New fluorescence technique for ultra-sensitive enzyme characterization developed - Screening catalytic activity at the single-molecule level
Enzymes are characterized by and valued for their ability
to catalyze a wide range of chemical reactions. Consequently,
precise analytical techniques for monitoring enzyme functions
are indispensable tools for the understanding of biological
phenomena at the molecular level, and for the development of
new pharmaceuticals. A research team at the Max Planck
Institute for Biophysical Chemistry in Göttingen, Germany,
headed by the Nobel laureate Manfred Eigen, published two
articles in PNAS (Proceedings of theNational Academy of
Sciences) introducing dual-color fluorescence
cross-correlation spectroscopy as a new method for an
ultra-sensitive characterization of enzyme activity (PNAS
95:1416-1420, abstract: http://www.pnas.org/cgi/content/abstract/95/4/1416)
and demonstrating its potential for high throughput screening
(PNAS 95:1421-1426, abstract: http://www.pnas.org/cgi/content/abstract/95/4/1421).
In recent years, fluorescence correlation spectroscopy (FCS)
has become an attractive analytical tool for the
investigation of biomolecular processes. Due to the interplay
of modern confocal optics, new dyes as efficient fluorescent
probes, sensitive photodetectors, and fast data processing,
FCS allows the observation of the dynamics of single
molecules in real time while they pass an open volume element
of about one femtoliter, i.e. only the size of a common
bacterial cell. This method was invented and worked out by
groups at the Cornell University, Ithaca, at the Karolinska
Institute, Stockholm, and at the Max Planck Institute,
Göttingen; nowadays it has found its way into several
laboratories and companies all over the world as a tool for
basic research as well as for industrial applications such as
drug screening.
Dual-color fluorescence cross-correlation spectroscopy
(dual-color FCS) was proposed by Rudolf Rigler, Stockholm and
Manfred Eigen, Göttingen, in the early nineties, and has
recently been implemented by Eigens group in
Göttingen. In contrast to conventional single-color FCS, the
new method uses two perfectly superimposed laser foci, and
detects correlated fluctuations that arise from single
molecules carrying two spectrally distinguishable fluorescent
labels. It allows precise and highly specific detection of
molecules on a large unspecific fluorescent background;
moreover, the method is fully compatible with biological
environments. Two important applications were addressed by
the authors: Real-time measurements of enzyme kinetics were
successfully performed, and the suitability of dual-color FCS
for high throughput screening has been demonstrated.
Kinetics of biomolecular interactions like nucleic acid
hybridization or protein aggregation were previously
investigated by Eigen and coworkers applying dual-color FCS.
Now, it has been extended to real-time analyses of enzyme
kinetics. As a first application, the kinetic parameters of
the cleavage reaction of a double-helix DNA catalyzed by a
specific endonucleolytic enzyme were determined. Furthermore,
catalytic activity down to enzyme concentrations of one
picomole per liter was detected with high reproducibility.
This is at least one order of magnitude more sensitive as
compared to other homogeneous endonucleolytic assays. As the
authors pointed out, this technique can, in principle, be
used for any reaction in which a (covalent or non-covalent)
linkage between two different fluorophores is either broken
or formed. This includes other hydrolytic enzymes like
proteases, esterases or glycolyases, as well as ligating
enzymes.
High-throughput screening with dual-color FCS was
demonstrated in the second article; this combination is
termed RAPID FCS (Rapid Assay Processing by Integration of
Dual-color Fluorescence Cross-correlation Spectroscopy).
While conventional FCS identifies molecules by their
diffusion properties, requiring a considerable amount of
analysis time, dual-color FCS simply counts doubly
labeled molecules; therefore, analysis times for simple
yes-or-no decisions are much shorter and data evaluation is
faster. As reported by the authors, analysis times of one
second per sample and less were achieved with endonucleolytic
assays; sample volumes could even be reduced to
submicroliters without decreasing the signal strength. RAPID
FCS can probe 104 to 105 samples per
day, and possibly more. Therefore, this technology is an
ideal tool for ultra-high throughput screening when combined
with nano-technology; and it will gain access to progressive
selection strategies in evolutionary biotechnology, in which
rare and specific binding or catalytic properties have to be
screened in large numbers of samples.
Journal
Proceedings of the National Academy of Sciences