Enzymes engage key roles in a wide variety of important physical and medical processes, which thus can be altered by manipulating the behavior of enzymes in charge. The capability for manipulation requires an exact understanding of enzymatic operation modes though, which can be increased by employing fluorescence spectroscopy techniques. To date several fluorescence-based assays using labeled substrates have been developed to examine different subclasses of hydrolases. We developed a method that circumvents the unspecific probe enzyme interactions and affinity problems occurring in common probes as those based on fluorescence resonance energy transfer (FRET) by taking advantage of the comparably strong electron donating properties of the naturally occurring nucleic acid guanosine (G). Combined with an appropriate fluorophore this compound shows efficient photoinduced electron transfer (PET) quenching reactions only upon contact formation. Thus, initially quenched enzyme substrates, e.g. specific nucleic acid sequences, can be designed that cause a distinct increase in fluorescence signal upon specific hydrolysis. Here we demonstrate the general validity of PET probes for the observation of various nucleases at the ensemble and single molecule level. The rapid response time of the probes enables real-time monitoring of enzyme activities and provides quantitative data which are compared to those of commonly available and recently published, more complex probes. Additionally the applicability of this method is demonstrated for peptidases via fluorophore tryptophan (Trp) interaction.

Date Published: 13 February 2009
PDF: 10 pages
Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 718503 (13 February 2009); doi: 10.1117/12.809249

Published in SPIE Proceedings Vol. 7185:
Single Molecule Spectroscopy and Imaging II
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)