Optimization of pH values to formulate the bireagent kit for serum uric acid assay.

A new formulation of the bireagent kit for serum uric acid assay was developed based on the effects of pH on enzyme stability. At 4 °C, half-lives of uricases from Bacillus fastidious and Arthrobacter globiforms were longer than 15 months at pH 9.2, but became shorter at pH below 8.0; half-lives of ascorbate oxidase and peroxidase were comparable at pH 6.5 and 7.0, but became much shorter at pH higher than 7.4. In the new formulation of the bireagent kit, Reagent A contained peroxidase, 4-aminoantipyrine, and ascorbate oxidase in 50 mM phosphate buffer at pH 6.5; Reagent B contained B. fastidious or A. globiforms uricase in 50 mM sodium borate buffer at pH 9.2; Reagents A and B were mixed at 4:1 to produce a final pH from 7.2 to 7.6 for developing a stable color. The new bireagent kit consumed smaller quantities of three enzymes for the same shelf life. With the new bireagent kit, there were linear responses of absorbance at 546 nm to uric acid up to 34 mM in reaction mixtures and a good correlation of uric acid levels in clinical sera with those by a commercial kit, but stronger resistance to ascorbate. Therefore, the new formulation was advantageous.

Microplate-based method to screen inhibitors of isozymes of cyclic nucleotide phosphodiesterase fused to SUMO.

The feasibility for microplate-based screening of inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE) was tested via the coupled action of a phosphatase on adenosine-5'-monophosphate and an improved malachite green assay of phosphate. Human full-length PDE4B2 and truncated mutant (152-528aa) were expressed in Escherichia coli via fusion to SUMO, which after purification through Ni-NTA column exhibited specific activities >0.017 U mg(-1). In the presence of proteins <30 mg L(-1), absorbance for 10 µΜ phosphate was measurable; a PDE isozyme of specific activity over 0.008 U mg(-1) after reaction for 20 min thus suited for microplate-based screening of inhibitors. By using Biotek ELX 800 microplate reader, affinities of two forms of PEDE4B2 for cAMP, rolipram and papaverine varied over three magnitudes and were consistent with those by routine assay, respectively. Hence, the proposed method was promising for high-throughput-screening of inhibitors of phosphate-releasing enzymes bearing specific activities over 0.008 U mg(-1).

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution: chemometrics and experimental models.

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (λ(A)) much larger than that of Product B (λ(B)) formed by Enzyme B action on Substrate B; λ(B) is close to the longest isoabsorbance wavelength of Product A and Substrate A (λ(0)); (b) absorbance at λ(A) and λ(0) is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with γ-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. γ-Glutamyltransferase releases p-nitroaniline from γ-glutamyl-p-nitroaniline with λ(0) at 344 nm and λ(A) close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing λ(B) at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of γ-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and ß-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with λ(0) at 405 nm and λ(A) at 458 nm, ß-D-galactosidase releases 4-nitrophenol from ß-D-(4-nitrophenyl)-galactoside with λ(B) at 405 nm. No interference from substrates/products made SDESA of ß-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising.

Comparison of Förster-resonance-energy-transfer acceptors for tryptophan and tyrosine residues in native proteins as donors.

Homogenous bioaffinity analysis with tryptophan/tyrosine residues in native proteins as FÖrster-resonance-energy-transfer (FRET) donors is feasible when suitable fluorophors can act as FRET acceptors in ligands (FRET probes) and FRET efficiency in complexes of proteins and FRET probes is high enough. In complexes of proteins and FRET probes, suitable acceptors should have excitation peaks around 335 nm and high rotation freedom, are preferred to have sufficient quantum yields and excitation valleys around 280 nm. In protein binding sites mimicked with mixtures of neutral phosphate buffer and organic solvents, quantum yields of candidate acceptors are altered inconsistently but their excitation peaks show tiny changes. Fluorophores as acceptors in such FRET probes are buried inside glutathione-S-transferase and have low rotation freedom, but are localized on streptavidin surface and display high rotation freedom; FRET efficiency in complexes of streptavidin and its FRET probes is much stronger than that in complexes of glutathione-S-transferase and its FRET probes. Specially, the quantum yield is about 0.70 for free 1-naphthylamine probe in neutral phosphate buffer, about 0.50 for 1-naphthylamine probe bound by streptavidin, and about 0.15 for that bound by glutathione-S-transferase. The quantum yield is about 0.06 for free dansylamide probe, about 0.11 for dansylamide probe bound by streptavidin and about 0.27 for that bound by glutathione-S-transferase. Therefore, 1-naphthylamine and dansylamide are effective acceptors when they localize on surfaces of complexes of proteins and FRET probes.

Integration of kinetic analysis of reaction curve with a proper classical approach for enzymatic analysis.

