E. I. Dementieva

Quenching of tryptophan fluorescence of firefly luciferase by substrates

The interaction of firefly luciferase with substrates (luciferin and MgATP) by steady-state and time-resolved fluorescence is studied. The efficient quenching of tryptophan fluorescence of the active enzyme takes place upon its binding with substrates. In the presence of ATP the quenching is of dynamic type and is caused by structural changes in the protein molecule upon ATP binding. A model is proposed in which the complex has smaller fluorescence quantum yield than the free enzyme because of partial quenching of tryptophan fluorescence by the new microenvironment. Quenching of tryptophan fluorescence by luciferin due to the efficient energy transfer from tryptophan to luciferin is discussed. The calculated distance between Trp-419 and luciferin for the L. mingrelica luciferase in the enzyme-substrate complex is less than 12 A.

Recombinant Firefly Luciferase in Escherichia coli: Properties and Immobilization

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.

Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin

Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 Å for P. pyralis and 12–17Å for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.

Transient increase of tryptophan fluorescence of enzyme caused by photoexcitation of ligand in luciferase–luciferin complex

An experiment was proposed and accomplished that was based on the hypothesis of the dissociation of the luciferase-luciferin complex in photoexcitation. A pump-probe experiment was performed with the use of picosecond laser pulses and was based on the effect of quenching of enzyme tryptophan fluorescence caused by luciferin binding. A photoinduced increase of the tryptophan fluorescence intensity was detected. Experimental results were interpreted on the basis of the assumptions on photoinduced dissociation of the luciferin-luciferase complex and Forster energy transfer from tryptophan to luciferin. Under the assumption on the photoinduced dissociation and stationary quenching of tryptophan fluorescence the rate of propagation of the conformational changes in the protein caused by the complex dissociation was estimated to be >20 m/s.

Bioluminescence and Chemiluminescence

Emission of energy in the form of visible light during chemical or biochemical processes, so-called chemiluminescence (CL) or bioluminescence (BL), is not a rare phenomenon in nature. There are numerous organisms, both terrestrial and aquatic (freshwater and marine forms), that emit light (Hastings, 1983).

Bioluminescent Analysis of β-Galactosidase and Its Application for Immunoassay and DNA-Probes

Abstract Bioluminescent method for β-galactosidase assay is proposed with sensitivity as high as 0.25 fmol/1 with the help of bioluminogenic substrate – firefly luciferin derivative. Method was applied to immunoassay of foot-and-mouth disease viral antigen and biotinylated DNA-probe analysis with the help of β-galactoside labelled conjugates. Methods proved to be very sensitive, rapid and simple in performance. Photographic detection and reprobing of the samples are possible.