Gaobo Long

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 β-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, β-D-galactosidase releases 4-nitrophenol from β-D-(4-nitrophenyl)-galactoside with λ(B) at 405 nm. No interference from substrates/products made SDESA of β-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 activity indexes for recognizing enzyme mutants of higher activity with uricase as model

BackgroundFor 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.ResultsDuring 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.ConclusionSpecific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

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.

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

BackgroundA 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.ResultsNHS 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.ConclusionThe new method is favorable to quantify molar equivalents of NHS esters of mPEG derivatives and thus control quality of their preparations.

Two glycosidases as label enzymes for concurrent enzyme-linked-immunosorbent-assay of two components via spectrophotometric-dual-enzyme-simultaneous-assay in one solution

For concomitant enzyme-linked-immunosorbent-assay (ELISA) of two analytes of interest in one well of a microplate via spectrophotometric-dual-enzyme-simultaneous-assay in one solution (SDESA), β-D-galactosidase on 4-nitro-1-naphthyl-β-D-galactopyranoside (4NNPG) and α-D-glucosidase on 4-nitrophenyl-α-D-glucopyranoside (4NPG) were tested as labels with their consistent optimum buffer (sodium phosphate buffer at pH 7.4). Hydrolysis of 4NNPG yielded 4-nitro-1-naphthol exhibiting the longest absorbance peak of 458 nm and the longest isoabsorbance wavelength of 400 nm for absorptivity equal to that of 4NNPG; hydrolysis of 4NPG produced 4-nitrophenol with the longest absorbance peak of about 405 nm. For SDESA, two enzyme reactions were initiated concurrently; absorbance of two chromogenic products was concomitantly measured in one solution via swift alternation between 450 and 405 nm with a Biotek ELX 800 microplate reader, or between 450 nm and a wavelength close to 400 nm with a spectrophotometer; the overlapped absorbance of chromogenic substances was resolved based on the linear additivity of absorbance. For separate assay, just one substrate was used to detect the corresponding label enzyme. During SDESA, initial rates of two glycosidases tolerated negligible interference from the substances involved; the use of the isoabsorbance wavelength of 400 nm to measure absorbance reduced the limit of quantification (LOQ) of α-D-glucosidase. Under the same conditions, the LOQ of either of the enzymes via SDESA was consistent with that via separate assay when activities of the other enzyme were varied over the quantifiable range. By ELISA via SDESA with penicillin G and clenbuterol in one sample as two analytes of interest, the content, the coefficient of variation, the limit of detection, LOQ and the quantification range of either of the analytes were consistent with those via separate assay when quantities of the other analyte were varied over the quantifiable range, respectively. Hence, ELISA via SDESA with those two glycosidases as labels has great promise.

Facile quantitative comparison of specific activities of fusion-tagged enzyme/mutants in cell lysates via prediction of their maximum adsorption by anti-tag antibody immobilized in microplate wells

The maximum activities of fusion-tagged enzyme/mutants from cell lysates adsorbed by an anti-tag antibody immobilized in microplate wells were predicted to serve as equivalents of their specific activities for comparison with a six-histidine (6His)-tagged esterase and its tagged mutant as models. In brief, (a) a fixed quantity of a monoclonal anti-6His antibody was immobilized in microplate wells; (b) the maximum activity of a tagged enzyme/mutant from a cell lysate for saturation binding to the immobilized antibody (Vs) was predicted from the response of activities of the adsorbed tagged enzyme/mutant to quantities of total proteins in wells from the same lysate; and (c) Vs of tagged enzyme/mutants in lysates served as equivalents of their specific activities for comparison. Prediction of Vs of a tagged enzyme needed initial rates for absorbance changes of over 0.090 in 30 min, the highest occupancy of over 40% of binding sites of the immobilized antibody, and sufficient abundance of the tagged enzyme in lysates. With 0.6 μg antibody for immobilization in wells and total proteins of 10.0 to 128 μg from cell lysates, Vs of the tagged esterase had a coefficient of variation below 10% when the apparent specific activities in lysates varied more than four times. The ratio of Vs of the tagged esterase to the tagged mutant had higher precision and consistency with the ratio of their apparent specific activities from a large number of independent lysates. Hence, Vs predicted for tagged enzyme/mutants in cell lysates was suitable for comparison and may be applicable to verify positive mutants in a library.

Method to screen aromatic ligands in mixtures for quantitative affinities to target using magnetic separation of bound ligands along with HPLC and UV photometry detection

AbstractA new screening method was tested to estimate the affinity of an aromatic ligand in a mixture through competitive binding against an exogenous reference ligand. The target protein was immobilized on magnetic particles and one or several ligand(s) in each reaction mixture were prepared by parallel combinatorial-synthesis without purification. Specifically, the binding of aromatic biotin derivatives to streptavidin immobilized on magnetic particles was used as the model. An approximation equation that works under the condition of binding ratios below 0.1 for the candidate ligand and the reference ligand was developed. It was found that the relative affinity of each biotinylated aromatic candidate ligand in mixtures was consistent with that by using HPLC-MS analyses or by a homogenous method using its purified counterpart. Hence, this new screening method using HPLC-UV is considered to be highly effective. FigureThe representative procedure to realize the new screening technique for quantitative affinity of an aromatic candidate ligand of unknown quantity in a mixture sample

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

BackgroundThe 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.ResultsThe 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.ConclusionsThis 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.

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 Forster 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.