Xiaolan Yang

An improved malachite green assay of phosphate: Mechanism and application

The classical malachite green (MLG) assay of phosphate, which added MLG after molybdate to the acidified reaction solutions of phosphate, tolerated interference from papaverine, sildenafil, and some similar hydrophobic amines. Resonance Rayleigh scattering signals, the alleviation of interference by poly(vinyl alcohol), and the precipitation of some yellow complexes supported that the irreversible aggregation of the complexes of a hydrophobic amine of interference and phosphomolybdate reduced the amounts of phosphomolybdate accessible to MLG and caused the interference. By adding MLG before molybdate to the acidified reaction solutions of phosphate, the complexes of phosphomolybdate and MLG were preferentially formed before the complexes of phosphomolybdate and such a hydrophobic amine effectively aggregated; thereby, an improved MLG assay of phosphate with the resistance to common hydrophobic amines was developed. Using the improved MLG assay of phosphate and a phosphatase to release phosphate from AMP, a spectrometric method successfully estimated the half-inhibition concentrations of papaverine on the recombinant human cyclic nucleotide phosphodiesterase (PDE) isozyme 4 and the mixture of PDE isozymes from rabbit brain. Therefore, the improved MLG assay of phosphate was a favorable and universal technique for developing spectrometric methods for characterizing and screening inhibitors of enzymes that release phosphate during their actions.

Uricases as therapeutic agents to treat refractory gout: Current states and future directions

Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I‐III Regulatory, Quality, Manufacturing Postmarketing Phase IV

Effects of Modification of Amino Groups with Poly (Ethylene Glycol) on a Recombinant Uricase from Bacillus fastidiosus

After modification with monomethoxyl-poly(ethylene glycol)-5000, a recombinant intracellular uricase from Bacillus fastidiosus ATCC 29604 showed residual activity of about 65%, a thermo-inactivation half-life >85 h, a circulating half-life about 20 h in rats in vivo, consistent effects of common cations, and consistent optima for reaction temperature and pH. Thus, this uricase can be formulated via modification with monomethoxyl-poly(ethylene glycol).

Site-Specific PEGylation of Therapeutic Proteins via Optimization of Both Accessible Reactive Amino Acid Residues and PEG Derivatives

Modification of accessible amino acid residues with poly(ethylene glycol) [PEG] is a widely used technique for formulating therapeutic proteins. In practice, site-specific PEGylation of all selected/engineered accessible nonessential reactive residues of therapeutic proteins with common activated PEG derivatives is a promising strategy to concomitantly improve pharmacokinetics, allow retention of activity, alleviate immunogenicity, and avoid modification isomers. Specifically, through molecular engineering of a therapeutic protein, accessible essential residues reactive to an activated PEG derivative are substituted with unreactive residues provided that protein activity is retained, and a limited number of accessible nonessential reactive residues with optimized distributions are selected/introduced. Subsequently, all accessible nonessential reactive residues are completely PEGylated with the activated PEG derivative in great excess. Branched PEG derivatives containing new PEG chains with negligible metabolic toxicity are more desirable for site-specific PEGylation. Accordingly, for the successful formulation of therapeutic proteins, optimization of the number and distributions of accessible nonessential reactive residues via molecular engineering can be integrated with the design of large-sized PEG derivatives to achieve site-specific PEGylation of all selected/engineered accessible reactive residues.

Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions

Bacillus fastidious uricase (BF uricase) containing 322 amino acid residues exhibited high stability under physiological conditions. Its crystal structure was solved to 1.4-Å resolution, showing homotetramer containing two homodimers. After the intersubunit antiparallel β-sheet in its homodimer, each subunit had a total of 18 C-terminus residues forming an α-helix (Q305-A313) and random coil (S314-L322) on surface to bury other two α-helices (I227-T238 and I244-R258). In comparison, reported crystal structures of Arthrobacter globiformis and Aspergillus flavus uricases had atomic coordinates of only some C-terminus residues, while the crystal structures of all the other uricases accessible before September 2014 missed atomic coordinates of all their C-terminus residues, after the intersubunit antiparallel β-sheets. In each homodimer of BF uricase, H-bonds were found between E311 and Y249 and between Y319 and D257; electrostatic interaction networks were found to surround D307 plus R310 and intersubunit R3, K312 plus D257, E318 plus K242, and L322 plus R258. Amino acid mutations that disrupted those interactions when R3 and D307 were reserved caused moderate decreases of activity at pH 9.2 while negligible decreases of activity at pH 7.4, but destroyed stability at pH 7.4 while slightly decreased stability at pH 9.2. Such structural information guided the fusion of 6His-tag to the C-terminus of the mutant L322D with SNSNSN as a linker to reserve the activity and stability. Hence, the folding of the C-terminus residues is crucial for thermal stability of BF uricase under physiological conditions; these new structural insights are valuable for molecular engineering of uricases.

