al., 2006, 2009), glutathione-S-tranferase (GST) (Liao, et al., 2003b; Zhao, L.N., et al., 2006), butylcholineesterase (Liao, et al., 2009; Yang, et al., 2011), LDH (Cheng, et al., 2008) and LDH-coupled ALT reaction systems (Yang, et al., 2010). Uricase of simple kinetics is a good example to study new methods for kinetic analysis of reaction curve; reactions of GGT and ADH suffer product inhibition and kinetic analyses of their reaction curves are complicated because they require unreported parameters. Hence, our new methods for kinetic analysis of reaction curve and the integration strategies for quantifying enzyme substrates and initial rates are demonstrated with uricase, GST and ADH as examples.
2.5.1 Uricase reaction
Uricase follows simple Michaelis-Menten kinetics on single substrate in air-saturated buffers, and suffers neither reversible reaction nor product inhibition (Liao, 2005; Liao, et al., 2005a, 2005b; Zhao, Y.S., et al., 2006). Uricase reaction curve can be monitored by absorbance at 293 nm. The potential interference from the intermediate 5-hydroxylisourate with uric acid absorbance at 293 nm can be alleviated by analyzing data of steady-state reaction in borate buffer at high reaction pH (Kahn & Tipton, 1998; Priest & Pitts, 1972). The integrated rate equation for uricase reaction with the predictor variable of reaction time is Equ.(4). Uricases from different sources have different $K_m$ (Liao, et al., 2005a, 2006; Zhang, et al., 2010; Zhao, Y.S., et al., 2006). Using Equ.(4), $K_m$ of Candidate utilis is estimated with reasonable reliability (Liao, et al., 2005a). Using Equ.(9) to estimate the ratio of $V_m$ to $K_m$, uricase mutants of better catalytic capacity and their sensitivity to xanthine are routinely characterized (data unpublished). Thus, we used uricases of different $K_m$ as models to test the two integration strategies for enzyme substrate assay and initial rate assay, respectively.
Uricase from Bacillus fastidiosus A.T.C.C. 29604 has high $K_m$ to facilitate predicting $A_b$ (Zhang, et al., 2010; Zhao, Y.S., et al., 2006, 2009). Reaction curves at low levels of uric acid with this uricase at 40 U/L are demonstrated in Fig. 3. Steady-state reaction is not reached within 30 s since reaction initiation; it is difficult to get more than 5 data with absorbance changes over 0.003 for kinetic analysis of reaction curve at uric acid levels below 3.0 µmol/L. At 40 U/L of this uricase, the absorbance after reaction for 5.0 min has negligible difference from that after reaction for 30 min for uric acid below 5.0 µmol/L. To quantify the difference between $A_0$ and $A_b$ after reaction for 5.0 min, the equilibrium method has an upper limit of about 5.0 µmol/L, while kinetic analysis of reaction curve with $K_m$ as a constant is feasible for $S_0$ of about 5.0 µmol/L. Thus, the change of absorbance over 0.050 between $A_0$ and the absorbance after reaction for 5.0 min can be the switch threshold to change from the equilibrium method to kinetic analysis of reaction curve.
This integration strategy for enzyme substrate assay gives the linear response from about 1.5 µmol/L up to 60 µmol/L uric acid at 40 U/L uricase (Fig.4, unpublished), and shows resistance to the action of xanthine at 30 µmol/L in reaction solutions (this level of xanthine always caused negative interference with all available kits commercialized for serum uric acid assay). Therefore, the integration strategy for uric acid assay is clearly superior to any other uricase method reported.
Uricases from Candida sp. with $K_m$ of 6.6 µmol/L (Sigma U0880) and Bacillus fastidiosus uricase from A.T.C.C. 29604 with $K_m$ of 0.22 mmol/L are used to test the integration strategy for initial rate assay. The use of uric acid at $S_0$ of 25 µmol/L to monitor reaction curves