Analytica Chimica Acta, accepted, 07/07/2015. This is the accepted version without proofing
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 9 of 26
4). We expected that if the recovered components were real, and not composite signals
caused by co-linearity in the data, that then the calculated anisotropy should be relatively
constant. This was the case, apart from Comp3, where the feature at Δλ= 45 nm yielded an r
value close to one, indicating that this feature was most likely scattering [1].
Figure 4: Wavelength dependence of anisotropy (dots) for Comp1-4 (a-d, respectively) with overlaid MCR
generated VV emission component (continuous line). Anisotropy values >0.5 (or negative values) indicate
regions of either very low spectral intensity (which lead to inaccurate r calculations) or scattering artefacts.
3.3 Component assignment: Component 4 (Comp4) corresponding to Trp–214 (assignment
based on peak position (SI, Fig. S-6), high emission intensity, and literature references)[16,
18, 30] dominated emission (Figure 2) with ~42–5% of the explained variance (Table 1).
Excitation profiles for Comp3 showed a weak band at ~300 nm and a strong, broad band at
~340 nm, and the corresponding emission profiles were mostly a broad band with a large Δλ
of ~110 nm except for the VV data, which also showed a narrow band at Δλ ~45 nm. This
narrow band was due to the Raman band of water as it had an anisotropy of ~1.0 and this
feature was also present in TSFS spectra of PBS (SI, Fig. S-7). Comp3, λ em ~440 nm,
originated from Trp RTP, and to prove this, the heavy atom method was used to increase the
phosphorescence signal [5]. When MCR was re-run, Comp3 scores increased dramatically
corresponding to the expected increase in RTP signal (SI, Fig. S-8/9). These results correlate
with previously published data on HSA and other proteins, and thus confirm unambiguously
the simultaneous observation/resolution of Trp phosphorescence and fluorescence.[5, 46]
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 9 of 26
4). We expected that if the recovered components were real, and not composite signals
caused by co-linearity in the data, that then the calculated anisotropy should be relatively
constant. This was the case, apart from Comp3, where the feature at Δλ= 45 nm yielded an r
value close to one, indicating that this feature was most likely scattering [1].
Figure 4: Wavelength dependence of anisotropy (dots) for Comp1-4 (a-d, respectively) with overlaid MCR
generated VV emission component (continuous line). Anisotropy values >0.5 (or negative values) indicate
regions of either very low spectral intensity (which lead to inaccurate r calculations) or scattering artefacts.
3.3 Component assignment: Component 4 (Comp4) corresponding to Trp–214 (assignment
based on peak position (SI, Fig. S-6), high emission intensity, and literature references)[16,
18, 30] dominated emission (Figure 2) with ~42–5% of the explained variance (Table 1).
Excitation profiles for Comp3 showed a weak band at ~300 nm and a strong, broad band at
~340 nm, and the corresponding emission profiles were mostly a broad band with a large Δλ
of ~110 nm except for the VV data, which also showed a narrow band at Δλ ~45 nm. This
narrow band was due to the Raman band of water as it had an anisotropy of ~1.0 and this
feature was also present in TSFS spectra of PBS (SI, Fig. S-7). Comp3, λ em ~440 nm,
originated from Trp RTP, and to prove this, the heavy atom method was used to increase the
phosphorescence signal [5]. When MCR was re-run, Comp3 scores increased dramatically
corresponding to the expected increase in RTP signal (SI, Fig. S-8/9). These results correlate
with previously published data on HSA and other proteins, and thus confirm unambiguously
the simultaneous observation/resolution of Trp phosphorescence and fluorescence.[5, 46]