Microarray enzyme substrate/inhibitor

The family of HDAC enzymes has been named after their first substrate identified, i.e., the nuclear histone proteins. Histone proteins (H2A, H2B, H3 and H4) form an octamer complex, around which the DNA helix is wrapped in order to establish a condensed chromatin structure. The acetylation status of histones is in a dynamic equilibrium governed by histone acetyl transferases (HATs), which acetylate and HDACs which are responsible for the deacetylation of histone tails (Fig. 1). Inhibition of the HDAC enzyme promotes the acetylation of nucleosome histone tails, favoring a more transcriptionally competent chromatin structure, which in turn leads to altered expression of genes involved in cellular processes such as cell prohferation, apoptosis and differentiation. Inhibition of HDAC activity results in the activation of only a limited set of pre-programmed genes microarray experiments have shown that 2% of all genes are activated by structmally different HDAC inhibitors [1-5]. In recent years, a growing number of additional nonhistone HDAC substrates have been identified, which will be discussed in more detail below. 

The second type of enzyme application in microfluidics is protein interaction studies. In order to study interactions between enzymes and substrates, peptides, or other proteins, an alternative to microtiter plates is the microarray. These arrays are very often used in conjunction with microfluidic sample and reagent delivery systems alternatively, microchannels can be used to deposit spots in an array. By immobilizing substrates or inhibitors on the array, it is possible to screen large numbers of molecules for their relative abilities to bind a particular enzyme. This is typically accomplished by tagging the enzyme with a fluorescent molecule and using the intensity of the array spots to determine the amount of enzyme bound (Fig. 3a). This is a promising... 

Having established the means to monitor the catalytic turnover of a fluorogenic substrate by a CYP450 enzyme in a microarray format, it is then relatively straightforward to set up competitive assays to determine the kinetic parameters for either unlabelled inhibitors or unlabelled substrates as follows (see Note 18) ... 

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