Analytica Chimica Acta, accepted, 07/07/2015. This is the accepted version without proofing
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 5 of 26
ranges). A weaker broad band (λ ex = 290–370/Δλ= 40–150 nm), corresponding to emission
band maxima at 444 nm (HH/VH) and 454–460 nm (VV/HV) spectra was ascribed to Trp–
214 RTP (vide infra).
TSFS spectra and the constituent emission and excitation maxima were all
significantly red-shifted. This was because most standard fluorescence spectrometers (as
used here) have polymer thin film polarizers for anisotropy measurements and these typically
cut-off below 290–300 nm (SI, Fig. S-3) which resulted in distorted MDF spectra. This
caused the excitation maxima of the recovered components to be red-shifted by amounts that
were equal to the real excitation maximum plus the spectral distance to the cut-off point. For
TSFS, emission maxima (λ ex +Δλ) were also shifted towards longer wavelengths by the same
spectral distance as the excitation maxima. This also reshaped the polarized TSFS spectra
compared to non-polarized spectra (SI, Fig. S-4). One advantage of this effect is that it
compensated for the lack of tri-linearity in TSFS data which facilitated reliable component
recovery using MCR (vide infra).
Figure 1: Changes in the aniso-TSFS contour plots for HSA 1mg mL
–1 in PBS undergoing thermal unfolding:
a) 10 °C, b) 50 °C, and c) after cooling from 7020 °C, and chemical unfolding at 20 °C with: d) 0 M, e) 2 M,
and f) 4 M GuHCl. The color bar on the right represents anisotropy.
Aniso-TSFS plots (Figure 1) have a distinct striped pattern, which highlighted the
significant anisotropy variation across the emission space. This was due to numerous factors
including: fluorophore type and number, variability in mobility of fluorophores located in
different protein domains, variations in intra-molecular FRET, and changing local chemical
environment. In contrast, aniso-TSFS plots of simple fluorophores with single state emission
show constant anisotropy values across the entire emission space [41]. HSA aniso-TSFS
patterns were very different for thermal compared to chemical denaturation because of the
different unfolding pathways. Univariate analysis of average anisotropy values (SI, Fig. S-5)
did not adequately explain which fluorophores contributed to the anisotropy and pattern
changes.
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 5 of 26
ranges). A weaker broad band (λ ex = 290–370/Δλ= 40–150 nm), corresponding to emission
band maxima at 444 nm (HH/VH) and 454–460 nm (VV/HV) spectra was ascribed to Trp–
214 RTP (vide infra).
TSFS spectra and the constituent emission and excitation maxima were all
significantly red-shifted. This was because most standard fluorescence spectrometers (as
used here) have polymer thin film polarizers for anisotropy measurements and these typically
cut-off below 290–300 nm (SI, Fig. S-3) which resulted in distorted MDF spectra. This
caused the excitation maxima of the recovered components to be red-shifted by amounts that
were equal to the real excitation maximum plus the spectral distance to the cut-off point. For
TSFS, emission maxima (λ ex +Δλ) were also shifted towards longer wavelengths by the same
spectral distance as the excitation maxima. This also reshaped the polarized TSFS spectra
compared to non-polarized spectra (SI, Fig. S-4). One advantage of this effect is that it
compensated for the lack of tri-linearity in TSFS data which facilitated reliable component
recovery using MCR (vide infra).
Figure 1: Changes in the aniso-TSFS contour plots for HSA 1mg mL
–1 in PBS undergoing thermal unfolding:
a) 10 °C, b) 50 °C, and c) after cooling from 7020 °C, and chemical unfolding at 20 °C with: d) 0 M, e) 2 M,
and f) 4 M GuHCl. The color bar on the right represents anisotropy.
Aniso-TSFS plots (Figure 1) have a distinct striped pattern, which highlighted the
significant anisotropy variation across the emission space. This was due to numerous factors
including: fluorophore type and number, variability in mobility of fluorophores located in
different protein domains, variations in intra-molecular FRET, and changing local chemical
environment. In contrast, aniso-TSFS plots of simple fluorophores with single state emission
show constant anisotropy values across the entire emission space [41]. HSA aniso-TSFS
patterns were very different for thermal compared to chemical denaturation because of the
different unfolding pathways. Univariate analysis of average anisotropy values (SI, Fig. S-5)
did not adequately explain which fluorophores contributed to the anisotropy and pattern
changes.