Enzymes experimental methodology

The experimental methodology for exploiting enzymes is very straightforward and can be performed with common organic equipment, and with routine experimental and work-up procedures. A range of reaction conditions can be applied, including the use of immobilized enzymes and organic solvents. 

Experimental methodologies utilizing radiolabeled compound are similar to those where authentic standard is available (see Section 15.6.3). Radiolabeled studies can be conducted using the following expressed enzyme and/or human liver microsomes for the evaluation of A m-V max chemical inhibition, and/or correlation analysis using a characterized bank of human liver microsomes (Tuvesson et al., 2005 Zhu et al., 2005 Zhang et al., 2007). 

The ability of SPR to probe both kinetic and thermodynamic processes, as well as to provide micro-structural information, make it a very important component of the experimental methodology available to probe molecular interactions occurring at surfaces. Furthermore, it allows some of the limitations of other techniques to be overcome. For example, other methods often require one of the partners to be labelled in some way in order to allow it to be detected. Fluorescent probes, radioactive labels, and attachment of independently detectable molecules (e.g. enzymes) have all been used for this purpose. These suffer from the drawback that they may interfere with the binding of the labelled partner to the unlabelled one, or cause unwanted structural perturbations. SPR observations can be based solely on the dielectric properties of molecules, or their intrinsic light absorption characteristics, and thus require no specific labelling. 

A notorious underestimation of the dynamic properties of microbial and cellular populations results from matching the duration of the respective batch cultivations to the relevant time constant of the biosystem under investigation. However, metabolic regulation of enzyme activities and fluxes often takes place on a time scale of seconds rather than days although the latter may also be true. It is therefore in the scope of promoting biotechnological research to adopt and develop appropriate experimental concepts, methodologies and equipment [397]. 

In the usual experimental protocols involving PAF, the objective often is to determine whether PAF is formed under stimulatory conditions or whether it has been metabolized by cells, cellular extracts, or specific enzymes. In most instances, the reactions can be followed only through the use of radiolabeled precursors and certain simple analytical procedures. The methodology to be... 

---