Acylation is rate-limiting in glycosylasparaginase-catalyzed hydrolysis of N4-(4'-substituted phenyl)-L-asparagines

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between N-acetyl-D-glucosamine and L-asparagine in the catabolism of glycoproteins. The mechanism has been proposed to resemble that of serine proteases involving an acylation step where a nucleophilic attack by a catalytic Thr residue on the carbonyl carbon of the N-glycosylic bond gives rise to a covalent beta-aspartyl-enzyme intermediate, and a deacylation step to give the final products. The question posed in this study was: Is the acylation step the rate-limiting step in the hydrolysis reaction as in serine proteases? To answer this question a series of mostly new substituted anilides was synthesized and characterized, and their hydrolysis reactions catalyzed by glycosylasparaginase from human amniotic fluid were studied. Five N4-(4'-substituted phenyl)-L-asparagine compounds were synthesized and characterized: 4'-hydrogen, 4'-ethyl, 4'-bromo, 4'-nitro, and 4'-methoxy. Each of these anilides was a substrate for the enzyme. Hammett plots of the kinetic parameters showed that acylation is the rate-limiting step in the reaction and that upon binding the electron distribution of the substrate is perturbed toward the transition state. This is the first direct evidence that acylation is the rate-limiting step in the enzyme-catalyzed reaction. A Br?nsted plot indicates a small, negative charge (-0.25) on the nitrogen atom of the leaving group anilines containing electron-withdrawing groups, and a small, positive charge (0.43) on the nitrogen atom of the leaving group anilines containing electron-donating groups. The free energy (incremental) change of binding (delta deltaGb) in the enzyme-substrate transition state complexes shows that substitution of a substituted phenyl group for the pyranosyl group in the natural substrate results in an overall loss of binding energy equivalent to a weak hydrogen bond, the magnitude of which is dependent on the substituent group. The data are consistent with a mechanism for glycosylasparaginase involving rapid formation of a tetrahedral structure upon substrate binding, and a rate-limiting breakdown of the tetrahedral structure to a covalent beta-aspartyl-enzyme intermediate that is dependent on the electronic properties of the substituent group and on the degree of protonation of the leaving group in the transition state by a general acid.     

Retention,direct detection,and quantitation of palladium (II) using high-performance liquid chromatography and evaporative light scattering detection

This study demonstrates the use of high-performance liquid chromatography and evaporative light scattering detection for the direct detection and quantitation of palladium II. After evaluating the effects of buffer concentration and pH, the separation of cobalt II, copper I, copper II, nickel II, and palladium II was accomplished using a Chromolith® Performance SI monolithic column with a hydrophilic interaction chromatography mode gradient elution. Typical validation parameters were evaluated to assess the method’s quantitative performance for palladium II which included specificity, accuracy, precision, linearity, stability, and limit of detection. This technique provides a unique and practical alternative method for the accurate quantitation of palladium II.     

Determination of sulfate and chloride ions in highly saline oilfield water by capillary electrophoresis using bilayer-coated capillaries and indirect absorption detection

Analysis of highly saline oilfield waters for anions presents challenges. Traditional analytical techniques used for such analysis tend to suffer from both poor sensitivity and selectivity due to the high concentrations of salt present in the samples. A capillary electrophoresis method was developed for the simultaneous determination of chloride and sulfate anions which is relevant to the oilfield analysis industry and of economic value. Due to the extremely high concentrations of chloride in highly saline oilfield waters, it is difficult to achieve baseline electrophoretic separation necessary for accurate quantitation. By using a capillary with a noncovalently bound bilayer coating using Polybrene, a cationic polymer and sodium dodecyl sulfate (SDS), an anionic surfactant and a buffer consisting of 50 mM TRIS, 30 mM SDS, 5% methanol and 26 mM chromium trioxide (CrO₃) at pH 6.7, baseline separation (R_s > 1.5) of chloride and sulfate was achieved. To mimic possible oilfield water samples, model water solutions of 5%, 10%, 15% and 20% chloride containing low ppm sulfate were prepared and successfully analysed using the method developed. In addition, the method was applied to determine chloride and sulfate anions in highly saline oilfield water samples. The accuracy of the method developed was verified by analysing NIST certified standards of chloride and sulfate. The results obtained for chloride and sulfate with the indirect CE-UV method were in close agreement (94–100% accuracy; <2.5% relative standard deviations) with those of the certified standard analysed by ion chromatography.     

Ion-molecule reactions of 2-methoxyethanol with some amines, carboxylic acids and amino acids under chemical ionization conditions

2-Methoxyethanol chemical ionization of amines, carboxylic acids and amino acids has been found to produce numerous adduct ions. The most intense adduct ions for amines are [M + H](+) and [M + 77](+), for carboxylic acids [M + 27](+), [M + 59](+) and [M + 77](+), and for amino acids [M + H](+), [M + 13](+), [M + 27](+) and [M + 77](+). Either the adduct ion [M + H](+) or [M + 77](+) was the most abundant ion found for amino acids. The proton affinities of amino acids are noticed to control the formation of the [M + H](+) and [M + 77](+) ions. The relative abundance of [M + 13](+) and [M + 27](+) ions varied for different amino acids being most intense for phenylalanine and aspartic acid. Copyright 1999 John Wiley & Sons, Ltd.     

Status of electroweak phase transition and baryogenesis

I review recent progress on the electroweak phase transition and baryogenesis, focusing on the minimal supersymmetric Standard Model as the source of new physics.