Yunsheng Zhao

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.

Characterization of n uricase from Bacillus fastidious A.T.C.C. 26904 and its application to serum uric acid assay by a patented kinetic uricase method

An intracellular uricase from Bacillus fastidious A.T.C.C. 26904 was characterized and evaluated for serum uric acid assay by a patented kinetic uricase method. The active uricase was 151 kDa by gel filtration through Sephadex G‐200. Both SDS/PAGE and matrix‐assisted laser‐desorption ionization–time‐of‐flight MS resolved a single polypeptide with a molecular mass of approx. 36.0 kDa. The N‐terminal sequence was AERTMFYGKGDV. The optimum pH for this uricase ranged from 9.0 to 10.5. At pH 9.2, the Km (Michaelis–Menten constant) was 204±14 μmol/l (n=8) and the Ki (inhibition constant) for xanthine was 41±7 μmol/l (n=5). By analysing the data monitored within 5 min at 0.03 unit/ml uricase, this kinetic uricase method gave linear response to uric acid in reaction solution from 1.3 to 60 μmol/l. Aside from other common errors, 30 μmol/l xanthine in the reaction solution caused no error in this kinetic uricase method, while it caused negative error in the indirect equilibrium method by peroxidase‐coupled assay of H2O2. Uric acid in clinical sera by this kinetic uricase method (Ck) closely and positively correlated with that from the indirect equilibrium method (Ce) (Ck=0.008+1.081× Ce, r>0.990, n=99). However, Bland–Altman analysis suggested inconsistency between Ck and Ce. These results indicated that this kinetic uricase method using this uricase was reliable for serum uric acid assay with enhanced resistance to xanthine besides other common errors.

Evaluation of a kinetic uricase method for serum uric acid assay by predicting background absorbance of uricase reaction solution with an integrated method

A patented kinetic uricase method was evaluated for serum uric acid assay. Initial absorbance of the reaction mixture before uricase action (A0) was obtained by correcting the absorbance at 293 nm measured before the addition of uricase solution, and background absorbance (Ab) was predicted by an integrated method. Uric acid concentration in reaction solution was calculated from ΔA, the difference between A0 and Ab, using the absorptivity preset for uric acid. This kinetic uricase method exhibited CV<4.3% and recovery of 100%. Lipids, bilirubin, hemoglobin, ascorbic acid, reduced glutathione and xanthine <0.32 mmol/L in serum had no significant effects. ΔA linearly responded to 1.2 to 37.5 µmol/L uric acid in reaction solution containing 15 µl serum. The slope of linear response was consistent with the absorptivity preset for uric acid while the intercept was consistent with that for serum alone. Uric acid concentrations in clinic sera by different uricase methods positively correlated to each other. By Bland-Altman analysis, this kinetic uricase method accorded with that by quantifying the total change of UV absorbance on the completion of uricase reaction. These results demonstrated that this kinetic uricase method is reliable for serum uric acid assay with enhanced resistance to both xanthine and other common errors, wider range of linear response and much lower cost.

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).

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.

Reversible Inactivation of an Intracellular Uricase from Bacillus fastidiosus via Dissociation of Homotetramer into Homodimers in Solutions of Low Ionic Strength

An intracellular uricase from Bacillus fastidiosus with high catalytic capacity and strong resistance to xanthine was inactivated in water but could be essentially re-activated in solutions of high ionic strength. By polyacrylamide gel electrophoresis (PAGE), gradient PAGE, sodium-dodecyl-sulfate-PAGE, gel-filtration through Sephadex G200, and activity staining with peroxidase and its chromatogenic substrate, this homotetrameric uricase in water was found to dissociate into inactive homodimers that could form active homotetramers again in solutions of high ionic strength. Sensitivity to low ionic strength of solutions complicates formulation of this uricase as a drug and its elimination requires protein engineering.

