For a linear response, an optimized competitive bioaffinity assay of a ligand requiresCRT> 3 ×CPT,CPT> 50 ×KdR, andKdR> 260 ×KdX(CRTandCPTare concentrations of the probe and protein whileKdXandKdRareKdfor the ligand and probe, respectively).

The screening of ligands in mixtures through magnetic recovery of target-ligand complexes needs site-specific immobilization of 6His-tagged targets on magnetic-submicron-particle. Ni2+-NTA-functionalized magnetic-subnnicron-particle is more readily-available than bis-sulfone-functionalized one. The noncovalent conjugates of 6His-tagged targets and Ni2+-NTA-functionalized magnetic-submicron-particle are stable at pH 8.0, but ligand affinities at pH 8.0 may be different from those at pH 7.4. Of full-length human cyclic nucleotide phosphodiesterase 4B2, the inhibition potency of tested neutral compounds was indeed different at pH 7.4 and 8.0. The noncovalent conjugate of 6His-tagged Escherichia coil alkaline phosphatase and Ni2+-NTA-functionalized magnetic-subnnicron-particle released less than 6% of the immobilized enzyme in 3 h at pH 7.4, which enabled the recovery of >90% target-ligand complexes from mixtures of binding reaction. For a 6His-tagged truncated mutant of human cyclic nucleotide phosphodiesterase 4B2 and Ni2+-NTA-functionalized magnetic-subnnicron-particle, the noncovalent conjugate showed similar stability at pH 7.4. After site-specific immobilization on Ni2+-NTA-functionalized and bis-sulfone-functionalized magnetic-submicron-particle, these two 6His-tagged enzymes reserved >90% of their original activities. The use of bis-sulfone-functionalized magnetic-subnnicron-particle caused nonspecific adsorption of those two 6His-tagged enzymes and much longer time for immobilization. Therefore, it was rational to use Ni2+-NTA-MSP for site-specific immobilization of 6His-tagged targets to screen ligands in mixtures.

Spectrophotometric-dual-enzyme-simultaneous-assay (SDESA) for enzyme-linked-immunosorbent-assay (ELISA) of two components in one well is a patented platform when a special pair of labels is accessible. With microplate readers, alkaline phosphatase on 4-nitro-1-naphthylphosphate (4NNPP) served as label A; Pseudomonas aeruginosa arylsulfatase (PAAS) and acetylcholinesterase (AChE) on their substrates derived from 4-nitrophenol/analogue served as candidate label B, and were compared for SDESA with an engineered alkaline phosphatase of Eschrichia coli (ECAP). For SDESA, the interference from overlapped absorbance was corrected based on linear additivity of absorbance to derive initial rates reflected by absorbance change at 450 nm for ECAP and at 405 nm for PAAS or AChE, after the correction of spontaneous hydrolysis. For SDESA with ECAP, AChE already had sufficient activity in an optimized buffer; PAAS was more favorable for substrate stability and product absorbance except for lower activity. Therefore, PAAS engineered for sufficient activity plus alkaline phosphatase is absorbing for ELISA via SDESA.

Using an automatic microplate reader,a high-throughput screening method was developed to recognize uricase mutants of higher activity. The expression vectors of wildtype uricase and its three mutants were used to transform Escherichia coli cells separately. After lysis of cells via ultrasonic treatment,crude enzymes in lysates served as samples to measure activity. Uricase activity was estimated by analyzing absorbance at 293 nm from 8 min to 28 min since reaction initiation. The concentrations of total protein in a lysate were determined by the Bradford assay. Uricase activity or specific activity was compared against a threshold to recognize a mutant of higher activity. The reliability of activity and specific activity for recognizing positive mutants were evaluated by Receiver-operation-curve( ROC). Any uricase mutant with 200% improvement of activity yielded area-under-the-curve( AUC) close to 1. 00. For a mutant with about 50% improvement of activity,ROC analysis of specific activity gave AUC close to 1. 00 while the analysis of activity gave smaller AUC. Thethreshold was determined by fixing the sensitivity at 90% in ROC and expressed as the mean plus 1. 7-fold standard deviation of specific activity of the starting material. A uricase mutant with 100% improvement of specific activity could be efficiently recognized as a positive candidate by the high-throughput method.

