Enzyme assay protease sensitivity

Due to their response mechanism the polyion-selective electrodes are not sensitive to the small fragments of polyionic macromolecules. Thus, if an enzyme cleaves the polyionic molecule these sensors can be used for detection of enzyme activity. Polycation protamine is rich in arginine residues that make it a suitable substrate for protease-sensitive electrochemical assays. Real-time detection of trypsine activity was demonstrated with the protamine-selective electrode as a detector [38],... 

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

In the second section of this chapter, strategies to identify substrates for biochemical protease assays are discussed. Section 2.3 focuses on theoretical and practical aspects of various fluorescence-based readouts for biochemical protease assays. Finally concrete experiments for the determination of enzyme kinetics relevant for the development of robust and sensitive biochemical protease assays are summarized in Section 2.4. Altogether this chapter offers guidelines for the development of biochemical protease assays for the purpose of protease inhibitor-directed drug discovery. 

There are other examples of HIV protease substrates that utilize the fluorescence energy transfer technique. Perhaps the strongest characteristic of these assays is that they provide a continuous readout of enzyme activity. Another advantage is their sensitivity (they use small concentrations of enzyme). The disadvantage of these assays is that they are susceptible to interference by some compounds (inhibition artifacts) because of inner and outer filter effects (see Notes 8-10). Other assays, for example, those based on radioactivity or high-performance liquid chromatography (HPLC) analysis of products are tedious to run, but are less susceptible to interference or inhibition artifacts. 

AlphaScreen applications include assays for enzymes such as protein kinases and proteases, immunoassays such as cAMP detection, and protein-protein and protein-DNA interactions. A recent literature example is a comparison of AlphaS-creen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. In this comparison, the AlphaScreen system showed the highest sensitivity and the broadest dynamic range [149]. Another recent publication concerns a high-throughput binding assay for a TNF receptor [175]. 

The first enzyme to catalyze prolyl isomerization was identified by Fischer and colleagues (1984). This discovery was possible because they developed an ingenious assay for prolyl isomerases based on the conformational specificity of chymotrypsin. This protease cleaves a chro-mogenic reporter group from a tetrapeptide (such as succinyl-Ala-Ala-Pro-Phe-4-nitroanilide) only when the Ala—Pro bond of this peptide is in the trans conformation. In aqueous solution the assay peptide exists as a 90 10 mixture of molecules with the Ala—Pro bond in trans and cis, respectively. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the peptide molecules are cleaved within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow because it is limited in rate by the cis —trans isomerization of the Ala—Pro bond. Acceleration of this reaction serves as a sensitive probe for prolyl isomerase activities. 

EPTC and Butvlate Fluorescein Diacetate Assay. Spectrophotometric determinations of the hydrolysis of fluorescein diacetate have been shown to be simple, rapid, and sensitive methods for determining microbial activity in soil (18). Essentially, the hydrolytic cleavage of diacetate from fluorescein is responsible for the reaction products including fluorescein, which may be detected spectrophotometrically at 490 nm. This method is somewhat nonspecific in that it is indicative of overall activity of several enzymes (protease, lipase, esterase) rather than of a specific class of enzymes. Enzyme activity may be influenced by subtle pH changes in the sample since abiotic hydrolysis of fluorescein diacetate may occur. Also, an associated lag phase in soil hydrolytic activity must be accounted for in each assay. 

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