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A new ion-exchange medium was prepared from magnetic particles of ∼1.0 µm through coating with small zwitterions and then functionalizing with ampholytic groups for the isoelectric point of ∼6.4 and denoted MSP-ZEWB. With Meyerozyma guilliermondii uricase (MGU) as the model of soluble proteins, the purification of a protein via ion-exchange was compared between MSP-ZEWB through elution in discontinuous mode and Toyopearl SP-650C as a classical ion-exchange medium through elution in continuous mode. MGU was adsorbed at pH 7.6 or 8.0 and eluted via competitive displacement by NaCl or electrostatic repulsions with an elution buffer at pH 10 to reverse the type of net charges of MGU. From MSP-ZEWB, MGU was eluted more rapidly with the elution percentages higher than those from Toyopearl SP-650C. For yielding a unit of MGU activity, MSP-ZEWB gave the elution solution volumes that were ∼50% of those obtained with Toyopearl SP-650C. The yields of MGU of the highest purity from MSP-ZEWB were higher than those from Toyopearl SP-650C, but the highest purification folds with both media were comparable. MSP-ZEWB regenerated for 16 times still showed the consistent purification efficacy. Therefore, the ion-exchange media bearing small zwitterion coats showed great promise for the purification of soluble proteins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Uricase after modification with monomethoxy poly(ethylene glycol) (m PEG) is currently the sole agent to treat refractory gout. For formulating Bacillus fastidious uricase, succinimidyl carbonate of m PEG-5000 ( SC-m PEG5k) and succinimidyl succinate of m PEG-5000 ( SS-m PEG5k) were compared. SC-m PEG5k 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-m PEG5k or SS-m PEG5k at pH 9.2. Oxonate and xanthine concentration-dependently protected the bacterial uricase from inactivation during PEGylation. With SC-m PEG5k 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-m PEG 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. [ABSTRACT FROM AUTHOR]

Abstract: The estimation of enzyme kinetic parameters by nonlinear fitting reaction curve to the integrated Michaelis–Menten rate equation ln(S 0/S)+(S 0−S)/K m=(V m/K m)×twas investigated and compared to that by fitting to (S 0−S)/t=V m−K m×[ln(S 0/S)/t] (Atkins GL, Nimmo IA. The reliability of Michaelis-Menten constants and maximum velocities estimated by using the integrated Michaelis-Menten equation. Biochem J 1973;135:779-84) with uricase as the model. Uricase reaction curve was simulated with random absorbance error of 0.001 at 0.075 mmol/l uric acid. Experimental reaction curve was monitored by absorbance at 293 nm. For both CV and deviation <20% by simulation, K m from 5 to 100 μmol/l was estimated with Eq. (1) while K m from 5 to 50 μmol/l was estimated with Eq. (2). The background absorbance and the error in the lag time of steady-state reaction resulted in negative K m with Eq. (2), but did not affect K m estimated with Eq. (1). Both equations gave better estimation of V m. The computation time and the goodness of fit with Eq. (1) were 40-fold greater than those with Eq. (2). By experimentation, Eq. (1) yielded K m consistent with the Lineweaver–Burk plot analysis, but Eq. (2) gave many negative parameters. Apparent K m by Eq. (1) linearly increased, while V m were constant, vs. xanthine concentrations, and the inhibition constant was consistent with the Lineweaver–Burk plot analysis. These results suggested that the integrated rate equation that uses the predictor variable of reaction time was reliable for the estimation of enzyme kinetic parameters and applicable for the characterization of enzyme inhibitors. [Copyright &y& Elsevier]