Analytica Chimica Acta, accepted, 07/07/2015. This is the accepted version without proofing
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 14 of 26
the fact that unfolding of subdomain IIIA exposed a smaller hydrophobic area to the solvent
compared to the unfolding of subdomain IIA, which leads to lower structural disruption [15,
55]. This behavior was obvious from the scores plots where the degree of change in Comp1
was greater than that for Comp2.
4. Conclusions
ARMES can be defined as the resolving of fluorophore emission from mixtures based
on the differential emission polarization of the constituent fluorophores caused by variations
in rotational correlation times or fluorescence lifetimes, which is achieved by the use of
multi-way chemometrics. The ARMES method in this iteration with its combined use of
aniso-TSFS and MCR based analysis provides a unique approach for studying protein
unfolding/folding/denaturation processes.
Since anisotropy in multi-fluorophore
macromolecules was fundamentally sensitive to both emission and excitation wavelengths we
get unique spectral data, which can be analyzed using MCR to show how the emission of
each fluorophore (or groups of closely related fluorophores) changes in terms of both
intensity and anisotropy. The anisotropy element in particular facilitates the resolution of
near identical fluorophores, which have overlapping emission spectra. ARMES can be
considered as being analogous to time-resolved emission spectroscopy (TRES). However, it
does not require the use of complicated time-resolved instrumentation, and furthermore it can
resolve overlapping fluorophores that have very similar lifetimes based on their local rigidity.
For HSA in solution, ARMES recovered four emitting species, three fluorescent and
one phosphorescent. This resolving of RTP from the stronger overlapping fluorescence was a
key advantage of this method. Furthermore, by following the MCR scores of the raw data
and the aniso-TSFS data we were able to easily follow the unfolding process and
unambiguously identify different processes, and changes in refolded HSA. Even with the
limitations of MCR recovered emission/excitation spectra, the method provides a unique tool,
which can be used for any multi-fluorophore macromolecular system in its present form.
As with many fluorescence techniques ARMES is non-destructive and relatively rapid,
but has distinct advantages in uses standard fluorescence spectrophotometers, requires no
extrinsic labels, and delivers a wealth of information simultaneously about all the
fluorophores. Another big advantage if the fact that it is also feasible to recover ARMES
data from samples in which there is interfering background signals from small molecule
fluorophores, enabling in-situ protein characterization, for example in the presence of cell
culture media [41]. The current limitations of the method as currently implemented are
largely related to the time taken to collect all four constituent HH, VV, HV, and VH
measurements and time required for data analysis. This limits its application for kinetic
measurements, and reduces the scope for high throughput screening applications. However,
these limitations might be overcome through revised spectrometer design and the automation
of the data analysis methods.
5. Supplemental information available
Supporting information is available and includes further details on the spectral and quantitative
analyses.
Acknowledgements
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 14 of 26
the fact that unfolding of subdomain IIIA exposed a smaller hydrophobic area to the solvent
compared to the unfolding of subdomain IIA, which leads to lower structural disruption [15,
55]. This behavior was obvious from the scores plots where the degree of change in Comp1
was greater than that for Comp2.
4. Conclusions
ARMES can be defined as the resolving of fluorophore emission from mixtures based
on the differential emission polarization of the constituent fluorophores caused by variations
in rotational correlation times or fluorescence lifetimes, which is achieved by the use of
multi-way chemometrics. The ARMES method in this iteration with its combined use of
aniso-TSFS and MCR based analysis provides a unique approach for studying protein
unfolding/folding/denaturation processes.
Since anisotropy in multi-fluorophore
macromolecules was fundamentally sensitive to both emission and excitation wavelengths we
get unique spectral data, which can be analyzed using MCR to show how the emission of
each fluorophore (or groups of closely related fluorophores) changes in terms of both
intensity and anisotropy. The anisotropy element in particular facilitates the resolution of
near identical fluorophores, which have overlapping emission spectra. ARMES can be
considered as being analogous to time-resolved emission spectroscopy (TRES). However, it
does not require the use of complicated time-resolved instrumentation, and furthermore it can
resolve overlapping fluorophores that have very similar lifetimes based on their local rigidity.
For HSA in solution, ARMES recovered four emitting species, three fluorescent and
one phosphorescent. This resolving of RTP from the stronger overlapping fluorescence was a
key advantage of this method. Furthermore, by following the MCR scores of the raw data
and the aniso-TSFS data we were able to easily follow the unfolding process and
unambiguously identify different processes, and changes in refolded HSA. Even with the
limitations of MCR recovered emission/excitation spectra, the method provides a unique tool,
which can be used for any multi-fluorophore macromolecular system in its present form.
As with many fluorescence techniques ARMES is non-destructive and relatively rapid,
but has distinct advantages in uses standard fluorescence spectrophotometers, requires no
extrinsic labels, and delivers a wealth of information simultaneously about all the
fluorophores. Another big advantage if the fact that it is also feasible to recover ARMES
data from samples in which there is interfering background signals from small molecule
fluorophores, enabling in-situ protein characterization, for example in the presence of cell
culture media [41]. The current limitations of the method as currently implemented are
largely related to the time taken to collect all four constituent HH, VV, HV, and VH
measurements and time required for data analysis. This limits its application for kinetic
measurements, and reduces the scope for high throughput screening applications. However,
these limitations might be overcome through revised spectrometer design and the automation
of the data analysis methods.
5. Supplemental information available
Supporting information is available and includes further details on the spectral and quantitative
analyses.
Acknowledgements