**1. Introduction**

This work was initiated by the problem of investigating the photophysical properties of complex protein molecules and performing the diagnostics of such molecules in water environment. At the present time, fluorescence spectroscopy (fluorimetry) is widely used to study complex organic compounds (COC) (Lakowicz, 1999). Together with spectrophotometry these methods form the basis for fast and nondestructive diagnostics of COC in the natural environment, i.e. they present the diagnostic methods *in vivo* and *in situ*. However, the conventional (linear) fluorescence spectroscopy methods can not provide complete information on fluorescent objects under study because of insufficient selectivity (fluorescence bands of most COC are broad and structureless at room temperature).

The capabilities of fluorescence spectroscopy can be enhanced by using the methods of laser fluorimetry, in particular nonlinear laser fluorimetry (Fadeev et al., 1999). This method allows one to get information on the molecular level and determine the photophysical parameters of molecules (absorption cross section, lifetime in excitation state, intersystem crossing and energy transfer rates, etc.). Furthermore, the parameters can be measured *in vivo* and *in situ* in the absence of *a priori* information, which is necessary for conventional spectroscopic methods (for example, molecular concentration (Banishev et al., 2009)).

The diagnostics of protein complexes is an intricate problem if a molecule contains more than one absorption/fluorescent center (Permyakov, 1992). The problem becomes much more complex if, in addition, the protein specimen (ensemble of molecules) is a mixture of several chemically nonidentical types of molecules (subensembles) which cannot be separated, i.e. their partial concentrations are unknown. The second situation is typical for the special kind of proteins, namely, fluorescent proteins (FPs) (Piatkevich et al., 2010). The solutions of FPs are usually mixtures of several types of molecules, which are chemically different and have their own set of photophysical properties (Verkhusha et al., 2004). In this case for unambiguous interpretation of experimental data it is necessary to make simultaneous measurements of a large number of parameters, i.e. to simultaneously apply (or, better, synthesize) several spectroscopic methods.

<sup>\*</sup> Corresponding Author

Laser Fluorescence Spectroscopy:

Application in Determining the Individual Photophysical Parameters of Proteins 185

Fig. 1. (a) The *NFl(F)* dependencies (see text): (1) in the absence of fluorescence saturation, and (1b) when saturation appears (for most COC at *F*>1023 cm−2s−1 (Fadeev et al., 1999)). (b) Photophysical processes in COC (Lakowicz, 1999), without accounting for intermolecular

The parameters of saturation curves depend on photophysical characteristics of molecules fluorophores, so that such characteristics can be extracted from these curves after resolving an inverse problem (Fadeev et al., 1999). This is a basement of nonlinear laser fluorimetry as a method for investigation of photophysical properties of COC. To solve the inverse problem, we should first calculate (either analytically or numerically) the theoretical saturation curves by using the fluorescence response formation model of an ensemble of fluorescent molecules under their excitation by laser radiation. In present work two models have been used: the conventional model of fluorescence response formation and the model

The conventional model (Banishev et al., 2008a, 2009) in describing the fluorescence response is represented as a system of equations that describes the kinetics of concentration of COC molecules at the corresponding energy states (Fig. 1(b)). In the case of monomolecular solutions of non-interactive organic compounds, we must give priority to

transition from the ground singlet state S0 (level 1) to the first excited state S1 (level 3)

the rates of radiative and radiationless transitions from S1 to S0; *K'32* is the rate of the

The model of the fluorescence response, which takes into account processes pointed out

above, can be described by the following set of kinetic equations (Fadeev et al., 1999):

*<sup>3</sup>* of the molecule in the S1 state (the fluorescence decay time);

*T=K'32/K3* of the molecule transition to the lower triplet state T1

*<sup>13</sup>*, which determines the probability of a molecule

*-1=K31+K'31+K'32; K31* and *K'31* are

the following photophysical parameters when defining the saturation curve:

stimulated by a photon flux with the density *F*;

(level 2) due to the intersystem crossing, where *K3*

interactions. Solid and dotted vertical lines are radiation and radiationless transitions

respectively, Si are singlet states and Ti are triplet states.

of localized donor-acceptor (LDA) pairs.


transition from S1 to T1.



In this chapter, a new approach based on the simultaneous use of nonlinear laser fluorimetry, spectrophotometry and conventional fluorimetry methods is presented. The approach allows us to *in vivo* determine the individual photophysical parameters of fluorophores in multi-fluorophore protein complexes. The approach has been applied for investigation of the photophysical properties of the protein molecules of different complexity. Two classes of proteins have been chosen, namely, serum albumins (by the examples of human and bovine serum albumins) and fluorescent proteins (by the example of monomeric red FP mRFP1). The following new results are presented.


Except for this key target, the methodological task has been put, namely, to demonstrate the unique capabilities of the nonlinear laser fluorimetry method (which is not, so far, well known in a wide circle of opticians) on the specific object.