Significance of combined tests of serum golgi glycoprotein 73 and other biomarkers in diagnosis of small primary hepatocellular carcinoma

BACKGROUND The most practical serum biomarker AFP is negative in 80% patients with small tumor in the early stage of primary hepatocellular carcinoma (PHC). OBJECTIVE To investigate significance of combined tests of serum golgi glycoprotein 73 (GP73), lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3), glypican 3 (GPC3), des-gamma-carboxy prothrombin (DCP), CA199, ferritin (FER) and carcinoembryonic antigen (CEA) in diagnosis of small PHC with negative AFP. METHODS A total of 50 cases of small PHC, 60 cases of non-PHC digestive system disorders and 40 cases of healthy controls were collected. Serum GP73, GPC3 and DCP were analyzed by enzyme-1inked immunosorbent assay. AFP-L3 was separated by microspincolumn; the levels of CA199, FER, CEA, AFP-L3 and total AFP were quantified by electrochemiluminescence immunoassays; percentage of AFP-L3 to total AFP was calculated. RESULTS Levels of those biomarkers except FER were all significantly different among three groups (P < 0.01). Positive detection rates of seven biomarkers in PHC were higher than other groups (P < 0.01). In individual detection, the highest sensitivity, accuracy rate and area under ROC curve were 72.0%, 86.7% and 0.826 by GP73 and the highest specificity was 98.0% by GPC3. In combined detection with those seven biomarkers, the sensitivity, specificity, accuracy rate and area under ROC curve were 82.0%, 95.0%, 90.1% and 0.884. CONCLUSIONS GP73 and AFP-L3 are superior biomarkers to aid the diagnosis of small PHC with negative AFP. The combined detection of biomarkers improved the diagnostic accuracy.

A new practical system for evaluating the pharmacological properties of uricase as a potential drug for hyperuricemia.

The use of uricase-deficient mammals to screen formulations of engineered uricases as potential drugs for hyperuricemia involves heavy costs and presents a technical bottleneck. Herein, a new practical system was investigated to evaluate the pharmacological significance of a bacterial uricase based on its ability to eliminate uric acid in plasma in vitro, its pharmacokinetics in vivo in healthy rats, and the modeled pharmacodynamics in vivo. This uricase, before and after modification with the monomethyl ether of poly(ethylene glycol)-5000, effectively eliminated uric acid in vitro in rabbit plasma, but its action was susceptible to xanthine inhibition. After intravenous injection of the modified uricase without purification, a bi-exponential model fit well to uricase activities in vivo in the plasma of healthy rats; the half-life of the modified uricase was estimated without interference from the unmodified uricase leftover in the sample and was nearly 100-fold longer than that of the unmodified uricase. Using a model of the elimination of uric acid in vivo taking into account of uricase pharmacokinetics and xanthine inhibition, modeled pharmacodynamics supported that the half-life of uricase and its susceptibility to xanthine are crucial for the pharmacological significance of uricase. Hence, this practical system is desirable for doing preliminary screening of formulations of engineered uricases as potential drugs for hyperuricemia.

Correlation of serum arylesterase activity on phenylacetate estimated by the integrated method to common classical biochemical indexes of liver damage

The correlation of serum arylesterase (PON1) activity on phenylacetate determined by an integrated method to classical biochemical indexes of liver damage was investigated for the use of PON1 activity to evaluate liver damage. PON1 reaction curve as absorbance at 270 nm for 0.20 mmol/L phenylacetate hydrolysis was analyzed by the integrated method to determine maximal PON1 reaction rate. Classical biochemical indexes of liver damage were determined routinely. The 95% confidence threshold of PON1 activity in sera from healthy individuals was 2.12 mkat/L [(4.73±1.31) mkat/L, n=105]. PON1 activity in clinical sera was closely correlated to serum albumin, total protein and the ratio of albumin to globulins, but was weakly correlated to both direct and total bilirubin in serum. There were no correlations of PON1 activity to γ-glutamyltransferase, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. Among 127 clinical sera with PON1 activity>2.12 mkat/L, there were 92% healthy individuals examined by albumin, 90% healthy individuals examined by total protein, 88% healthy individuals examined by total bilirubin, 86% healthy individuals examined by direct bilirubin and 64% healthy individuals examined by the ratio of albumin to globulins, respectively. In each group of healthy individuals judged by classical biochemical indexes of close correlation to PON1 activity, percentage of healthy individuals examined by PON1 activity was always >80%. These results suggested PON1 activity on phenylacetate estimated by the integrated method was also suitable for the evaluation of liver damage.