Analytica Chimica Acta, accepted, 07/07/2015. This is the accepted version without proofing
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 1 of 26
Anisotropy Resolved Multidimensional Emission Spectroscopy (ARMES):
a new tool for protein analysis.
Radu Constantin Groza, Boyan Li, and Alan G. Ryder.*
Nanoscale BioPhotonics Laboratory, School of Chemistry, National University of Ireland,
Galway, Galway, Ireland.
* Corresponding author: Email: alan.ryder@nuigalway.ie, Phone: +353-91-492943.
Postal address: Nanoscale Biophotonics Laboratory, School of Chemistry, National
University of Ireland, Galway, University Road, Galway, Ireland.
Running Title: Protein analysis using multidimensional fluorescence anisotropy.
Abstract:
Structural analysis of proteins using the emission of intrinsic fluorophores is complicated by
spectral overlap. Anisotropy resolved multidimensional emission spectroscopy (ARMES)
overcame the overlap problem by the use of anisotropy, with chemometric analysis, to
resolve the emission of each fluorophore. Total synchronous fluorescence scan (TSFS)
provided information about all the fluorophores that contributed to emission while anisotropy
provided information about the mobility of each fluorophore. Here the utility of ARMES
was demonstrated via study of the chemical and thermal denaturation of human serum
albumin (HSA).
Multivariate curve resolution (MCR) analysis of the constituent polarized emission
ARMES data resolved contributions from four emitters: fluorescence from tryptophan (Trp),
solvent exposed tyrosine (Tyr), Tyr in a hydrophobic environment, and room temperature
phosphorescence (RTP) from Trp. The MCR scores, anisotropy, and literature validated
these assignments and showed all the expected transitions during HSA unfolding. This new
methodology for comprehensive intrinsic fluorescence analysis of proteins is applicable to
any protein containing multiple fluorophores.
Keywords: Fluorescence, Anisotropy, Multidimensional, Protein, Unfolding, Chemometrics.
corrections. DOI: 10.1016/j.aca.2015.06.011
.
Page 1 of 26
Anisotropy Resolved Multidimensional Emission Spectroscopy (ARMES):
a new tool for protein analysis.
Radu Constantin Groza, Boyan Li, and Alan G. Ryder.*
Nanoscale BioPhotonics Laboratory, School of Chemistry, National University of Ireland,
Galway, Galway, Ireland.
* Corresponding author: Email: alan.ryder@nuigalway.ie, Phone: +353-91-492943.
Postal address: Nanoscale Biophotonics Laboratory, School of Chemistry, National
University of Ireland, Galway, University Road, Galway, Ireland.
Running Title: Protein analysis using multidimensional fluorescence anisotropy.
Abstract:
Structural analysis of proteins using the emission of intrinsic fluorophores is complicated by
spectral overlap. Anisotropy resolved multidimensional emission spectroscopy (ARMES)
overcame the overlap problem by the use of anisotropy, with chemometric analysis, to
resolve the emission of each fluorophore. Total synchronous fluorescence scan (TSFS)
provided information about all the fluorophores that contributed to emission while anisotropy
provided information about the mobility of each fluorophore. Here the utility of ARMES
was demonstrated via study of the chemical and thermal denaturation of human serum
albumin (HSA).
Multivariate curve resolution (MCR) analysis of the constituent polarized emission
ARMES data resolved contributions from four emitters: fluorescence from tryptophan (Trp),
solvent exposed tyrosine (Tyr), Tyr in a hydrophobic environment, and room temperature
phosphorescence (RTP) from Trp. The MCR scores, anisotropy, and literature validated
these assignments and showed all the expected transitions during HSA unfolding. This new
methodology for comprehensive intrinsic fluorescence analysis of proteins is applicable to
any protein containing multiple fluorophores.
Keywords: Fluorescence, Anisotropy, Multidimensional, Protein, Unfolding, Chemometrics.