8) column (150 x 2.1 mm, 5 microm). The mobile phase used for the plasma and amniotic fluid was 10 mM acetate/citrate buffer-3.7 mM aqueous octanesulfonic acid (87.5:12.5, v/v) at a flow-rate of 0.2 ml/min. The mobile phase used for the tissue samples was 30 mM acetate/citrate buffer with 5 mM octanesulfonic acid-acetonitrile (99:1, v/v). Both aqueous mobile phase portions were pH adjusted to 3.08. All separations were done using an Agilent 1100 Series HPLC system with UV detection of 254 nm. The assay was validated for each matrix over a range of 0.25-100 microg/ml over 3 days using five replicates of three spiked concentrations. The relative standard deviation and percent error for each validation data set was <15% for middle and high quality control (QC) points and <20% for all low QC points. All calibration curves showed good linearity with an R(2)>0.99. The extraction efficiency for recovery of acyclovir from all matrices was >80%.     

Glycosphingolipid glycosyltransferase assay using sephadex G-50 chromatography in aqueous phase

An assay method for glycosphingolipid glycosyltransferase activity using simple Sephadex G-50 gel permeation chromatography in an aqueous solvent has been developed. An acceptor glycosphingolipid and a donor radioactive nucleotide sugar were incubated with an enzyme source. The reaction mixture was loaded onto a Sephadex G-50 column previously equilibrated with 0.3% (w/v) Triton X-100, 0.1 M sodium chloride, and 0.02% (w/v) sodium azide. The radiolabeled reaction product was eluted by the same solvent in the excluded volume and was collected directly into a liquid scintillation vial, separated from other radioactive compounds. This assay method was utilized to determine the activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:GM3 ganglioside sialyltransferase, which catalyzes the synthesis of GD3 ganglioside, and proved to be as reliable and sensitive as previously published assay procedures. In addition, this assay can be carried out in less time and is simpler than previously reported procedures.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.     

Paper chromatographic system for the identification of glycerol in bacterial cell walls

Ikawa, Miyoshi (University of New Hampshire, Durham), James W. Morrow, and Sheila J. Harney. Paper chromatographic system for the identification of glycerol in bacterial cell walls. J. Bacteriol. 92:812-814. 1966.-The solvent system consisting of isopropanol-5% boric acid (7:1, v/v) separates glycerol from the other carbohydrate constituents which are found in hydrolysates of bacterial cell walls. This system is useful for the identification of glycerol even when anhydroribitol and rhamnose are both present, and has been found to be applicable on cell wall hydrolysates as well as on synthetic mixtures.     

How do organic solvents affect peroxidase structure and function?

The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.     

Double-stranded structure for hyaluronic acid in ethanol-aqueous solution as revealed by circular dichroism of oligomers

The sigmoidal nature of circular dichroism (CD) changes for hyaluronic acid solutions as a function of solvent composition or temperature is studied as a function of chain length by using oligomers. We find a chain length effect with approximately nine disaccharides required for the structural transition as a function of organic solvent, which proves that the transition is cooperative with large transition enthalpy and entropy. The transition also depends on sample concentration as expected for strand association, and this was investigated in detail for oligomers 12 and 16 disaccharides long. Indeed, it was possible to prevent completely the transition in mixed solvent with sufficient dilution of these oligomers, which demonstrates strand association. The CD data in mixed solvent as a function of oligomer concentration were fit with various models for association of two and more strands. Simplex methods were used to investigate the vector space of unknowns for the models, and two-strand models were shown to consistently give a better fit. A cooperative two-strand zipper model which allows relative sliding of the chains had the smallest fitting error and produced the following thermodynamic parameters (in terms of a duplex of disaccharide units) for the ordered structure in an aqueous solution containing 45% v/v ethanol, 12.5 mM NaH2PO4, and 7.5 mM H3PO4: enthalpy of growth, -1.0 +/- 0.3 kcal mol-1; entropy of growth, -2.3 +/- 1.3 eu mol-1; enthalpy of initiation, -20 +/- 3 kcal mol-1; entropy of initiation, -71 +/- 15 eu mol-1. The results are consistent with a double-stranded and helical structure for hyaluronic acid in solutions of reduced dielectric constant.     