To determine inhibition constant (K(i)) of tight-binding inhibitor, the putative method estimated an apparent K(i) from the response of initial rates to total concentrations of the inhibitor considering its depletion during binding for conversion into the true K(i), but was impractical with glutathione S-transferase of sophisticated kinetics. A fluorometric titration assay of dissociation constant (K(d)) was thus proposed. Schistosoma japonicum glutathione S-transferase (SjGST) action on a nonfluorescent divalent pro-inhibitor and glutathione yielded a divalent product in active site to act as a tight-binding inhibitor, whose binding quenched fluorescence of SjGST at 340 nm under the excitation at 280 nm. K(d) was estimated from the response of fluorescence of SjGST at 340 nm to total concentrations of the divalent product considering its depletion during binding. By fluorometric titration assay, K(d) of two tested nonfluorescent divalent products varied from subnanomolar to nanomolar, but both were resistant to change of SjGST levels and consistent with their apparent K(i) estimated via the putative method. Hence, fluorometric titration assay of K(d) of nonfluorescent tight-binding inhibitors/ligands was effective to GST and may be universally applicable to common enzymes/proteins; affinities of tight-binding inhibitors of GST can be approximated by their apparent K(i) estimated via the putative method.

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.

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.

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.

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.

目的:合成4-硝基-1-萘磷酸酯（4-nitro-1-naphthyl phosphate,NNPP）为显色底物测定牛小肠黏膜碱性磷酸酶（alka-line phosphatase,ALP）。方法:4-硝基-1-萘酚（4-nitronaphthol,4-NNP）与三氯氧磷反应,经硅胶柱纯化制得NNPP;检测产物吸收跟踪水解过程测定初速度,双倒数法测定米氏常数（Michaelis-Menten constant,Km）。结果:ALP水解NNPP产物最大差吸收峰接近460 nm,等吸收波长接近405 nm。与p-硝基苯酚相比,在pH 6.0~7.0间4-NNP消光系数为其3倍以上,在pH 7.0以上为其2倍以上。ALP对NNPP的Km约12μmol/L而p-硝基苯基磷酸酯的Km约35μmol/L,产物磷酸相对NNPP的竞争性抑制常数接近20μmol/L。ALP催化NNPP水解效率接近水解p-硝基苯基磷酸酯的40%。结论:NNPP可用于测定ALP活性,且有望与作用于p-硝基苯酚类显色底物的其它酶联用实现单通道两种酶同步测定。

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.

This study investigated xylenol-orange-assay-of-hydrogen-peroxide(XOAHP) for measuring uricase activities in cell lysates and recognizing mutants of higher activity.Four mutants of the intracellular uricase of Bacillus fastidiosus were combined into pairs so that ratios of their catalytic capacities varied from 1.3 to 4.1.The pET28a vector containing mutant gene was transformed into Escherichia coli BL21(DE3).Each of thirty clones was transferred into 1.0 mL liquid medium for amplification,and subject to induced expression for 16 h at 18 ℃.Cells were then harvested and lyzed via sonication treatment to get the supernatant as lysate.In Tris-HCl buffer at pH 8.9,XOAHP was effective to measure uricase activity with uric acid of no more than 0.33 mmol/L.SDS-PAGE showed small differences in expression efficiency of mutants.The activity concentration was positively related to the level of total proteins in lysates of tested mutants.Receiver-operation-curve(ROC) analysis showed the higher the ratio of catalytic capacities of uricase mutants in pairs the chloser to 1.00 of the area-under-thecurve(AUC),with an AUC 0.95 for a pair of mutants at the ratio of about 1.8 between their catalytic capacities.With a threshold of the difference between the activity concentration of a candidate as 1.4 times that of the standard deviation of the starting material,positive mutants of catalytic capacities 1.8 times that of the starting material could be effectively recognized.Therefore,XOAHP plus a proper threshold is effective for high-throughput-screening of mutant library of uricase.

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.

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.

At pH from 5.5 to 7.6, absorptivity of 4-nitro-1-naphthol at 450 nm is over 2.1-fold of that of para-nitrophenol at 405 nm and over 9.6-fold of that of ortho-nitrophenol at 415 nm. On 4-nitro-1-naphthyl-β-d-galactopyranoside at pH 7.4, catalytic efficiency of Escherichia coli β-d-galactosidase is 3-fold of that on para-nitrophenyl-β-d-galactopyranoside and about 40% of that on ortho-nitrophenyl-β-d-galactopyranoside, and produces a lower quantification limit of penicillin G by enzyme-linked-immunoabsorbent-assay. Hence, 4-nitro-1-naphthol is favorable to prepare chromogenic substrates of hydrolytic enzymes of neutral or slightly acidic optimum pH.

Abstract 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.