Imaging of Enzyme Activities

Razgulin A. Ma N. Rao J. Strategies for in vivo imaging of enzyme activity An overview and recent advances. Chemical Society Reviews, 2011,40,4186-4216. 

The imaging of enzyme activity at insulating supports can be achieved via feedback or generation-collection modes as described in Secs. I.C and I.D. 

Imaging of enzyme activities Thrombin, a serine protease, plays an important role in thrombogenesis, cleaving fibrinogen to form fibrin monomers, which subse-... 

Oyamatsu, D., Hirano, Y., Kanaya, N., Mase, Y., Nishizawa, M., Matsue, T. Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate-probe distance. Bioelectrochemistry 2003, 60, 115-121. 

The concentrations of the enzyme substrate have to be selected with some care. In the GC experiment, it should be well above the Michaelis-Menten constant of the enzyme for this substrate. The presence of the UME with its sheath will also limit the diffusion of the enzyme substrate towards the active regions. This may lead to an underestimation of enzyme activity on the surface or to great distorsion in recorded images. Using electrodes with a small RG is a good idea for GC experiments. This question is explained in Procedure 52 (see in CD accompanying this book). 

The [Eu(Tc)] assay for H202 can also be used for the determination of enzyme activities. This comprises all enzymes that produce (oxidases) or consume (catalase, peroxidases) H2O2. For example, the activity of glucose oxidase can be directly imaged after the addition of [Eu(Tc)] as indicator and glucose as substrate. Steady-state and time-resolved imaging schemes can be applied for the read-out of a microwell plate-based assay. The best results were obtained by the RLI method (Fig. 19) [115]. 

A related technique was demonstrated by Shiku et al. (91), who formed alkylsilane monolayers at glass substrates and used electrochemical generation of OH radicals at the tip via the Fenton reaction to locally destroy the SAM. Diaphorase could then be patterned on the surface by physical adsorption to the undamaged hydrophobic areas or via covalent linkages to the radical-attacked areas. The former process was imaged in feedback mode with a ferrocenyl mediator and showed a decreased current over the disk of destroyed SAM and a constant background of enzyme activity over the rest... 

The in vivo imaging studies carried out with cysteine protease-directed probes [50] underscored the need for a generally applicable methodology for in vivo analysis of enzyme activities. Indeed, as exemplified by many protease families, most enzymes are subject to multiple mechanisms for... 

---