Validated liquid chromatographic-ultraviolet method for the quantitation of tadalafil in human plasma using liquid-liquid extraction

A highly selective, sensitive and rapid HPLC method has been developed and validated to quantify tadalafil in human plasma. The tadalafil and internal standard (loratadine, I.S.) were extracted by liquid-liquid extraction technique followed by an aqueous back-extraction allowing injection of an aqueous solvent in the HPLC system. The chromatographic separation was performed on a reverse phase BDS Hypersil C-18 column (250 mm x 4.6 mm, 5 microm, Thermo Separation Co., USA) with a mobile phase of acetonitrile and aqueous solution containing 0.012 M triethylamine+0.020 M orthophosphoric acid (50/50, v/v). The analytes were detected at 225 nm. The assay exhibited a linear range of 5-600 ng/mL for tadalafil in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL. The within- and between batch precision (expressed as coefficient of variation, C.V.) did not exceed 10.3% and the accuracy was within -7.6% deviation of the nominal concentration. The recovery of tadalafil from plasma was greater than 66.1%. Stability of tadalafil in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and 30 days storage in a freezer. This validated method is applied for the clinical study of the tadalafil in human volunteers.     

Kinetic resolution of racemic alpha-methyl-beta-propiothiolactone by lipase-catalyzed hydrolysis

Kinetic resolution of racemic alpha-methyl-beta-propiothiolactone (rac-MPTL) using lipases in organic solvent was studied. The lipase from Pseudomonas cepacia (PCL) showed the highest (S)-enantioselectivity (E > 100), and cyclohexane containing 1% (v/v) buffer was identified as the best reaction medium for maintaining high enantioselectivity as well as high reaction rate. While the substrate inhibition was not observed up to 300 mM rac-MPTL, severe product inhibition was observed even at 50 mM racemic 3-mercapto-alpha-methyl propionic acid (rac-MMPA), which made the use of high substrate concentration difficult. To overcome the product inhibition, the products, (R)-MMPA, were neutralized by addition of a dilute basic solution. Although the resolution reaction proceeded further by the base titration, the enantioselectivity of the reaction decreased as a result of nonenantioselective hydrolysis of rac-MPTL in the basic solution. Under these conditions, 200 mM rac-MPTL was successfully resolved to above 95% ee(S) with 53% conversion.     

Enzymatic production of 4-hydroxyphenylacetaldehyde by oxidation of the amino group of tyramine with a recombinant primary amine oxidase

In this study, an efficient enzymatic process for the synthesis of 4-hydroxyphenylacetaldehyde (4-HPAA) from tyramine was developed using whole cells of recombinant Escherichia coli co-expressing primary amine oxidase (PrAO) from E. coli and catalase (CAT) from Bacillus pumilus. The reaction conditions for the synthesis of 4-HPAA were systematically optimized starting from a monophasic aqueous buffer. The optimum reaction temperature, pH, and biocatalyst loading were 33 °C, 7.5, and 20 g/L wet cells, respectively. Substrate feeding strategies were employed to alleviate substrate inhibition, providing a 14.8 % increase in yield. A biphasic catalytic system was explored to avoid product inhibition and thus further improve the 4-HPAA yield. Ethyl acetate was found to be the best organic solvent, and the optimum volume ratio of the organic phase to the aqueous phase was 40 % (v/v). Under the optimized conditions on a 1 L scale, a yield of 76.5 % was obtained with a substrate concentration of 120 mM. Thus, the bioconversion was more efficient in the ethyl acetate/buffer biphasic system than in the monophasic aqueous system, and the yield of 4-HPAA was improved 1.89-fold.     

