Flavin Fluorescence Research Articles

Simultaneous assessment of NAD(P)H and flavins with multispectral fluorescence lifetime imaging microscopy at a single excitation wavelength of 750nm.

Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications. We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells. We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids. The use of a multispectral detector in combination with an excitation at a single wavelength of 750nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins. The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.

Identification by methods of steady-state and kinetic spectrofluorimetry of endogenous porphyrins and flavins sensitizing the formation of reactive oxygen species in cancer cells.

The question about acceptor molecules of optical radiation that determine the effects of photobiomodulation in relation to various types of cells still remains the focus of attention of researchers. This issue is most relevant for cancer cells, since, depending on the parameters of optical radiation, light can either stimulate their growth or inhibit them and lead to death. This study shows that endogenous porphyrins, which have sensitizing properties, may play an important role in the implementation of the effects of photobiomodulation, along with flavins. For the first time, using steady-state and kinetic spectrofluorimetry, free-base porphyrins and their zinc complexes were discovered and identified in living human cervical epithelial carcinoma (HeLa) cells, as well as in their extracts. It has been shown that reliable detection of porphyrin fluorescence in cells is hampered by the intense fluorescence of flavins due to their high concentration (micromolar range) and higher (compared to tetrapyrroles) fluorescence quantum yield. Optimization of the spectral range of excitation and the use of extractants that provide multiple quenching of the flavin component while increasing the emission efficiency of tetrapyrroles makes it possible to weaken the contribution of the flavin component to the recorded fluorescence spectra.

Design of a line-of-sight fluorescence-based imaging diagnostic for classification of microbe species

Fluorescence imaging of certain biochemicals, including flavins and pyridine nucleotides, has utility in characterizing the metabolic state of tissue and in discriminating between microbial species. There is significant clinical utility in this class of imaging techniques but most measurements reported to date require specialized training and equipment rendering most implementations unsuitable for routine medical imaging. Here, a low-cost and robust imaging technique is designed using ultraviolet-induced fluorescence of pyridine nucleotides (primarily NADH) and flavins (primarily FAD) in microbial samples. The diagnostic is optimized to distinguish between different microbial species based on previously reported spectral data using a ratiometric imaging approach. A detailed performance analysis is provided that relates the measured fluorescence intensity ratio (FIR) to the relative concentration ratio of NADH to FAD using a simplified spectroscopic model. Analysis suggests the technique is sensitive to changes in the NADH/FAD concentration ratio over several orders of magnitude, with better than 10% FIR precision on a per-pixel basis for microbial smears as thin as 10 s of microns at a resolution of 30 mm−1 and exposures of 20 ms. Representative microbe samples from eight species were imaged to demonstrate the proposed technique. Results show that the FIR varies by an order of magnitude across different species but the intra-species variation is only ∼5% for the conditions used here. An additional imaging band may be necessary to classify species that contain red pigments or bacteriochlorophyll. Radiative trapping was discussed as a possible limitation of the technique, but no clear evidence for radiative trapping was observed here. Overall, the results suggest that the proposed approach is feasible for rapid, low-cost, and robust characterization of microbial samples.

Flavin fluorescence lifetime and autofluorescence optical redox ratio for improved visualization and classification of brain tumors.

Modern techniques for improved tumor visualization have the aim to maximize the extent of resection during brain tumor surgery and thus improve patient prognosis. Optical imaging of autofluorescence is a powerful and non-invasive tool to monitor metabolic changes and transformation in brain tumors. Cellular redox ratios can be retrieved from fluorescence emitted by the coenzymes reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Recent studies point out that the influence of flavin mononucleotide (FMN) has been underestimated. Fluorescence lifetime imaging and fluorescence spectroscopy were performed through a modified surgical microscope. We acquired 361 flavin fluorescence lifetime (500-580 nm) and fluorescence spectra (430-740 nm) data points on freshly excised different brain tumors: low-grade gliomas (N=17), high-grade gliomas (N=42), meningiomas (N=23), metastases (N=26) and specimens from the non-tumorous brain (N=3). Protein-bound FMN fluorescence in brain tumors did increase with a shift toward a more glycolytic metabolism (R=-0.87). This increased the average flavin fluorescence lifetime in tumor entities with respect to the non-tumorous brain. Further, these metrics were characteristic for the different tumor entities and showed promise for machine learning based brain tumor classification. Our results shed light on FMN fluorescence in metabolic imaging and outline the potential for supporting the neurosurgeon in visualizing and classifying brain tumor tissue during surgery.

