Xiaolei Hu

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.

Comparison of modification of a bacterial uricase with N‐hydroxysuccinimide esters of succinate and carbonate of monomethoxyl poly(ethylene glycol)

Uricase after modification with monomethoxy poly(ethylene glycol) (mPEG) is currently the sole agent to treat refractory gout. For formulating Bacillus fastidious uricase, succinimidyl carbonate of mPEG‐5000 (SC‐mPEG5k) and succinimidyl succinate of mPEG‐5000 (SS‐mPEG5k) were compared. SC‐mPEG5k possessed higher purity, comparable reaction rate constant with glycine but lower hydrolysis rate, and stronger effectiveness to modify amino groups. The uricase possessed two types of amino groups bearing a 25‐fold difference in reactivity with SC‐mPEG5k or SS‐mPEG5k at pH 9.2. Oxonate and xanthine concentration‐dependently protected the bacterial uricase from inactivation during PEGylation. With SC‐mPEG5k at a molar ratio of 200 to uricase subunits and oxonate of 50 µM, the PEGylated uricase (1) retained about 73% of the original activity, (2) displayed about 10% reactivity to rabbit anti‐sera recognizing the native uricase, (3) elicited IgG in rats accounting for about 5% of that by the native uricase, (4) exhibited circulation half‐life time of about 25 H in cock plasma in vivo, and (5) concurrently maintained uric acid at lowered levels for over 20 H. Hence, PEGylation with SC‐mPEG under the protection of a competitive inhibitor was a practical approach to formulation of the bacterial uricase; protection of enzymes by competitive inhibitors during PEGylation may have universal significance.

Fluorometric Titration Approach for Calibration of Quantity of Binding Site of Purified Monoclonal Antibody Recognizing Epitope/Hapten Nonfluorescent at 340 nm

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

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.

Selective and sensitive homogenous assay of serum albumin with 1-anilinonaphthalene-8-sulphonate as a biosensor.

Homogenous selective assay of albumin (ALB) in clinical sera was tested with 1-anilinonaphthalene-8-sulphonate (ANS) as Förster-resonance-energy-transfer (FRET) acceptor of tryptophan residues and biosensor of ALB. Between the excitation at 280 and 350 nm, the ratio of the fluorescence at 470 nm of free ANS in ethanol was about 1.9 while that of the complexes of ALB and ANS was about 3.9, supporting FRET in complexes of ANS and ALB. ANS below 1.0 mM saturated one site of ALB with Kd of about 0.13 μM in 20 mM sodium phosphate buffer at pH 7.0. For selective assay of ALB, 0.30 μM ANS was used to quantify fluorescence of the complexes at 470 nm under the excitation at 280 nm. ALB from 1.8 to 25 nM was quantified, whose lower limit was below 1% than that by bromocresol green assay while one-third than that by immunoturbidimetric assay. Globular proteins at comparable levels gave negligible signals. This new method showed reasonable resistance to other interfering substances in clinical sera. Quantities of ALB in clinical sera by this method were consistent with those by bromocresol green assay and immunoturbidimetric assay. Hence, homogenous assay of ALB with ANS as FRET biosensor was effective.

Achievement of linear response for competitive bioaffinity assays of ligands: criteria of optimized interaction systems

For a linear response in a competitive bioaffinity assay of a ligand, an optimized system requires CRT greater than 3-fold of CPT, CPT greater than 50-fold of KdR, and KdR greater than 260-fold of KdX, based on chemometrics for bioaffinity interactions (CRT and CPT are the concentrations of the probe and the biomacromolecule and KdX and KdR are the dissociation constants of complexes with the ligand and the probe, respectively). These criteria were tested for a competitive bioaffinity assay of biotin. The probe was a conjugate of monomethyl-poly-(ethylene glycol)-5000, 1-naphthyl-ethylenediamine and biotin. The complex of the probe with streptavidin was quantified by the fluorescence at 430 nm based on Forster resonance energy transfer with tryptophan residues as the intrinsic donors. By fluorometric titration, KdR of the probe was 5.4 ± 1.4 nM (n = 4). At 1.5 μM probe plus 0.50 μM streptavidin, there was a linear decrease of fluorescence at 430 nm for biotin concentrations ranging from ∼36 to ∼500 nM; the linear response slope was consistent with that for the fluorescence at 430 nm to the concentration of the complex of streptavidin and the probe. Biotin at 81 and 414 nM was estimated with variation coefficients below 7%. These proposed criteria may be universally applicable for linear responses in competitive assays of ligands.

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

Abstract 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.

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.