An integration strategy to estimate the initial rates of enzyme reactions with much expanded linear ranges using uricases as models.

A new strategy was proposed to estimate the initial rates of reactions catalyzed by Michaelis-Menten enzymes via integrating the classical initial rate method for low activities with an improved integrated method for high activities. Between these two individual methods, this integration strategy required: (a) the consistent linear response slopes, acquired with an optimized preset substrate concentration (PSC) to derive the initial rates from the maximal reaction rates estimated by the improved integrated method; (b) an overlapped region of the initial rates measurable with consistent results, realized with an optimized reaction duration to record reaction curves for analyses by the improved integrated method; (c) a switch cutoff, preset as the instantaneous substrate concentration slightly above that after a given lag time when the enzyme activity was just below the upper limit for the linear response of the classical initial rates. By simulation with uricases at a given initial substrate concentration (S(0)), the optimized PSC was 93% S(0), the optimized reaction duration at S(0) from 0.35-fold to 11.0-fold Michaelis-Menten constant (K(m)) was within 6.0 min and the switch cutoff was available at the given S(0) after 30-s lag time, all of which were combined to produce 300-fold linear ranges. By experimentation with one uricase of K(m) at 6.6 microM and the other uricase of K(m) at 220 microM under optimized conditions, this integration strategy with S(0) at 75 microM produced 100-fold linear ranges. Thus, this integration strategy exhibited much expanded linear ranges and practical efficiency over wide ratios between S(0) and K(m).

Design, synthesis and evaluation of novel quinazoline-2,4-dione derivatives as chitin synthase inhibitors and antifungal agents.

A series of novel 1-methyl-3-substituted quinazoline-2,4-dione derivatives were designed, synthesized, and characterized by (1)H NMR, (13)C NMR and MS spectral data. Their inhibition against chitin synthase (CHS) and antifungal activities were evaluated in vitro. Results showed compounds 5b, 5c, 5e, 5f, 5j, 5k, 5l, and 5o had strong inhibitory potency against CHS. Compound 5c, which has the highest potency among these compounds, had a half-inhibition concentration (IC50) of 0.08mmol/L, while polyoxin B as positive drug had IC50 of 0.18mmol/L. These IC50 values of compounds 5i, 5m, 5n, and 5s were greater than 0.75mmol/L, which revealed that those compounds had weak inhibition activity against CHS. Moreover, most of these compounds exhibited moderate to excellent antifungal activities. In detail, to Candida albicans, the activities of compound 5g and 5k were 8-fold stronger than that of fluconazole and 4-fold stronger than that of polyoxin B; to Aspergillus flavus, the activities of 5g, 5l and 5o were16-fold stronger than that of fluconazole and 8-fold stronger than that of polyoxin B; to Cryptococcus neoformans, the minimum-inhibition-concentration (MIC) values of compounds 5c, 5d, 5e and 5l were comparable to those of fluconazole and polyoxin B. The antifungal activities of these compounds were positively correlated to their IC50 values against CHS. Furthermore, these compounds had negligible actions to bacteria. Therefore, these compounds were promising selective antifungal agents.

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.

The measurement of serum cholinesterase activities by an integration strategy with expanded linear ranges and negligible substrate-activation.

OBJECTIVES To measure serum cholinesterase (SCHE) with an integration strategy. DESIGN AND METHODS At 54.0 micromol/L butyrylthiolcholine, SCHE initial rates were calculated with 50.0 micromol/L butyrylthiolcholine and maximal rates via an improved integrated method if substrate consumptions within 5.0 min were over 60%, or were determined by the classical initial rate method. RESULTS The linear range was from 16 to 1560 nkat/L, and SCHE in clinic sera showed negligible substrate-activation. CONCLUSION This strategy was effective.

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.