For enzymatic analysis to quantify a substrate or enzyme, kinetic analysis of reaction curve can be integrated with a proper classical approach. For their integration, they should have consistent slopes and intercepts of linear response and an overlapped region of analyte quantities measurable under optimized conditions. To quantify a substrate after optimizations of tool enzyme activity and reaction duration, the equilibrium method works when the reaction is completed within the reaction duration; otherwise, kinetic analysis of reaction curve applies providing at least seven data with sufficient consumption of substrate. To quantify an enzyme after optimizations of initial substrate concentration and reaction duration, the classical initial rate method works when an estimated initial rate locates within the linear range; otherwise, kinetic analysis of reaction curve applies after the conversion of the quantification index with optimized parameters. This integration strategy has ideal linear ranges and practical efficiency for quantifying an enzyme at moderate substrate levels and for quantifying a substrate at moderate cost on tool enzyme; it has promise to simultaneous assays of multiple enzymes in one reaction vessel each time and ,thus, potential applications to concurrently quantify multiple serum enzymes, screen inhibitors against multiple enzyme targets, and detect multiple serum components by enzymeimmunoassay.

Estimation of affinities of ligands in mixtures via magnetic recovery of target-ligand complexes and chromatographic analyses: chemometrics and an experimental model.

BACKGROUND: The combinatorial library strategy of using multiple candidate ligands in mixtures as library members is ideal in terms of cost and efficiency, but needs special screening methods to estimate the affinities of candidate ligands in such mixtures. Herein, a new method to screen candidate ligands present in unknown molar quantities in mixtures was investigated. RESULTS: The proposed method involves preparing a processed-mixture-for-screening (PMFS) with each mixture sample and an exogenous reference ligand, initiating competitive binding among ligands from the PMFS to a target immobilized on magnetic particles, recovering target-ligand complexes in equilibrium by magnetic force, extracting and concentrating bound ligands, and analyzing ligands in the PMFS and the concentrated extract by chromatography. The relative affinity of each candidate ligand to its reference ligand is estimated via an approximation equation assuming (a) the candidate ligand and its reference ligand bind to the same site(s) on the target, (b) their chromatographic peak areas are over five times their intercepts of linear response but within their linear ranges, (c) their binding ratios are below 10%. These prerequisites are met by optimizing primarily the quantity of the target used and the PMFS composition ratio.The new method was tested using the competitive binding of biotin derivatives from mixtures to streptavidin immobilized on magnetic particles as a model. Each mixture sample containing a limited number of candidate biotin derivatives with moderate differences in their molar quantities were prepared via parallel-combinatorial-synthesis (PCS) without purification, or via the pooling of individual compounds. Some purified biotin derivatives were used as reference ligands. This method showed resistance to variations in chromatographic quantification sensitivity and concentration ratios; optimized conditions to validate the approximation equation could be applied to different mixture samples. Relative affinities of candidate biotin derivatives with unknown molar quantities in each mixture sample were consistent with those estimated by a homogenous method using their purified counterparts as samples. CONCLUSIONS: This new method is robust and effective for each mixture possessing a limited number of candidate ligands whose molar quantities have moderate differences, and its integration with PCS has promise to routinely practice the mixture-based library strategy.

Ampholytic ion-exchange materials coated with small zwitterions for high-efficacy purification of ionizable soluble biomacromolecules.

For the purification of soluble proteins and nucleic acids through ion-exchange, the ampholytic ion-exchange materials (AIEMs) were designed, which possessed both short aliphatic carboxyl and short aliphatic amines/imidazole at optimized ratios on solid supports coated with high density of small zwitterions; under optimized conditions, the soluble ionizable biomacromolecules were adsorbed on those AIEMs via electrostatic attractions and eluted effectively through electrostatic repulsions. As the proof-of-concept, magnetic submicron particles bearing short aliphatic carboxyl and the coats of small zwitterion served as the starting solid supports, which were conjugated with lysine alone, or with lysine plus glycine or N,N­dimethylethylenediamine, to yield magnetic AIEMs whose surfaces possessed zero net charges at different pH. Such magnetic AIEMs exhibited ideal efficacy to release acid red 13 as an anion at the elution pH optimized for strong electrostatic repulsions; those magnetic AIEMs were proven absorbing under optimized conditions for the purification of soluble proteins stable at pH close to their isoelectric points and solid-phase extraction of nucleic acids in applicable biological mixtures. Therefore, the designed AIEMs are promising for the high-efficacy purification of ionizable soluble biomacromolecules.

Data for ampholytic ion-exchange materials coated with small zwitterions for high-efficacy purification of ionizable soluble biomacromolecules.

Data in this article are associated with the research article "Ampholytic ion-exchange materials coated with small zwitterion for high-efficacy purification of ionizable soluble biomacromolecules" (Rao et al., 2018) [1]. This article provided data on how to design ampholytic ion-exchange material (AIEM) for the purification of ionizable soluble biomacromolecules for both high activity yields and favorable homogeneity, with two uricases as protein models and a plasmid as DNA model. Data were made publicly available for further analyses.

Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities.

By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.

Facile characterization of the immobilization of streptavidin on magnetic submicron particles with a fluorescent probe of streptavidin.

