Measurement Of Residual Activity Research Articles

Digital Microfluidics in Newborn Screening for Mucopolysaccharidoses: A Progress Report.

Newborn screening (NBS) for mucopolysaccharidosis type I (MPS I, Hurler syndrome) is currently conducted in about two-fifths of the NBS programs in the United States and in a few other countries. Screening is performed by measurement of residual activity of the enzyme alpha-l-iduronidase in dried blood spots using either tandem mass spectrometry or digital microfluidic fluorometry (DMF). In this article, we focus on the development and practical experience of using DMF to screen for MPS I in the USA. By means of their responses to a questionnaire, we determined for each responding program that is screening for MPS I using DMF the screen positive rate, follow-up methods, and classification of confirmed cases as either severe or attenuated. Overall, the results show that at the time of reporting, over 1.3 million newborns in the US were screened for MPS I using DMF, 2094 (0.173%) of whom were screen positive. Of these, severe MPS I was confirmed in five cases, attenuated MPS I was confirmed in two cases, and undetermined phenotype was reported in one case. We conclude that DMF is an effective and economical method to screen for MPS I and recommend second-tier testing owing to high screen positive rates. Preliminary results of NBS for MPS II and MPS III using DMF are discussed.

Structural changes associated with the inactivation of lipoxygenase by pulsed light

Pulsed light (PL) is a non-thermal technology able to inactivate enzymes. Lipoxygenase (LOX) causes enzymatic rancidity in some foods. This study aimed to determine the structural changes associated with the PL inactivation of LOX and used measurements of residual activity, temperature, spectropolarimetry, fluorescence, spectrophotometry, free sulfhydryl and carbonyl contents and electrophoresis. LOX inactivation was non-log-linear, with a slight temperature rise. PL significantly increased the concentration of carbonlys and free-sulfhydryls of LOX and caused loss of ellipticity and intrinsic fluorescence, a red shift in the intrinsic fluorescence peak and an increase in 1-anilino-8-naphthalenesulfonate fluorescence. The turbidity of LOX sample increased during inactivation and electrophoretic bands faded. Phase diagram analysis showed no evidence of formation of intermediates. In summary, the inactivation of LOX by PL followed a Weibull kinetics, is exclusively photochemical and is an all-or-none process where the protein gets oxidized, decreases its α-helix content, unfolds and aggregates.

Determinations of 171Er half-life and some 171Tm transition energies

The neutrons have been captured by Erbium nuclei which were received by using clinical electron linear accelerator. In this experiment, the possibility of the neutron capture process has been observed because of emitted neutrons appearing in the experimental area. In particular, neutron capture of 170Er nucleus has been observed. After the neutron capture of 170Er nucleus, the unstable 171Er has been formed and decayed into the 171Tm. By using this reaction path, some transition energies of 171Tm obtained from the residual activity measurements and the half-life of 171Er have been determined, and they are in agreement with adopted values in the literature.

Catalyst deactivation by coke formation in microporous and desilicated zeolite H-ZSM-5 during the conversion of methanol to hydrocarbons

Desilication has become a standard method to prepare mesoporous zeolite catalysts that display improved lifetime or resistance toward deactivation in hydrocarbon transformation processes involving coking. In this contribution, the deactivation behaviors of a regular, commercial ZSM-5 catalyst and its mesoporous, desilicated counterpart have been investigated and compared. After partial deactivation in the conversion of methanol to hydrocarbons at elevated reaction pressure, the catalyst beds were fractionated from top to bottom of the reactor. These fractions were analyzed by several spectroscopic techniques, gas adsorption measurements, thermogravimetry, coke analysis by a catalyst dissolution/extraction protocol, and measurements of residual activity for the individual catalyst fractions. Remarkable differences between the desilicated and the parent H-ZSM-5 catalysts are seen, in particular with respect to the mode of deactivation along the catalyst beds. It appears that the more uniform deactivation of the desilicated catalyst is due to a complex interplay among alterations of porosity, activity, and rate of deactivation upon desilication.

N-terminal Purification Tag Alters Thermal Stability of the Carboxylesterase EstGtA2 from G. thermodenitrificans by Impairing Reversibility of Thermal Unfolding

The novel thermostable carboxylesterase EstGtA2 from G. thermodenitrificans (accession no. AEN92268) was functionally expressed and purified using an N-terminal fusion tag peptide. We recently reported general properties of the recombinant enzyme. Here we report preliminary data on thermal stability of EstGtA2 and of its tagged form. Conformational stability was investigated using circular dichroism and correlated with residual activity measurements using a colorimetric assay. The tag peptide had no considerable impact on the apparent melting temperature: T(m) value = 64.8°C (tagged) and 65.7°C (cleaved) at pH 8. After thermal unfolding, the tag-free enzyme rapidly recovered initial activity at 25°C (1.2 Umg(-1)), which was corroborated by substantial refolding (83%) as determined by far-UV CD transitions. However, after thermal unfolding, the purification tag drastically decreased specific activity at 25°C (0.07 Umg(-1)). This was corroborated by the absence of refolding transition. Although the purification tag has no undesirable impact on activity before thermal unfolding as well as on Tm, it drastically hinders EstGtA2 refolding resulting in a major loss of thermal stability.

Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms

In response to stressful conditions like supra-optimal salinity in the growth medium or temperature, many microorganisms accumulate low-molecular-mass organic compounds known as compatible solutes. In contrast with mesophiles that accumulate neutral or zwitterionic compounds, the solutes of hyperthermophiles are typically negatively charged. (2 R)-2-(α- d-Mannopyranosyl)glycerate (herein abbreviated as mannosylglycerate) is one of the most widespread solutes among thermophilic and hyperthermophilic prokaryotes. In this work, several molecules chemically related to mannosylglycerate were synthesized, namely (2 S)-2-(1- O-α- d-mannopyranosyl)propionate, 2-(1- O-α- d-mannopyranosyl)acetate, (2 R)-2-(1- O-α- d-glucopyranosyl)glycerate and 1- O-(2-glyceryl)-α- d-mannopyranoside. The effectiveness of the newly synthesized compounds for the protection of model enzymes against heat-induced denaturation, aggregation and inactivation was evaluated, using differential scanning calorimetry, light scattering and measurements of residual activity. For comparison, the protection induced by natural compatible solutes, either neutral (e.g., trehalose, glycerol, ectoine) or negatively charged (di- myo-inositol-1,3′-phosphate and diglycerol phosphate), was assessed. Phosphate, sulfate, acetate and KCl were also included in the assays to rank the solutes and new compounds in the Hofmeister series. The data demonstrate the superiority of charged organic solutes as thermo-stabilizers of enzymes and strongly support the view that the extent of protein stabilization rendered by those solutes depends clearly on the specific solute/enzyme examined. The relevance of these findings to our knowledge on the mode of action of charged solutes is discussed.

Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability

Fungal cellobiohydrolases from the glycosyl hydrolase family 7 are key enzymes in crystalline cellulose hydrolysis. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these cellulases for industrial application. We report here a successful expression of the single-module cellobiohydrolase Cel7B from a thermophilic fungus Melanocarpus albomyces in Saccharomyces cerevisiae (Sc Cel7B). An automated, robotic thermostability screening method, based on residual activity measurements on a small soluble substrate methylumbelliferyl-lactoside (MULac), was then set-up to screen the first generation random mutant libraries. Out of the nine positive thermostable mutants, we picked three based on structural considerations, each containing a single amino acid change (A30T, G184D or S290T). Cel7B A30T and S290T mutants showed improved unfolding temperature ( T m) by 1.5 and 3.5 °C, respectively. In addition, the temperature optimum ( T opt) on a soluble substrate had improved by 5 °C for the A30T mutant. Interestingly, the best enzyme variant on microcrystalline cellulose (Avicel) hydrolysis was the Cel7B S290T, which could hydrolyse Avicel at 70 °C two times more effectively than the Sc Cel7B. Overall the consensus mutation S290T, located in the hydrophobic core of Cel7B, led to a cellobiohydrolase variant having also application potential in hydrolysis of polymeric substrates at elevated temperatures.

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.

Experimental studies on excitation functions of the proton-induced activation reactions on silver

Excitation functions were measured for the production of 106m,105Ag, 103,101,100Pd, 105,102,101m,100,99Rh and 97Ru via proton-induced activation reactions on natural silver using a stacked foil technique in the energy range 11-80 MeV. The residual activity measurements were carried out nondestructively by the high-resolution HPGe γ-ray spectroscopy. Thick target integral yields were deduced using the measured cross-sections from the respective threshold energies of the investigated reactions up to 80 MeV. The present work gives new results for the investigated radionuclides. The data in MENDL-2P deduced with the theoretical model code ALICE-IPPE are consistent in shape with the measured values, but show disagreement in magnitude.

Characterization of mutant xylanases using fourier transform ion cyclotron resonance mass spectrometry: stabilizing contributions of disulfide bridges and N-terminal extensions.

