Kinetics of chromium nucleotide isomer interconversion

The rates of chromium nucleotide isomer interconversion were studied as a function of pH, ionic strength, and temperature. Nucleotide isomers were separated using high voltage electrophoresis and gel filtration chromatography. The rate of conversion of monodentate adenosine 5°-monophosphate-chromium salt (CrADP) to the bidentate complex increased with increasing pH, temperature, and ionic strength. Optimal stability for CrADP complexes was found to be at pH 3.5 with a temperature of 4°C. It was found that at pH values above 7.0, the chromium complexes rapidly decomposed even at 4°C. It was found that the conversion of monodentate CrADP to binentate CrADP required the removal of one proton by the solvent. The activation energy for the conversion was found to be 7.3 kcal/mol at pH 6.5. The kinetics of the isomer interconversion are described in terms of possible conversion mechanisms.     

High-performance liquid chromatographic system for separating sugar phosphates and other intermediary metabolites

A simple method for separating intermediates of carbohydrate metabolism, including the difficult-to-resolve sugar phosphates, using anion-exchange high-performance liquid chromatography is described. A gradient of decreasing borate concentration (10 to 0 mM) and increasing ionic strength (0 to 150 mM NH₄Cl) separates phosphorylated sugars based on the strength of the ester complex that they form with borate anion. Lyophillized samples are reconstituted in a low ionic strength aqueous medium (5 mM triethanolamine-HCl, pH 8.1) and chromatographed on a commercially available anion-exchange column (Hamilton PRP-X100). The process requires only 3 h and permits nanomole detection of the phosphorylated intermediates of the glycolytic and pentose shunt pathways.     

An optimized procedure for the separation of amino acid phenylthiohydantoins by reversed-phase HPLC

An optimization procedure for the separation of 24 PTH-amino acids by high-performance liquid chromatography on an inexpensive Merck Superspher Si 60 RP-8, (4.0 x 250 mm) column with PTH-Nle as an internal standard is described. The effects of pH, ionic strength, temperature and gradient were investigated. Using conventional HPLC equipment, the practical detection limit is about 5 pmol.     

High-performance liquid chromatography–ionspray mass spectrometry for the specific determination of digoxin and some related cardiac glycosides in human plasma

An original method based upon high-performance liquid chromatography coupled to ionspray mass spectrometry (HPLC–ISP-MS) has been developed for the identification and quantification in plasma of several cardiac glycosides, namely digoxin, digitoxin, lanatoside C and acetyldigitoxin. After single-step liquid–liquid extraction by chloroform–2-propanol (95:5, v/v) at pH 9.5 using oleandrin as an internal standard, solutes are separated on a 4 μm NovaPak C₁₈ (Waters) column (150×2.0 mm, I.D.), using a gradient of acetonitrile–2 mM NH₄COOH, pH 3 buffer (flow-rate 200 μl/min, post-column split 1:3). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with an ISP interface. In most instances the major ion observed is not [M+H]⁺ as expected, but [M+NH₄]⁺. The mean retention times (min) are: lanatoside C, 5.74; digoxin, 6.00; digitoxin, 8.08, oleandrin, 8.30, acetyldigitoxin, 8.66 and 9.01 (isomers α and β, respectively). The lower limits of detection in single ion monitoring mode range from 0.15 ng/ml (α- and β-acetyldigitoxin) to 0.60 ng/ml (lanatoside C), making the method less sensitive than radioimmunoassay, whereas it is much more specific.     

The monomer -- dimer association of rabbit lver aryl sulfatase A and its relationship to the anomalous kinetics.

The polymerization of aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) has been studied by frontal gel chromatography on Sephadex G-200 and Bio-Gel A-5m under various conditions of pH, ionic strength, and temperature. The aryl sulfatase A molecule exists as a monomer and as a dimer at pH 7.5 and pH 4.5, respectively. The extent of dissociation is markedly pH-, protein concentration-, and ionic strength-dependent. Only a small effect of temperature was observed. The enthalpy change (ΔH^o) for the dissociation was ?2.5 ± 1 kcal/mol at pH 5.5–5.6, and the entropy change for dissociation of the enzyme dimer to two monomeric units was ?47 cal mol^?1 deg^?1. Sulfate ion has little effect on the extent of dissociation of the enzyme at pH 5.6. The present studies suggest that the dissociation of rabbit liver aryl sulfatase A is regulated by the ionization of amino acid residues whose apparent pK is between pH 5 and 6. The driving force for the association of the subunits of the enzyme is primarily ionic and/or ionic/hydrogen bond formation. The small enthalpy change and the fact that dissociation is strongly favored by an increase in the ionic strength suggest that hydrophobic interactions play only a minor role in stabilizing the dimeric quaternary structure relative to the monomeric state. The monomeric form of the enzyme exhibits the anomalous kinetics often observed with sulfatase A but the dimer does not show anomalous kinetics. Since aryl sulfatase A is probably in the dimeric form in the lysosome, the anomalous kinetics of the enzyme are unlikely to be of physiological importance in the intact lysosome.     

Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry

A rapid, sensitive method using liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed and evaluated for the simultaneous quantitative determination of caffeine metabolites 1U, 1X and AAMU in human urine. This method involved a simple dilution of urine samples. The chromatographic separation was achieved on a C₁₈ reversed-phase column using a gradient of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was performed in the negative ion, selected ion monitoring mode. This method yielded acceptable accuracy and precision within the range 0.25–50 μg/ml. This analytical method was applied to investigate the N-acetylator phenotype of HIV-infected patients and compared with high-performance liquid chromatography with UV detection. Its specificity was better, which appeared to be absolutely necessary to prevent errors in metabolic ratios and phenotype interpretation.     

High-performance liquid chromatographic analyser for guanidino compounds using benzoin as a fluorogenic reagent

An automatic analyser based on high-performance liquid chromatography has been developed for the quantification of biogenic guanidino compounds in human physiological fluids. Fourteen guanidino compounds are mutually separated within 35 min on a cation-exchange column with a stepwise gradient elution of pH and/or ionic strength in the mobile phase and then converted automatically to their fluorescent derivatives with benzoin. The method in this system is simple, rapid and sensitive; the lower limits of detection are 5–50 pmol for monosubstituted guanidino compounds, 1 nmol for creatine and 20 nmol for creatinine in 100 μl of injection volume.     

The effect of ionic strength on the kinetics of fluoride binding to cytochrome c peroxidase.

The kinetics of the reaction between cytochrome c peroxidase and fluoride was investigated as a function of ionic strength over the pH range 4 to 8. The ionic strength was varied between 0.01 and 0.10 m. At 0.01 m ionic strength, the reaction rates were determined between pH 2.7 and 9.2. A consideration of the ionic strength and pH dependence of the association rate constant for the fluoride-cytochrome c peroxidase reaction leads to the conclusion that hydrofluoric acid is the only significant reactive form of the ligand between pH 2.5 and 8. Above pH 8, binding of fluoride anion contributes to the apparent association rate even though the pH-independent rate constant for fluoride anion binding is more than 3 × 10⁵ times smaller than the rate constant for hydrofluoric acid binding.     

Separation and quantification of muropeptides with high-performance liquid chromatography

About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.     

Kinetic studies on the conformational isomerization reaction of the four diastereomers of beta,gamma-bidentate CrATP

The rates of the conformational isomerization reaction of the diastereomers of beta,gamma-bidentate CrATP were studied as a function of pH, buffer concentration, ionic strength, and temperature. The progress of the reaction was monitored by quenching the reaction at various times, and then isolating the individual diastereomers and quantitating the percent of each. This was accomplished using the reverse-phase high-performance liquid chromatography separation technique developed in this laboratory [K. J. Gruys, and S. M. Schuster, Anal. Biochem. 125, 66-73 (1982)]. The rate constants for this isomerization were then determined by obtaining the best computer fit of the data to a reversible binary mechanism (i.e., A in equilibrium B) using interative descent methods. The reaction rate was shown to be dependent on pH, temperature, and ionic strength, but independent of buffer concentration. Keq. constants were independent of all variables except ionic strength. The results from this study are interpreted in terms of a reaction mechanism involving a preequilibrium ionization of the diastereomers followed by a rate-limiting interconversion process.     

Reversed-phase ion-pair liquid chromatographic procedure for the simultaneous analysis of S-adenosylmethionine, its metabolites and the natural polyamines

A method using reversed-phase ion-pair liquid chromatography with dual detection was developed for the simultaneous determination of the S-adenosylmethionine (SAM) analogues and the natural polyamines. The separation is obtained with a gradient elution and by adjusting the concentration of octanesulfonic acid used as ion-pairing agent, the ionic strength of the eluent, the pH and the acetonitrile content of the eluents. The SAM analogues are analyzed by UV detection at 254 nm and the polyamines by fluorescence detection after post-column derivatization with o-phthalaldehyde. The method allows the determination of the SAM analogues and the polyamines in one single run by direct injection of tissue extracts. The procedure is applied to the study in rats and in hepatoma tissue culture cells of the biochemical effects of α-difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase.     

Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection

Positional and geometric isomers of mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms were separated on commercially available reversed-phase columns in gradient systems composed of acetonitrile and water, utilizing photodiode array detection. The biological samples were hydrolyzed with 2 M NaOH for 35–40 min at 85–90°C. After cooling, the hydrolysates were acidified with 4 M HCl and the free fatty acids were extracted with dichloromethane. The organic solvent was removed in a gentle stream of argon. The fatty acids were determined after pre-column derivatization with dibromacetophenone in the presence of triethylamine. The reaction components were mixed and reacted for 2 h at 50°C. Separations of derivatized fatty acids were performed on two C₁₈ columns (Nova Pak C₁₈, 4 μm, 250×4.6 mm, Waters) by binary or ternate gradient programs and UV detection at 254 and 235 nm. The geometric and positional isomers of some unsaturated fatty acids were substantially retained on the C₁₈ columns and were distinct from some saturated fatty acids, endogenous substances in biological samples or background interference. Only slight separation of critical pairs of cis-9 C_18:1/cis-11 C_18:1 and cis-6 C_18:1/trans-11 C_18:1 was obtained. A ternate gradient program can be used for complete fractionation of a mixture of conjugated linoleic acid isomers (CLA) from cis-9, cis-12 and trans-9, trans-12 isomers of C_18:2. The CLA isomers in the effluent were monitored at 235 nm. The CLA isomers were differentiated from saturated and unsaturated fatty acids using a photodiode array detector. The utility of the method was demonstrated by evaluating the fatty acid composition of duodenal digesta, rapeseed and maize oils.     

Influence of network structure on the degradation of photo-cross-linked PLA-b-PEG-b-PLA hydrogels

Triblock copolymers of functionalized poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) (PLA-b-PEG-b-PLA) have been widely investigated as precursors for fabricating resorbable polymeric drug delivery vehicles and tissue engineering scaffolds. Previous studies show degradation and erosion behavior of PLA-b-PEG-b-PLA hydrogels to rely on macromer chemistry as well as structural characteristics of the cross-linked networks. In this research, the degradation kinetics of diacrylated PLA-b-PEG-b-PLA copolymers as soluble macromers and cross-linked gels are directly compared as a function of macromer concentration, buffer pH, and ionic strength. The pseudo first-order rate constants for degradation of soluble macromers increase with water concentration and show a minimum at intermediate pH values, but are insensitive to ionic strength. The degradation rate constants for covalently cross-linked gels display a greater sensitivity to local water concentration and a minimum at lower pH values than corresponding soluble macromers. In addition, ionic strength significantly affects the rate of gel degradation due to the direct correlation between the degree of network ionization and gel water content.     

A high salt tolerant neutral protease from Aspergillus Oryzae: Purification, characterization and kinetic properties

A neutral high salt tolerant protease from Aspergillus oryzae CICIM F0899 which could be used for soy sauce production and other relevant applications under high-salt conditions was purified to homogeneity through ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography with overall recovery of 2%. Its molecular weight was estimated to be 50 kDa by SDS-PAGE. The optimum pH and temperature for activity of the extracellular protease of A. oryzae CICIM F0899 were shown to be between 7.0–9.0, and 50°C, respectively. The protease behaved high salt tolerance in 18% NaCl and retained 72% of initial activity after 14 days, indicating the high stability. The enzyme activity was inhibited by metal ions such as Al³⁺ and Ag⁺, and slightly activated by Mn²⁺ and Cu²⁺. A kinetic model incorporating the Debye-Hückel limiting law was proposed for A. oryzae CICIM F0899 protease hydrolysis of casein at ionic strength NaCl from 0.10 to 3.18 M. It was found that, with the higher ionic strength, the Michaelis constant K _m of the protease monotonically increased while the turnover number k _cat decreased in accordance with first order kinetic model. The high-salt tolerant protease has been demonstrated to be promising for the soy sauce production process.     

