A. Yu. Chikishev

Oxyluciferin fluorescence is a model of native bioluminescence in the firefly luciferin—luciferase system

Abstract The steady state and subnanosecond time-resolved fluorescence properties of oxyluciferin and its structural analogues luciferin (LH2), 6′-methoxyluciferin (MeOLH2) and 2-cyano-6-hydroxybenzothiazole (BT) were investigated in aqueous (pH 1–10) and ethanol solutions. The structures of the three fluorescence emitters were identified as follows: blue, enol-phenolate; yellow—green, phenol; red, phenolate of keto form. The dissociation rate of the excited luciferin 6′-hydroxyl group is shown to be lower than the rate of emission by the phenol form itself and, consequently, although ground and excited pK values differ considerably (8.5 and −0.5 respectively), blue fluorescence is observed in non-polar and strong acidic aqueous solutions. Fluorescence decay curves for oxyluciferin and its analogues (blue and yellow—green emission maxima) were obtained in water (pH 7.8) and ethanol (96%).

Investigation of the interaction between firefly luciferase and oxyluciferin or its analogues by steady state and subnanosecond time-resolved fluorescence

Abstract Fluorescence excitation and emission spectra and fluorescence decay curves of firefly luciferase complexes with oxyluciferin (LO), luciferin (LH2), 6′-methoxyluciferin (MeOLH2) and 2-cyano-6-hydroxy-benzothiazole (BT) were obtained in aqueous (pH 2–10), aqueous—ethanolic and ethanolic solutions. The KD values of luciferase—fluorophore complexes were determined at pH 7.8 (11.3±0.3 μM for LH2, 1.5±0.5 μM for MeOLH2 and 13±1 μM for BT) and proved to be similar to the kinetically determined Km value for LH2 and Ki values for MeOLH2 and BT. The free rotation of the excited fluorophore indicates that enzyme—fluorophore complexes dissociate immediately after excitation. The different fluorescence properties of the enzyme—product (EP) complex isolated from the full reaction mixture and the complex of enzyme with synthesized oxyluciferin (E*LO) indicate structural differences in the oxyluciferin-binding site of luciferase at the time of EP and E*LO formation, which may be due to significant conformational changes in luciferase during the reaction. The microenvironment of excited luciferase-bound LO, LH2 and MeOLH2 shows more similarity to that in aqueous solution than that in ethanol. The proposed dissociation mechanism of bioluminescence is as follows: as soon as the electronically excited product (LO*) is formed, dissociation of the enzyme—product complex occurs, followed by LO* deactivation in an aqueous microenvironment (formed by the enzyme amino acid residues) and the rebinding of the deactivated product with luciferase.

Time-resolved spontaneous and coherent raman scattering of Ni-octaethylporphyrin in excited electronic states

Abstract The results are presented of a detailed analysis of the vibrational structure of the excited triplet electronic state T d of Ni-octaethylporphyrin (Ni-OEP) in solution by means of spontaneous and coherent Raman scattering spectroscopy. The application of these two methods generated complementary information about the characteristics of the T d state.

ATR-FTIR and FT Raman spectroscopy and laser cleaning of old paper samples with foxings

Samples of old paper are studied using ATR-FTIR and FT Raman spectroscopy. It is demonstrated that the methods of laser spectroscopy make it possible to distinguish between the foxing localization regions and the region of homogeneous aging (yellowing). A significant bleaching of old paper and an almost complete erasure of the foxing stains can be reached using laser irradiation.

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.

Conformational difference between ricin and ricin agglutinin in solution and crystal.

Ricin and ricin agglutinin are ribosome-inactivating proteins of the second type (RIP II). Both ricin and ricin agglutinin consist of Aand B-subunits connected to each other with a disulfide bond. The Aand B-subunits are responsible for the N-glycosidase enzymatic activity of the protein and for toxins binding to the cell, respectively. Proteins of this class have similar structures and mechanisms of toxic activity [1]. They are among the most toxic proteins.

Photoinduced formation of thiols in human hair

Raman, scanning electron, and optical microscopy of hair and spectrophotometry of soluble hair proteins are used to study the effect of UV-vis radiation on white hair. The samples of a healthy subject are irradiated using a mercury lamp and compared with non-irradiated (control) hair. The cuticle damage with partial exfoliation is revealed with the aid of SEM and optical microscopy of semifine sections. Gel filtration chromatography shows that the molecular weight of soluble proteins ranges from 5 to 7kDa. Absorption spectroscopy proves an increase in amount of thiols in a heavier fraction of the soluble proteins of irradiated samples under study. Raman data indicate a decrease in the amount of SS and CS bonds in cystines and an increase in the amount of SH bonds due to irradiation. Such changes are more pronounced in peripheral regions of hair. Conformational changes of hair keratins presumably related to the cleavage of disulfide bonds, follow from variations in amide I and low-frequency Raman bands. An increase in the content of thiols in proteins revealed by both photometric data on soluble proteins and Raman microspectroscopy of hair cuts can be used to develop a protocol of the analysis of photoinduced hair modification.

Polarization-Sensitive CARS of the Amide I Band of Pure and Liganded Chymotrypsin

Polarization-sensitive coherent anti-Stokes Raman scattering (PSCARS) is used to investigate the secondary structure of the protein chymotrypsin, both free and bound to antranilic acid. Advantage is taken of the extreme sensitivity of the PSCARS spectra to the orientation of the analyser. Clear changes are observed in the protein spectra as a result of binding to antranilic acid. It is concluded that PSCARS can be fruitfully applied to detect changes in the bonds of the peptide backbone of enzymatic proteins as a result of substrate binding.

Subglobular motion and proton transfer model in the α-chymotrypsin molecule

Abstract The relation between the internal dynamics of the protein molecule and the active site functioning during catalysis is considered within the framework of a simple electromechanical model. The time of the functionally important proton transfer is estimated. The role of damping of subglobular motion is discussed.

Optoacoustic measurements of the porosity of paper samples with foxings

The goal of this work is to investigate the porosity of old paper with foxings. Initially, paper samples are characterized using the conventional absorption and Fourier transform infrared spectroscopies. Optoacoustic method based on laser excitation of short ultrasound transients is used for porosity measurements. The paper porosity is calculated using the theory of two-component mixture with the experimentally determined sound speeds in paper samples saturated with liquids. An increase in the porosity in foxing stains on paper in comparison with the foxing-free fragments is clearly demonstrated.