Plasma riboflavin fluorescence as a diagnostic marker of mesenteric ischemia-reperfusion injury in rats

Due to the delayed and vague symptoms, it is difficult to early diagnose mesenteric ischemia injuries in the dynamics of acute illness, leading to a 60–80 % mortality rate. Here, we found plasma fluorescence spectra can rapidly assess the severity of mesenteric ischemia injury in animal models. Ischemia-reperfusion damage of the intestine leads to multiple times increase in NADH, flavins, and porphyrin auto-fluorescence of blood. The fluorescence intensity ratio between blue-fluorophores and flavins can reflect the occurrence of shock. Using liquid chromatography and mass spectroscopy, we confirm that riboflavin is primarily responsible for the increased flavin fluorescence. Since humans absorb riboflavin from the intestine, its increase in plasma may indicate intestinal mucosa injury. Our work suggests a self-calibrated and reagent-free approach to identifying the emergence of fatal mesenteric ischemia in emergency departments or intensive care units.

Characterization of two bacterial multi-flavinylated proteins harboring multiple covalent flavin cofactors

In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium. Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli, and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology.

An upper limit to the fluorescence quantum yield for reduced flavins – a spectroscopic approach to better understand photoflavoproteins

Flavins are blue‐light absorbing chromophores with rich redox activity, found in oxidized, one electron‐ and two electron‐reduced forms. Some, namely flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are used by enzymes as catalytic cofactors. Conflicting literature regarding reduced flavin fluorescence emission includes a plethora of results, from some finding there is negligible fluorescence to others observing significantly blue‐shifted emission. This discrepancy is of consequence because mechanistic kinetic models of photoflavoproteins derived from optical spectroscopy rely on a proper assignment of this emission. Reduced flavins are common redox cofactors in cells, and any fluorescence‐based imaging of this molecule in vivo requires an accurate estimate of its emission quantum yield and spectrum. Here, the photo‐induced reduction of HPLC‐purified FMN and other relevant flavins, in aqueous solutions (at pH 5.0 and 8.5) under aerobic and anaerobic conditions, has been studied via steady‐state absorption and fluorescence measurements. An exogenous reducing agent, ethylenediaminetetraacetic acid (EDTA), was used to photo‐reduce the flavins under investigation. Facile and complete reduction of FMN (~105‐fold) is achieved under anaerobic conditions and insignificant amounts of reduced fluorescence emission is observed, allowing for calculation of the upper limit of the quantum yield of reduced flavin. The low quantum yield (~10‐4) obtained is an important finding when considering flavoproteins, especially when studying them spectroscopically.

How many molecules of mitochondrial complex I are in a cell?

Mitochondrial complex I is the only enzyme responsible for oxidation of matrix NADH and regeneration of NAD+ for catabolism. Nuclear and mtDNA mutations, assembly impairments, and enzyme damage are implicated in inherited diseases, ischemia-reperfusion injury, neurodegeneration, and tumorogenesis.Here we introduce a novel method to measure the absolute content of complex I. The method is based on flavin fluorescence scanning of a polyacrylamide gel after separation of complexes by Clear Native electrophoresis. Using mouse primary astrocytes as an example, we calculated an average value of 2.2 × 105 complex I molecules/cell. Our method can be used for accurate quantification of complex I content.

