HPLC-based enzyme assays

Having in hands the above mentioned alkaloid Hbraries, the next step in the whole strategy was to develop HPLC-based enzyme assays in order to monitor the protein-dependent conversion of putative substrates into the corresponding products, followed by their identification by MS and NMR analysis. 

A new hydroxynitrile lyase (HNL) was isolated from the seed of Japanese apricot Prunus mume). It accepts benzaldehyde and a large number of unnatural substrates for the addition of HCN to produce the corresponding (7 )-cyanohydrins in excellent optical and chemical yields. A new high-performance liquid chromatography (HPLC)-based enantioselective assay technique was developed for the enzyme, which promotes the addition of KCN to benzaldehyde in a buffered solution (pH 4.0). Asymmetric synthesis of (7 )-cyanohydrins by a new HNL is described (Figure 8.4). ... 

Fig. 5.2 Analytical set-up for on-line enzyme assays based on ESI-MS. PI Carrier/HPLC pump. P2 HPLC pump delivering enzyme solution. P3 HPLC pump delivering substrate solution. 1 Mixing union. 2 Microcoil reactor. In case of on-line coupling to HPLC, the HPLC column is inserted...

Fig. 5.9 Design of the chip-based enzyme ESI-MS assay. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron). I Sample components/inhibitors injected by flow injection or eluting from capillary HPLC column. E Infusion pump delivering the enzyme cathepsin B. S infusion pump delivering the substrate Z-FR-AMC. Micro-chip design Vrije Universiteit Amsterdam. Micro-chip production Micronit Microfluidics BV (Enschede, The Netherlands).

GTPCH (EC 3.5.4.16) converts the substrate GTP to 7,8-dihydroneopterin triphosphate (H2NTP) and formate. GTPCH activity is determined by measuring neopterin, the completely oxidized and dephosphorylated H TP-product of the enzyme reaction. Conversion of H2NTP to neopterin is carried out after the enzymatic reaction in presence of iodine at pH 1.0, followed by dephosphorylation with alkaline phosphatase at pH 8.5-9.0. Neopterin is detected fluorimetrically at 350/440 nm upon HPLC separation. The assay is based with some modifications on the methods published by Viveros et al. and Hatakeyama and Yoneyama [15,16]. 

The of the MF + C02/formyl-MF couple is —497 mV and that of HVH2 is —414 mV [184]. Thus, in vitro H2 is not a good electron donor for Reaction (48). However, an HPLC-based assay in the direction of C02-reduction, using Ti(III)-citrate ( 0 = —480 mV) as an artificial electron donor, has been described [185]. In the opposite direction, the enzyme is easily assayed spectrophotometrically using the artificial electron acceptor methylviologen ( = —446 mV) ... 

E89 Powers, D., Kringle, R., Fame, N., Slickers, K., Sylvestre, E. and Rand, R. (1982). Specificity of the Ektachem analyzer enzyme-based creatinine assay compared with Jaffe and HPLC methods. Clin. Chem. 28, 1570, Abstr. 107. 

PNAE was the first enzyme detected in the ajmahne pathway. Detection and prehminary characterization were first reported in 1983. This enzyme turned out to be an extraordinary highly substrate selective protein which has been reported several times. In order to achieve a comprehensive purification of PNAE, an HPLC-based assay was developed for... 

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