8 Analytical Protocols in Phycobiliproteins Analysis
183
Study of energy transfer between tetrapyrrole chromophores during photosynthesis is an important topic in PBPs ‘research. The energy transfers in PBPs have
been investigated by steady-state fluorescence and fluorescence polarization spectroscopy, as well as by fluorescence lifetime and kinetic absorption spectroscopy
(Li et al. 2019). Single-molecule fluorescence spectroscopy was used for analysis
of switching of PC between different conformations and the role of this process in
energy transmission to photosystem I (Gwizdala et al. 2018). UV/VIS absorption,
fluorescence anisotropy, and CD spectroscopy are also useful tools for studying the
effects of ionic strength, pH, and protein concentration on oligomerization states
of PBPs (Thoren et al. 2006). Additionally, analytical ultracentrifugation enables
analysis of PC at smaller concentrations (≤0.01 mg/mL), and the determination of
equilibrium constants between different oligomerization states (monomers, trimers,
and hexamers) of PC (Berns and MacColl 1989).
8.4 PBPs Quantification and Stability Measurements
Tetrapyrrole chromophores are responsible for the typical color of various PBPs
and have characteristic light absorption properties: PE, pink-purple, λ max = 540–
570 nm; PC, blue, λ max = 610–620 nm, and APC, bluish-green, λ max = 650–655 nm
(Dufossé 2018). The quantity of PBPs/chromophores in solution is routinely determined by measurements of absorption in the visible spectral region (usually at the
wavelength(s) of PBSs maximum absorption). In general, four sets of equations,
combining the extinction coefficients, are used for the estimation of PBPs concentrations. The first was described by Bennett and Bogorad (1973) and the concentrations
are given in mg/mL:
[PC] = (A 615 − 0.474 A 652 ) /5.34;
[APC] = (A 652 − 0.205 A 615 ) /5.09;
[PE] = (A 562 − 2.41[PC] − 0.849 [APC]) /9.62.
The second set was proposed by Kursar et al. (1983) with concentrations expressed
as μg/mL:
[APC] = 181.3 A 651 − 22.3 A 614 ;
[PC] = 151.1 A 614 − 99.1 A 651 ;
[PE] = 155.8 A 498.5 −40.0 A 614 − 10.5 A 651 .
For expression of the hexameric form concentration in mol/L, the authors assumed
the molar mass of 210 kDa (APC), 225 kDa (PC), and 250 kDa (for PE [(αβ) 6 γ]).
For expression of chromophores concentration in mol/L, the number of tetrapyrroles
per hexamer was assumed to be 12 (APC), 18 (PC), and 40 (for PE [(αβ) 6 γ]).
183
Study of energy transfer between tetrapyrrole chromophores during photosynthesis is an important topic in PBPs ‘research. The energy transfers in PBPs have
been investigated by steady-state fluorescence and fluorescence polarization spectroscopy, as well as by fluorescence lifetime and kinetic absorption spectroscopy
(Li et al. 2019). Single-molecule fluorescence spectroscopy was used for analysis
of switching of PC between different conformations and the role of this process in
energy transmission to photosystem I (Gwizdala et al. 2018). UV/VIS absorption,
fluorescence anisotropy, and CD spectroscopy are also useful tools for studying the
effects of ionic strength, pH, and protein concentration on oligomerization states
of PBPs (Thoren et al. 2006). Additionally, analytical ultracentrifugation enables
analysis of PC at smaller concentrations (≤0.01 mg/mL), and the determination of
equilibrium constants between different oligomerization states (monomers, trimers,
and hexamers) of PC (Berns and MacColl 1989).
8.4 PBPs Quantification and Stability Measurements
Tetrapyrrole chromophores are responsible for the typical color of various PBPs
and have characteristic light absorption properties: PE, pink-purple, λ max = 540–
570 nm; PC, blue, λ max = 610–620 nm, and APC, bluish-green, λ max = 650–655 nm
(Dufossé 2018). The quantity of PBPs/chromophores in solution is routinely determined by measurements of absorption in the visible spectral region (usually at the
wavelength(s) of PBSs maximum absorption). In general, four sets of equations,
combining the extinction coefficients, are used for the estimation of PBPs concentrations. The first was described by Bennett and Bogorad (1973) and the concentrations
are given in mg/mL:
[PC] = (A 615 − 0.474 A 652 ) /5.34;
[APC] = (A 652 − 0.205 A 615 ) /5.09;
[PE] = (A 562 − 2.41[PC] − 0.849 [APC]) /9.62.
The second set was proposed by Kursar et al. (1983) with concentrations expressed
as μg/mL:
[APC] = 181.3 A 651 − 22.3 A 614 ;
[PC] = 151.1 A 614 − 99.1 A 651 ;
[PE] = 155.8 A 498.5 −40.0 A 614 − 10.5 A 651 .
For expression of the hexameric form concentration in mol/L, the authors assumed
the molar mass of 210 kDa (APC), 225 kDa (PC), and 250 kDa (for PE [(αβ) 6 γ]).
For expression of chromophores concentration in mol/L, the number of tetrapyrroles
per hexamer was assumed to be 12 (APC), 18 (PC), and 40 (for PE [(αβ) 6 γ]).