Fluorescence Anisotropy of Tyrosinate Anion Using One-, Two- and Three-Photon Excitation

Abstract

We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250–310 nm), two-photon (570–620 nm) and three-photon (750–930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pKa 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λmax 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565–580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at −60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos6θ to cos2θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.

Fluorescence Anisotropy Sensor Comprising a Dual Hollow-Core Antiresonant Fiber Polarization Beam Splitter

“…Indeed, in the case of two photon excitation (2PE), r f is 4/7 due to a higher photoselection for the two-photon absorption process [29]; above is a result of the 2PE's probability being proportional to cos 4 θ instead of cos 2 θ, as in the case of 1PE [29][30][31]. Similar results have been observed for the three-and four-photon excitation, which follow the cos 6 θ and cos 8 θ photoselection rules and have their r f equal to 2/3 and 8/11, respectively [20,[32][33][34].…”

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

“…A vast number of biological applications of fluorescence anisotropy have been reported to date due to the comparable timescale of rotational diffusion of biopolymers and the fluorescence lifetime of many fluorophores. Anisotropy measurement can provide information on the shape and size of proteins and have been used to measure protein-protein associations, the fluidity of membranes, binding and conformational dynamics [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]. However, fluorescence anisotropy is highly dependent on the environment (solvent viscosity), the size and shape of the fluorophore and the flexibility of the protein [ 2 ].…”

Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 2 Caused by CYS59TYR BOLA3 Mutation

“…In order to further examine the potential involvement of Tyr59 in cluster-binding in place of Cys59, we performed fluorescence emission spectroscopy, which has been extensively applied to discriminate between a tyrosine having a protonated or a deprotonated phenolic side-chain group (i.e., phenol- or phenoxide-tyrosine hereafter, respectively) in proteins [ 47 , 48 , 49 , 50 ], with the phenoxide state of the tyrosine being the only one able to coordinate a metal cofactor. A phenoxide-tyrosine has an emission maximum at 345 nm [ 51 ] while a phenol-tyrosine has an emission maximum at 303 nm [ 52 ].…”