A. A. Banishev

Laser fluorimetry of proteins containing one and two tryptophan residues

The true molecular photophysical parameters’ values of tryptophan residues in single-tryptophan-containing and two-tryptophan-containing proteins (by the example of human serum albumin and bovine serum albumin) have been determined for the first time with the use of the authors’ suggested algorithm. The algorthm is based on the simultaneous use of nonlinear and kinetic-laser fluorimetry. The obtained result opens up new approaches in the monitoring of living systems and other objects, containing single fluorophores and localized pairs of fluorophores.

A nanosecond laser fluorimeter

A method for determining the fluorescence decay times in the range 1–10 ns using a nanosecond laser fluorimeter is described. The fluorimeter has the following performance characteristics: a width of exciting pulses of 10 ns, a width of gate pulses of 10 ns, and a step of gate-pulse displacement of 2.5 ns. It is shown that this instrument allows determination of fluorescence decay times with an accuracy of 1–1.5 ns.

Localized donor-acceptor pairs of fluorophores: Determination of the energy transfer rate by nonlinear fluorimetry

A laser fluorescence method has been developed for determining the energy transfer rate. It is based on the model of collective states of localized donor-acceptor pairs of fluorophores proposed in our previous work and involves nonlinear fluorimetry (other photophysical parameters of the donor and acceptor are simultaneously determined). The proposed approach has been tested on mRFP1 fluorescent (red) protein.

Determination of photophysical parameters of red fluorescent protein mRFP1 under ultraviolet excitation by methods of laser fluorimetry.

We investigate photophysical processes that take place in macromolecules of a fluorescent protein mRFP1 under UV excitation [when the energy transfer in a localized donor-acceptor (LDA) pair, which is presented in the molecules of the protein, becomes apparent]. We used a special approach based on the fluorescence laser spectroscopy technique. The energy transfer rates in LDA pairs and photophysical parameters of fluorophores (chromophores) of three spectral forms, which coexist in the ensemble of the macromolecules of this protein, were determined under pulse UV laser excitation.

Determination of the quantum yield of singlet-triplet conversion in complex organic compounds by the method of laser fluorimetry

The possibilities of the nonlinear laser fluorimetry method for determination of the quantum yield into the triplet state are demonstrated. The values of the absorption cross section and the triplet-state quantum yield are determined from the fluorescent saturation curves for 6-aminophenolenone and rhodamine 6G (used as a test material).

Non-linear fluorimetry of fluorescent proteins

The results of development of the method of fluorescent proteins investigation are discussed. Photophysical parameters and concentrations of the chromophores of protein mRFP1 have been determined with use of non-linear fluorimetry.