**5. Steady-state fluorescence and absorption spectroscopy**

#### **5.1 Human and bovine serum albumins**

The absorption spectra of the proteins and (for comparison) the corresponding equimolar solutions (solution of tryptophan, tyrosine, and phenylalanine at the same ratio as they are contained in protein) are shown in Fig. 4. It is seen that the parameters of the absorption bands of proteins do not coincide with the corresponding parameters for the equimolar solutions.

Fig. 4. Solid line is an absorption spectrum of (a) human and (b) bovine serum albumin. Circles are absorption spectrum of an equimolar solution with relative contents of tryptophan, tyrosine, and phenylalanine as (a) 1:18:31 and (b) 2:20:27.

The measurements of the fluorescence emission spectra of the proteins (data not showed) revealed that the fluorescence of both proteins is blue shifted relative to the tryptophan fluorescence (353 nm) in a buffer solution (Banishev et al., 2008a). This is due to a decrease in the tryptophan environment polarity in the proteins. The maximum of the HSA fluorescence (332 nm) is blue shifted in comparison with BSA (342 nm). Since the fluorescence spectrum of the tryptophan residues reflects the polarity of their nearest environment, and since the properties of the environments of Trp-212 in BSA and Trp-214 in HSA are similar (Eftink et al., 1977), such a shift can be related to the fact that BSA contains tryptophan Trp-134 located in the environment with a higher polarity (in comparison with Trp-212). Thus, the total fluorescence spectrum of BSA is red shifted. This result will be necessary for choosing the registration wavelength in measuring the acceptor and donor fluorescence when the nonlinear and kinetic curves will be measured (Section 6.1).

protein. Irradiation of the protein molecules by the light from the green spectral range provokes a conversion of G form molecules to the molecules of GH form; as a result, the protein solution will represent a mixture of only two forms, namely, GH and R. In current work, for conversion the radiation of an Ar laser (LG-106-M1) at 488 nm (300mW) was used. An increment of the protein solution pH to 9 leads to a practically total transfer of the GH form molecules to the G form molecules (in that case, the protein solution is a mixture of the G and R forms). These procedures will be used as a way to decrease the amount of simultaneously existing forms in the mRFP1 solution when the method of the nonlinear

The absorption spectra of the proteins and (for comparison) the corresponding equimolar solutions (solution of tryptophan, tyrosine, and phenylalanine at the same ratio as they are contained in protein) are shown in Fig. 4. It is seen that the parameters of the absorption bands of proteins do not coincide with the corresponding parameters for the equimolar

Fig. 4. Solid line is an absorption spectrum of (a) human and (b) bovine serum albumin. Circles are absorption spectrum of an equimolar solution with relative contents of

fluorescence when the nonlinear and kinetic curves will be measured (Section 6.1).

The measurements of the fluorescence emission spectra of the proteins (data not showed) revealed that the fluorescence of both proteins is blue shifted relative to the tryptophan fluorescence (353 nm) in a buffer solution (Banishev et al., 2008a). This is due to a decrease in the tryptophan environment polarity in the proteins. The maximum of the HSA fluorescence (332 nm) is blue shifted in comparison with BSA (342 nm). Since the fluorescence spectrum of the tryptophan residues reflects the polarity of their nearest environment, and since the properties of the environments of Trp-212 in BSA and Trp-214 in HSA are similar (Eftink et al., 1977), such a shift can be related to the fact that BSA contains tryptophan Trp-134 located in the environment with a higher polarity (in comparison with Trp-212). Thus, the total fluorescence spectrum of BSA is red shifted. This result will be necessary for choosing the registration wavelength in measuring the acceptor and donor

tryptophan, tyrosine, and phenylalanine as (a) 1:18:31 and (b) 2:20:27.

fluorimetry will be implemented (see Section 6).

**5.1 Human and bovine serum albumins** 

solutions.

**5. Steady-state fluorescence and absorption spectroscopy** 

#### **5.2 Monomeric red fluorescent protein and its mutants at residue 66**

One can see from the absorption spectra of the mRFP1 (Fig. 5(a)) that in the wavelength range from 370 to 650 nm, there exist three absorption bands, which are explained by the presence of three spectral forms in the solution (Verkhusha et al., 2004), i.e. R, G and GH forms (the corresponding absorption maxima at 584, 503 and 380 nm). One can also see that the absorption bands of G and R forms overlap. Excitation of fluorescence in the absorption band of each form indicates that G and GH forms do not fluoresce, the R form fluoresces with maxima at 607 nm. The excitation of the protein solution by irradiation at a wavelength of 270 nm (tryptophan absorption band) leads to the appearance in the signal spectrum not only of an UV band (maximum at 330 nm), which corresponds to the tryptophan fluorescence in the protein matrix, but also of a band in the visible region of wavelengths (maximum at 607 nm) corresponding to fluorescence of the chromophore mRFP1 R form (Fig. 5(b)). The chromophore of GH and G forms is non-fluorescent (Campbell et al., 2002).