Synergism between Metarhizium anisopliae (Deuteromycota: Hyphomycetes) and Boric Acid against the German Cockroach (Dictyoptera: Blattellidae)

Mortality of German cockroaches, Blattella germanica (L.), caused by Metarhizium anisopliae (Metschnikoff) Sorokin strain AC-1 alone and in combination with different formulations of boric acid, was evaluated in laboratory bioassays. Topical application of M. anisopliae alone (8.96 × 10⁹ conidia/m²) required 28 days to cause >92% cockroach mortality (LT₅₀ = 10 days). In contrast, in combination with boric acid (topically applied as a dust or in drinking water), M. anisopliae killed cockroaches significantly faster than without boric acid. M. anisopliae conidial dust (8.96 × 10⁸ conidia/m²) with either 12.5% (w/w) boric acid dust or 0.1% (w/v) boric acid in drinking water killed 100% of the cockroaches in only 8 days (LT₅₀ = 5 days) and 10 days (LT₅₀ = 6 days), respectively, without compromising the fungus emergence from cadavers. Replacement of M. anisopliae with flour dust or heat-killed M. anisopliae conidia eliminated this effect, demonstrating that it was not the consequence of greater boric acid ingestion due to more extensive cockroach grooming upon exposure to M. anisopliae conidia. Moreover, injections of a low dose of M. anisopliae, which caused only 30% mortality, together with sublethal concentrations of boric acid into the cockroach hemocoel resulted in a doubling of mortality. Statistical analysis demonstrated a synergistic interaction between these two insecticides.     

Synthesis and high-performance liquid chromatography of maltulose and cellobiulose

The title compounds were prepared in ～90% overall yield by treatment of the appropriate aldodisaccharide (maltose or cellobiose) with boric acid and triethylamine in dilute, aqueous (pH 11, 70°) solution. When sodium hydroxide was substituted for triethylamine, the yields were unaltered, but reaction rates were increased. In addition, in the sodium hydroxide system, reactions at higher concentrations of sugar (up to 40% w/v) could be performed with only slight decreases in overall yields. New methods were developed for the high-performance liquid-chromatographic analysis (Zorbax-NH₂ column, modified with Column Life Extension Agent) of the sugars involved in these isomerizations. Multigram quantities of pure ketodisaccharides were prepared with a simple semi-preparative l.c. system.     

Crystallization and preliminary X-ray diffraction studies of Streptomyces phospholipase A2 in a calcium-binding form

Phospholipase A2 (PLA2) as a calcium-binding form, produced by Streptomyces violaceoruber, was crystallized in a form suitable for the diffraction analysis using the vapor diffusion method. Crystals were grown in 0.1 M Tris-HCl buffer (pH 8.5), 20 mM Ca2+ containing 50-60% (v/v) 2-methyl-2,4-pentanediol as a precipitant. They belong to the monoclinic space group P2(1), with the cell dimensions a=38.3 A, b=54.3 A, c=30.6 A, and beta=90.2 degrees. The crystals diffract the X-ray well and the diffraction intensity data were collected up to 1.6 A resolution. The crystal volume per unit mass, V(M) is 2.35 A3 Da(-1) with one molecule in the asymmetric unit, which corresponds to a solvent content of 47.7%.     

Rapid sequencing gel electrophoresis using glycerol-tolerant sodium taurine medium

We describe a new glycerol-tolerant sodium taurine (ST) medium for rapid sequencing gel electrophoresis by substituting the standard conductive media of Tris-boric acid-ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris-taurine-EDTA (TTE) and other low-ionic-strength media of sodium boric acid (SB). Low-ionic-strength and cost-effective ST media gave glycerol tolerance up to 50% (v/v) glycerol-containing DNA sample solution, shorter running time, and better resolution to separate small DNA oligonucleotides (20-45 mer) in 12% denaturing sequencing gel electrophoresis.