Monitoring of Reaction Kinetics Using MS

At the end of this chapter, the approach of Brivio and coworkers of using microreactor chips in the vacuum chamber of a MALDI-MS machine is worth mentioning [78,79]. This approach allows real on-line monitoring of reaction kinetics by MALDI-MS, but with only one time point at a time. The procedure for measuring a complete reaction curve is too time consuming and complex to be of practical use, but with dedicated and reconfigured M S equipment may be an option for on-hne studies. 

Metal-modified silicas were exposed to excess BuOOH vapor in order to generate the supported feri-butylperoxide complexes, followed by evacuation to remove PrOH and unreacted BuOOH. Reaction kinetics were monitored as the uptake of cyclohexene from the gas phase, using a ThermoNicolet Nexus FTIR spectrometer to measure the intensity of the o(C=C) mode. In situ spectra were recorded in custom-made glass reactors under vacuum. Formation of cyclohexene oxide was confirmed by GC/MS on an HP 6890 equipped with a DBI capillary column (J W Scientific). 

Molecular mass determination by MS is also useful for analysis of substrate specificities and cleavage patterns of enzymes, including chitinases, chitosanases, lysozymes, and chitin deacety-lases, as summarized with a few representative examples in Table 11.4. Continuous infusion of reaction mixtures into an ESI mass spectrometer (so called real time monitoring) was nsed to analyze the hydrolysis of D (z = 4 - 6) by several chitosanases and some mutants (Dennhart et al. 2008). ESl-MS is also snitable for simnltaneous measurement of individual kinetic constants of enzymes in mixtures of substrates, as reported for a bacterial sulfotransferase with A (z = 2 - 5) (Pi and Leary 2004). 

On-line MS methods enable continuous kinetic profiles to be obtained but they cannot easily accommodate complex sample preparation steps. In the 1980s, enzymatic reactions were monitored by a popular - at that time - ionization technique, namely fast atom bombardment (FAB)-MS [12, 13]. Heidmann etal. [14] used FAB-MS to identify conjugation products of reactive quinones with glutathione by conducting dynamic mass spectral analysis. Soon after the introduction of ESI to MS, its potential in the monitoring of biochemical reactions was recognized, especially in the detection of labile intermediates (cf. [15,16]). Nowadays ESI and MALDI are prime tools for the analysis of biomolecules. Both techniques are also suitable for the investigation of biocatalytic processes with diverse temporal resolutions [17]. 

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