Influence of nanoenvironment in reverse micelles on the fluorescence of flavins

Riboflavin (RF) and flavin adenine dinucleotide (FAD) are two chromophores in ubiquitous blue-light photoreceptors where molecular motions are partially constrained. However, most previous studies on their fluorescence were performed in solutions. In this work, we studied their fluorescence in Aerosol-OT (AOT) and cetyltrimethylammonium bromide (CTAB) reverse micelles (RMs) by using steady-state UV–visible spectroscopy and femtosecond transient absorption spectroscopy. Compared with in aqueous solution, fluorescence of RF in AOT RMs remains nearly constant, but it is quenched in CTAB RMs induced by intersystem crossing promoted by bromide anion. In addition, fluorescence of FAD in AOT RMs is strengthened because the stacked conformation is less favorable in AOT RMs than in aqueous solution. Moreover, the fluorescence quenching of FAD in CTAB RMs by intersystem crossing mainly affects the unstacked FAD because the intramolecular charge transfer in stacked FAD is faster than intersystem crossing by more than 40 folds. These results determine the dependence of fluorescence of flavins on the ions, polarity and steric constraints in nanoenvironment.

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min−1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus.

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.

Time-Resolved Imaging of Mitochondrial Flavin Fluorescence and Its Applications for Evaluating the Oxidative State in Living Cardiac Cells.

Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence. Here, we describe detection and measurement of endogenous mitochondrial flavin fluorescence directly in living cardiac cells using fluorescence lifetime imaging microscopy (FLIM) after excitation with 473nm picoseconds (ps) laser. Time-correlated single photon counting (TCSPC) method is employed.

Dependence of tuber fluorescence on potato storage duration

Fluorescent control methods are very sensitive and able to capture the ultra-weak reactions of tubers to lighting. However, they have not been developed due to the high sensitivity and complexity of the analysis of diverse information from natural samples. An important biological feature of tubers is repair, i.e. an ability to renew the cover tissue in places of mechanical damage. To assess the quality of tubers, natural, and wound periderms, a spectrofluorimeter was used. Optical measurements were performed. The spectra were recorded at a low voltage on a photovoltaic multiplier with a slit width of monochromators – 5 nm, in which the object fluorescence was illuminated by light with a variable wavelength. Results of the spectral reaction to these excitations were recorded. Tubers of potato varieties of different maturity groups were used for research. The relationship between the intensity of fluorescence of vitamins (max 468 nm) and flavins (max 520 nm) most fully reflects the state of tubers in different periods of storage. The aim of the study was to assess the quality of tubers by applying the fluorescent method to create a database for the development of portable devices that do not destroy the control of tubers during their storage.

Temporal metabolic partitioning of the yeast and protist cellular networks: the cell is a global scale-invariant (fractal or self-similar) multioscillator.

.Britton Chance, electronics expert when a teenager, became an enthusiastic student of biological oscillations, passing on this enthusiasm to many students and colleagues, including one of us (DL). This historical essay traces BC’s influence through the accumulated work of DL to DL’s many collaborators. The overall temporal organization of mass-energy, information, and signaling networks in yeast in self-synchronized continuous cultures represents, until now, the most characterized example of in vivo elucidation of time structure. Continuous online monitoring of dissolved gases by direct measurement (membrane-inlet mass spectrometry, together with NAD(P)H and flavin fluorescence) gives strain-specific dynamic information from timescales of minutes to hours as does two-photon imaging. The predominantly oscillatory behavior of network components becomes evident, with spontaneously synchronized cellular respiration cycles between discrete periods of increased oxygen consumption (oxidative phase) and decreased oxygen consumption (reductive phase). This temperature-compensated ultradian clock provides coordination, linking temporally partitioned functions by direct feedback loops between the energetic and redox state of the cell and its growing ultrastructure. Multioscillatory outputs in dissolved gases with 13 h, 40 min, and 4 min periods gave statistical self-similarity in power spectral and relative dispersional analyses: i.e., complex nonlinear (chaotic) behavior and a functional scale-free (fractal) network operating simultaneously over several timescales.