To characterize streptavidin immobilization on magnetic submicron particles (MSPs), residual streptavidin after magnetic removal of immobilized streptavidin was quantified with N-biotinyl-N'-(1-naphthyl)-ethylenediamine (BNEDA) based on Förster resonance energy transfer. Residual BNEDA after magnetic removal of bound BNEDA was measured by its own fluorescence. Streptavidin was immobilized at about 12 mg per gram of MSPs and easily retained over 50% of its original activity. These assays facilitated optimized streptavidin immobilization on MSPs.

Facile one-step coating approach to magnetic submicron particles with poly(ethylene glycol) coats and abundant accessible carboxyl groups.

PURPOSE: Magnetic submicron particles (MSPs) are pivotal biomaterials for magnetic separations in bioanalyses, but their preparation remains a technical challenge. In this report, a facile one-step coating approach to MSPs suitable for magnetic separations was investigated. METHODS: Polyethylene glycol) (PEG) was derived into PEG-bis-(maleic monoester) and maleic monoester-PEG-succinic monoester as the monomers. Magnetofluids were prepared via chemical co-precipitation and dispersion with the monomers. MSPs were prepared via one-step coating of magnetofluids in a water-in-oil microemulsion system of aerosol-OT and heptane by radical co-polymerization of such monomers. RESULTS: The resulting MSPs contained abundant carboxyl groups, exhibited negligible nonspecific adsorption of common substances and excellent suspension stability, appeared as irregular particles by electronic microscopy, and had submicron sizes of broad distribution by laser scattering. Saturation magnetizations and average particle sizes were affected mainly by the quantities of monomers used for coating magnetofluids, and steric hindrance around carboxyl groups was alleviated by the use of longer monomers of one polymerizable bond for coating. After optimizations, MSPs bearing saturation magnetizations over 46 emu/g, average sizes of 0.32 µm, and titrated carboxyl groups of about 0.21 mmol/g were obtained. After the activation of carboxyl groups on MSPs into N-hydroxysuccinimide ester, biotin was immobilized on MSPs and the resulting biotin-functionalized MSPs isolated the conjugate of streptavidin and alkaline phosphatase at about 2.1 mg/g MSPs; streptavidin was immobilized at about 10 mg/g MSPs and retained 81% ± 18% (n = 5) of the specific activity of the free form. CONCLUSION: The facile approach effectively prepares MSPs for magnetic separations.

Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model.

BACKGROUND: For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity. RESULTS: During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity. CONCLUSION: Specific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

Resonant-Mie-scattering of aggregates of phosphomolybdate and papaverine for measuring activities and screening inhibitors of cyclic nucleotide phosphodiesterase isozymes.

A resonant-light-scattering (RLS) method was proposed to quantify phosphate for screening inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE). In acidified mixtures of phosphate, papaverine and molybdate, there were aggregates exhibiting micrometre sizes, no absorbance peaks over 360 nm but strong RLS peaks at 392 nm; Mie scattering thus accounted for the RLS signals. When papaverine was added before molybdate to acidified samples of phosphate, RLS signals at 392 nm were stable from 5 to 25 min since the addition of molybdate; after optimization, phosphate from 0.40 to 3.60 µM was quantifiable. This RLS method tolerated 60 mg L(-1) proteins besides common PDE inhibitors and dimethyl sulfoxide in acidified samples of phosphate; the integration of this RLS method with the coupled action of a phosphomonoesterase on PDE product was thus rational to measure PDE activities without the removal of proteins in samples. By quantifying activities of a truncated mutant of human PDE4B2 via this RLS method, Michaelis-Menten constant, inhibition constants of rolipram, papaverine and theophylline varied over three magnitudes and were consistent with those estimated by an improved malachite green assay of phosphate, respectively. Hence, this novel RLS method was promising for screening inhibitors of PDE isozymes.

Facile spectrophotometric assay of molar equivalents of N-hydroxysuccinimide esters of monomethoxyl poly-(ethylene glycol) derivatives.

BACKGROUND: A new method is developed to quantify molar equivalents of N-hydroxysuccinimide (NHS) esters of derivatives of monomethoxyl poly-(ethylene glycol) (mPEG) in their preparations with NHS acetate ester as the reference. RESULTS: NHS ester of succinic monoester or carbonate of mPEG of 5,000 Da was synthesized and reacted with excessive ethanolamine in dimethylformamide at 25°C for 15 min. Residual ethanolamine was subsequently quantified by absorbance at 420 nm after reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS) at pH 9.2 for 15 min at 55°C followed by cooling with tap water. Reaction products of ethanolamine and NHS esters of mPEG caused no interference with TNBS assay of residual ethanolamine. Reaction between ethanolamine and NHS acetate ester follows 1:1 stoichiometry. By the new method, molar equivalents of NHS esters of carbonate and succinic monoester of mPEG in their preparations were about 90% and 60% of their theoretical values, respectively. During storage at 37°C in humid air, the new method detected spontaneous hydrolyses of the two NHS esters of mPEG more sensitively than the classical spectrophotometric method based on absorbance at 260 nm of NHS released by reaction with ammonia in aqueous solution. CONCLUSION: The new method is favorable to quantify molar equivalents of NHS esters of mPEG derivatives and thus control quality of their preparations.