Purification,properties, and mode of action of hemicellulase II produced by Ceratocystis paradoxa

An extra-cellular endo-hemicellulase (HC-II) from a culture isolate of the fungal plant pathogen Ceratocystis paradoxa (CP₂) was purified 147-fold by ammonium sulphate precipitation, DEAE-Sephadex chromatography, iso-electric focusing at pH 3–10, and gel-permeation chromatography. The resulting enzyme preparation, which contained traces of invertase, gave a single protein-band on disc electrophoresis at pH 8.4, and was active towards sucrose, hemicellulose, and carboxymethylcellulose (CMC). HC-II randomly degraded hemicelluloses from several different sources, to xylose and to arabinose-xylose and xylose oligosaccharides of d.p. 3–6 and 2–5, respectively, and also produced a degraded hemicellulose which precipitated from the digest solution. The precipitated hemicellulose contained less arabinose and uronic acid than the original hemicellulose. When redissolved by alkali-treatment, it was susceptible to further degradation by hemicellulases HC-I and HC-II. CMC was degraded by HC-II, mainly to D-glucose and cellobiose, with trace amounts of unidentified higher oligosaccharides, while cellobiose remained unattacked. Xylotriose (Xyl₃) was the lowest homologue of the xylose oligosaccharides attacked by HC-II at a significant rate, yielding xylobiose [Xyl₂; β-D-Xylp-(1→4)-D-Xyl] and xylose. AraXyl₃AraXyl₅ were mainly hydrolysed to AraXyl₂, xylose, and Xyl₂ or Xyl₃. HC-II had a temperature optimum of 80°, and was stable for 1 h at temperatures up to 70°. The pH optimum was 5.1, and HC-II was stable between pH 5–10. The K_m was 0.267 mg of hemicellulose B/ml. The effects of mercury(II) ions and high concentrations of xylose on the activity of HC-II were also investigated.     

Inversion of sucrose with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min^?1 g^?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydantoins

Eighteen side-chain-protected amino acids, routinely employed in solid-phase peptide synthesis, were derivatized to their phenylthiohydantoins (PTH) by one cycle of the Edman degradation. All of these side-chain-protected PTH amino acids elute, with almost-baseline resolution, in less than 18 min by high-performance liquid chromatography, utilizing a biphasic gradient of acetonitrile in 0.01 n sodium acetate, pH 4.5, or a linear gradient of 0 to 100% acetonitrile with the exception of the coelution of a O-benzyl-threonine and carbobenzoxy-lysine phenylthiohydantoin amino acids. The derivatized amino acids were subjected to reverse-phase chromatography on a Zorbax ODS column and monitored at 254 nm. None of the PTH amino acids coelute with side-chain-protected PTH amino acid counterparts, although PTH-tosyl-histidine undergoes deprotection to PTH-histidine in the Edman degradation. A protected decapeptide attached to a chloromethylated polystyrene resin was degraded on a solid-phase sequencer in 16 h. The PTH amino acids resulting from the automated Edman degradation on the decapeptide were fully resolved and quantified in less than 3 h demonstrating that automated high-performance liquid chromatography can keep pace with both the automated sequencer and synthesizer which requires minimally 2–3 h for attachment of each residue to the growing peptide chain.     

The sulphatase of ox liver XIX. On the nature of the polymeric forms of sulphatase a present in dilute solutions

Weight-average elution volumes of sulphatase A (an arysulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10²⁸ to 10²¹ M⁻³ on increasing the pH from 4.5 to 5.6 at ionic strength 0.1 at any particular pH value studied it was relatively insensitive to temperature variation so that ΔH° is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A.The equilibrium results are used to define the degree of association of sulphatase A likely to be encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species.The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negativity cooperative between the monomer units in the tetramer.In 2 mM sodium taurodeoxycholate and 0.035 M MnCl₂, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M MnCl₂ but the aggregation number is greater in the latter.     

Optimization of reaction conditions during enzyme immobilization on soluble carboxyl-containing carriers

The kinetics of 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide interaction with carboxylic groups in low and high molecular weight compounds have been investigated for choosing the optimum conditions for enzyme immobilization on carboxylic carriers. Optimum pH values depended on the pK_a of an acid. The ionic strength of the reaction mixture influenced the process of activation with carbodiimide and a decrease in reaction rate accompanied an increase in ionic strength. Optimum conditions for the coupling process with the use of carbodiimide activation are suggested. The process of fibrinolysin immobilization on the soluble copolymer of acrylic acid and acrylamide with carbodiimide as a coupling agent was also studied.