Staff Members of the Institute of Biochemistry, TU Graz http://www ...

2.1. Fluorescent suicide inhibitors for in-gel analysis of lipases and esterases
Fluorescent suicide inhibitors have been developed as activity recognition probes
(ARPs) for qualitative and quantitative analysis of active lipolytic enzymes in complex
biological samples (industrial enzyme preparations, serum, cells, tissues). Since
inhibitor binding to the active sites of lipases and esterases is specific and
stoichiometric, accurate information can be obtained about the type of enzyme and the
moles of active protein (active sites) in electrophoretically pure or heterogeneous
enzyme preparations.
Labelling of enzymes with an ARP specific for serine hydrolases
BG
Inactive
Inactive
RG NBD
RG: Reacti v e p h osphonate gr o up,
irreversibly inhibits the catalytic serine
Active
BG
BG: Binding group: is specifically recognized
by the active enzyme
Inactive
Inactive
Inhibited
RG
NBD
NBD : fluorescent tag
λ : 488 nm; λ : 540 nm
ex
em
Fluorescent inhibitors are currently applied to the proteomic analysis of the lipolytic
enzymes in human and animal cells. These studies are performed in the framework of
the joint project GOLD (Genomics of Lipid-associated Disorders-coordinated by KFU
Graz) which aims at discovering novel genes, processes and pathways that regulate lipid
homeostasis in humans, mice and yeast. This is one of the projects in the field of
functional genomics (GEN-AU, GENome research in AUstria) funded by the Austrian
Federal Ministry for Education, Science and Culture (bm:bwk).
Activity-based enzyme stain
Total protein stain
The lipolytic proteome of
mouse adipose tissue
Proteins of brown adipose tissue from fasted wild-type mice were labelled with a
fluorescent lipase inhibitor followed by 2-D electrophoretic separation. The
fluorescently tagged enzymes were detected with a laser scanner. A total protein stain
(Sypro rubyTM) is shown as a control. Fluorescent spots were cut out and after tryptic
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