Fig. 5. (a) Absorption (solid) and fluorescence excitation (dotted, registration at 607 nm) spectra of mRFP1. The spectra are normalized to the signal at 584 nm. (b) Emission of mRFP1 (excitation by 270 nm). (c) Absorption spectra of the mutants with polar and (d) nonpolar substitutions at position 66. The spectra are normalized to optical density at 270 nm.

Absorption spectra of the variants of mRFP1 with mutation at position 66 are shown in the Fig. 5(c, d).The absorption spectra structure of the mutants is similar to one for mRFP1. One can see that for all proteins in the wavelength range from 370 to 650 nm, the spectrum contains three bands with the maxima at 360-420 nm, 450-540 nm and 550-600 nm depending on the protein. These absorption peaks are corresponding to the GH, G and R forms. There only signal in the red spectral range were observed in the fluorescence emission and excitation spectra (i.e. the G form was found to be non-fluorescent) for all proteins except mRFP1/Q66F. For mRFP1/Q66F, the additional band in fluorescence excitation (maxima at 502 nm) and emission (maxima at 512 nm) spectra was detected,

Laser Fluorescence Spectroscopy:

Application in Determining the Individual Photophysical Parameters of Proteins 197

similarity of the properties of Trp-214 in HSA and Trp-212 in BSA (see Section 5.1), one can assume that, in the system of two tryptophans of BSA, Trp-212 serves as the donor of the energy (the energy transfer occurs via Forster mechanism), and Trp-214 is the acceptor (i.e.,

Fig. 6. (a) HSA and (b) BSA emission spectra at several values of photon flux density (see text); (1) is fluorescence and (2) is water Raman scattering band. (c) Saturation and (d) kinetic curves for BSA; fluorescence was registered at 390 (squares) and 310 (circles) nm. Lines are plotted using model (1b) and Eqs. (2b, c) for parameters from the Table 2.

The kinetic curves (see Eq. 6) and the fluorescence saturation curves of BSA are shown in Fig. 6(c, d). For BSA, the saturation curves depend on the registration wavelength in the wavelength range 310–390 nm. This is due to the fact that the BSA fluorescence band is a superposition of the bands of two tryptophans possessing different spectral properties. A similar difference in the curves for HSA is negligible. For the determination of the photophysical parameters of HSA fluorophore from fluorescence saturation and kinetic curves, the model (1a) and Eq. (2a) have been used. The same calculation procedure was done for BSA experimental curves, but with using the model (1b) and Eqs. (2b,c); the fluorescence signal was measured at 310 nm (when registered the fluorescence saturation and kinetic curves of the donor) and 390 nm (for similar curves of the acceptor). The resulting values of the parameters of protein fluorophores are presented in the Table 2.

As one can see from the Table 2 that the values of photophysical parameters *σ* and *τ* of Trp-214 in HSA and Trp-212 in BSA are similar. This result should have been expected based on a comparison of the structures of these proteins. The rates of energy transfer in BSA from excited donor to unexcited acceptor (*KDA*) and to excited acceptor (*KSS*) are small in

reasons: (i) in BSA, the tryptophan residues in the D–A pair are located at a distance that is

*-1*). This can be due to following

comparison with the rate of intramolecular relaxation (

its fluorescence spectrum is presumably shifted towards long wavelengths).

which attributed to the chromophore of G form. The spectral characteristics of the proteins are presented in the Table 1.

Note that the presence of the three protein forms can be qualitatively seen in the absorption spectrum (Fig. 5). However, as is described in (Banishev et al., 2009), the quantitative determination of the individual photophysical parameters of their chromophore with the help of only conventional methods are problematic. This is explained by the fact that the preparative separation of the forms is rather difficult and, as a result, it is hard to find their partial concentrations. At that rate, for example, for calculating the molar extinction coefficient (or absorption cross section) of chromophore from absorption spectra, the total protein concentration (total concentration of all forms) is used. As a result, the extinction coefficients are artificially underestimated (Kredel at al., 2008). At present, the only method that used for determining the individual extinction coefficient of chromophore of each spectral form can be found in (Ward, 2005). However, as it was pointed out in (Kredel et al., 2008), the values measured by the method are inaccurate in case of red FPs. Although, the procedure which enables to reduce the experimental errors has been proposed by (Kredel et al., 2008), the problem remains still topical.


Table 1. The position (nm) of the maximum of the absorption, fluorescence excitation and emission spectra of the proteins.