Structural properties and thermal stability of Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) and its three recombinant mutants were characterized using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry and hydrogen/deuterium (H/D) exchange reactions. TRX II has been previously stabilized by a disulfide bridge C110-C154 and other site-directed mutations (TRX II mutants DS2 and DS5). Very recently, a highly thermostable mutant was introduced by combining mutations of DS5 with an N-terminal disulfide bridge C2-C28 (mutant DB1). Accurate mass measurements of TRX II, DS2, DS5, and DB1 verified the expected DNA-encoded protein sequences (average mass error 1.3 ppm) and allowed unequivocal assignment of the disulfides without chemical reduction and subsequent alkylation of the expected cross-links. Moreover, H/D exchange reactions provided means for the detection of a major heat-induced conformational change comprising two interconverting conformers of very different H/D exchange rates as well as allowed the apparent melting temperatures (T(m)) to be determined (62.6, 65.1, 68.0, and 82.2 degrees C for TRX II, DS2, DS5, and DB1, respectively). Residual activity measurements verified that the enzymes inactivated at significantly lower temperatures than expected on the basis of the apparent T(m) values, strongly suggesting that the inactivation takes place through minor conformational change other than observed by H/D exchange. ESI FT-ICR analyses also revealed molecular heterogeneity in DS5 and DB1 due to the propeptide incorporation. Resulting unintentional N-terminal extensions were observed to further improve the stability of the DB1 mutant. The extension of six amino acid residues upstream from the protein N-terminus increased stability by approximately 5 degrees C.

Multi-step analysis of Hg2+ ion inhibition of jack bean urease

We performed a multi-step analysis of the inhibition of jack bean urease by Hg2+ ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg2+-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg2+, progress curve analysis, and spectroscopic assay of thiol groups in urease–Hg2+ complexes with a cysteine selective agent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). Hg2+ ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg2+ inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg2+ of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg2+ inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant Ki=1.9 nM (±10%).

Multi-step analysis of Hg 2+ ion inhibition of jack bean urease

We performed a multi-step analysis of the inhibition of jack bean urease by Hg 2+ ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg 2+-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg 2+, progress curve analysis, and spectroscopic assay of thiol groups in urease–Hg 2+ complexes with a cysteine selective agent 5,5 ′-dithiobis(2-nitrobenzoic acid) (DTNB). Hg 2+ ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg 2+ inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg 2+ of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg 2+ inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant K i=1.9 nM (±10%).

A general model for genetic regulation of turnover of glycosaminoglycans suggests a possible procedure for prediction of severity and clinical progress of mucopolysaccharidoses

Mucopolysaccharidoses are rare genetic diseases from the group of lysosomal storage disorders caused by deficiency of enzymes involved in degradation of mucopolysaccharides (glycosaminoglycans, GAGs). Within each mucopolysaccharidosis, there is a continuous spectrum of clinical features from the very severe to the more mildly affected individuals. Surprisingly, in most cases, it is not possible to predict severity and clinical progress (i.e., the natural history) of the disease on the basis of detection of particular mutations or residual activity of the deficient enzyme. In this article, the reasons for such an unexpected difficulty are discussed. A model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been proposed previously by others, however, in that model it was assumed that substrate concentration in the lysosome is not regulated, thus the residual activity of a hydrolase would be the only determinant of the rate of substrate accumulation. On the other hand, both a general model for genetic regulation of turnover of GAGs and results of very recent studies strongly suggest that expression of genes coding for enzymes involved in GAG synthesis is precisely regulated and may vary between individuals. Therefore, we propose that apart from measurement of residual activity of the enzyme involved in degradation of GAGs, the efficiency of synthesis of these compounds should also be estimated. If the hypothesis presented in this article is true, the ratio of the synthesis of glycosaminoglycans to the residual activity of the deficient enzyme should be of considerable prognostic value.

Ionically Bound Peroxidase from Peach Fruit

Soluble, ionically bound peroxidase (POD) and polyphenoloxidase (PPO) were extracted from the pulp of peach fruit during ripening at 20°C. Ionically bound form was purified 6.1-fold by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme showed only one peak of activity on Sephadex G-100 and PAGE revealed that the enzyme was purified by the procedures adopted. The purified enzyme showed a molecular weight of 29000 Da, maximum activity at pH 5.0 and at 40ºC. The calculated apparent activation energy (Ea) for the reaction was10.04 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 75ºC with a fast inactivation at 75ºC. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (0, 10, 20 %, w/w), with an activation energy (Ea) for inactivation increasing with sucrose concentration from 0 to 20% (w/w). The Km and Vmax values were 9.35 and 15.38 mM for 0-dianisidine and H2O2, respectively. The bound enzyme was inhibited competitively by ferulic, caffeic and protocatechuic acids with different values of Ki,. L-cysteine, p-coumaric and indolacetic acid and Fe++ also inhibited the enzyme but at a lower grade. N-ethylmaleimide and p-CMB were not effective to inhibit the enzyme demonstrating the non-essentiality of SH groups.

Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand d-tubocurarine enhances the affinity for small methanesulfonylfluoride

d-Tubocurarine, a reversible peripheral inhibitor of cholinesterases accelerates methanesulfonylation of Drosophila melanogaster wild type and W359L mutant. The kinetic evaluation of the process was performed in a step-by-step analysis. The second order overall sulfonylation rate constants, determined from classical residual activity measurements, were used in the subsequent analysis of progress curves. The latter were obtained by measuring the hydrolysis of acetylthiocholine in a complex reaction system of enzyme, substrate, irreversible and reversible inhibitor. The underlying kinetic mechanisms, from such a complex data, could only be untangled by targeted inspection and successive incorporation of reaction steps for which experimental evidence existed. The study showed that the peripheral ligand d-tubocurarine, by binding at the entrance into the active site of the two investigated enzymes (Goličnik et al., Biochemistry 40 (2001) 1214), enhances the affinity for small methanesulfonylfluoride, rather to speeding up the formation of a stable covalent enzyme-inhibitor complex. The specific arrangements at the rim of the active site of each individual enzyme dictate the actual events which can be detected by kinetic means.

Analysis of ore samples employing photon activation of the metastable states of gold and silver

The analytical method described here uses inelastic gamma scattering to produce 197 m Au(T 1/2=7.8 s), 107 m Ag(T 1/2=44.2 s) and 109 m Ag(T 1/2=39.8 s) with the subsequent measurement of residual activity. Bremsstrahlung radiation from linear electron accelerator with maximum γ quanta of 6 to 9 MeV is optimum for this purpose. Induced activity from gold and silver was measured with a HPGe. To obtain maximum sensitivity for detection, it is advantageous to measure both gamma and X-ray activity. Possible interferences were studied in detail, using the pure element spectra and also literature data. The most evident advantage of the method is its rapidity and relatively low cost of analysis.

Uncertainties of DS86 and prospects for residual radioactivity measurement.

Residual radioactivity data of 152Eu, 60Co and 36Cl have been accumulated and it has been revealed in the thermal neutron region that a systematic discrepancy exists between the measured data and activation calculation based on the DS86 neutrons in Hiroshima. Recently 63Ni produced in copper samples by the fast neutron reaction 63Cu(n,p)63Ni has been of interest for evaluation of fast neutrons. Reevaluation of atomic-bomb neutrons and prospects based on residual activity measurements have been discussed.

Peroxidase from peach fruit: thermal Stability

Peroxidase from peach fruit was purified 28.9-fold by DEAE-cellulose, Sephadex G-100 and hydroxylapatite chromatography. The purified enzyme showed only one peak of activity with an optimum pH of 5.0 and temperature of 40ºC. The calculated activation energy (Ea) for the reaction was 7.97 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 80ºC with a fast inactivation at 80ºC. PAGE of the inactivation course at 70ºC showed only one band of activity. Different sugars increased the heat stability of the activity in the following order: sucrose>lactose>glucose>fructose. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (10 to 40%, w/w) with the Ea for inactivation increasing with sucrose concentration from 0 to 20% (w/w). After inactivation at 70ºC and 75ºC the enzyme was able to be reactivated by up to 40% of the initial activity when stored at 30ºC.

Species specificity of anticoagulant activity of activated human protein C. Involvement of factor V as well as protein S

Activated protein C (APC) possesses species specificity in its anticoagulant activity. Human APC exerts only weak activity in rat plasma compared with that in human plasma. The present study was undertaken to estimate the difference in interaction of human and rat factors with human APC and to assess the cause of the species specificity. Human or rat protein S (PS), factor V, or factor VIII was used to supplement human plasma depleted of each respective factor, and the anticoagulant activity of human APC was measured in term of the elongation of activated partial thromboplastin time (APTT). The activity of human APC in rat PS- or factor V-supplemented plasma was weaker than that in the human PS- or factor V-supplemented plasma. Furthermore, using purified human and rat factor V, human APC showed weaker inactivation of rat factor V than human factor V. Equal anticoagulant activity was observed in human or rat factor VIII-supplemented plasma. And there was a little difference in the interaction of APC with its inhibitors in human or rat plasma during a few minutes of incubation as judged by measurement of residual activity by an enzyme capture assay. From these results factor V as well as PS seems to play a major role in the species specificity of APC.

An International Collaborative Study to Investigate Standardisation of Hirudin Potency

An international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of alpha-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard alpha-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200-222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15-15.97%. This corresponds to specific activities of approximately 14,300-15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000-16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard alpha-thrombin by all laboratories. This study has important implications for hirudin standardisation.