Flavin-based metabolic cycles are integral features of growth and division in single yeast cells

The yeast metabolic cycle (YMC) is a fascinating example of biological organization, in which cells constrain the function of specific genetic, protein and metabolic networks to precise temporal windows as they grow and divide. However, understanding the intracellular origins of the YMC remains a challenging goal, as measuring the oxygen oscillations traditionally associated with it requires the use of synchronized cultures growing in nutrient-limited chemostat environments. To address these limitations, we used custom-built microfluidic devices and time-lapse fluorescence microscopy to search for metabolic cycling in the form of endogenous flavin fluorescence in unsynchronized single yeast cells. We uncovered robust and pervasive metabolic cycles that were synchronized with the cell division cycle (CDC) and oscillated across four different nutrient conditions. We then studied the response of these metabolic cycles to chemical and genetic perturbations, showing that their phase synchronization with the CDC can be altered through treatment with rapamycin, and that metabolic cycles continue even in respiratory deficient strains. These results provide a foundation for future studies of the physiological importance of metabolic cycles in processes such as CDC control, metabolic regulation and cell aging.

Spectrally and spatially resolved laser-induced photobleaching of endogenous flavin fluorescence in cardiac myocytes.

Naturally occurring endogenous fluorescence of flavins, arising in response to excitation by visible light, offers broad opportunity to investigate mitochondrial metabolic state directly in living cells and tissues, including in clinical settings. However, photobleaching, the loss of the autofluorescence intensity following prolonged exposure to light is an inherent phenomenon occurring during the fluorescence acquisition, which can have a negative impact on the recorded data, particularly in the context of measurement of metabolic modulations in pathophysiological conditions. In the presented study, we present a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally‐resolved confocal imaging. We demonstrate significant nonuniform photobleaching related to different bleaching rates of individual flavin components, resolved by linear spectral unmixing of the recorded signals. Induced photodamage was without effect on the cell morphology, but lead to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. These findings point to the necessity of inducing limited photobleaching during metabolic screening in all studies involving visible light excitation and fluorescence acquisition in living cells. © 2018 The Authors Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry

Porphyrins and flavins as endogenous acceptors of optical radiation of blue spectral region determining photoinactivation of microbial cells

It is shown that exposure of suspensions of gram-positive Staphylococcus aureus, gram-negative Escherichia coli and yeast-like fungi Candida albicans to laser radiation of blue spectral region with 405 and 445 nm causes their growth inhibition without prior addition of exogenous photosensitizers. It is experimentally confirmed that compounds of flavin type capable of sensitizing the formation of reactive oxygen species can act as acceptors of optical radiation of blue spectral region determining its antimicrobial effect along with endogenous metal-free porphyrins (the role of endogenous porphyrins has been confirmed earlier by a number of researchers).The participation of these compounds in the antimicrobial effect of laser radiation is supported by the registration of porphyrin and flavin fluorescence in extracts of microbial cells upon excitation by radiation used to inactivate the pathogens. In addition, the intensity of the porphyrin fluorescence in extracts of microbial cells in the transition from radiation with λ = 405 nm to radiation with λ = 445 nm decreases by 15–30 times, whereas the photosensitivity of the cells under study in this transition decreases only 3.7–6.2 times. The contribution of porphyrin photosensitizers is most pronounced upon exposure to radiation with λ = 405 nm (absorption maximum of the Soret band of porphyrins), and flavins – upon exposure to radiation with λ = 445 nm (maximum in the flavin absorption spectrum and minimum in the absorption spectrum of porphyrins). The ratio between the intensity of the porphyrin and flavin components in the fluorescence spectrum of extracts depends on the type of microbial cells.

Identification of eukaryotic UDP-galactopyranose mutase inhibitors using the ThermoFAD assay

Aspergillus fumigatus is a human pathogen responsible for deadly infections in immune-compromised patients. A potential strategy for treating A. fumigatus infections is by targeting the biosynthesis of cell wall components, such as galactofuranase, which is absent in humans. Galactofuranose biosynthesis is initiated by the flavoenzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM requires the reduced form of the flavin for activity, which is obtained by reacting with NADPH. We aimed to identify inhibitors of UGM by screening a kinase inhibitor library using ThermoFAD, a flavin fluorescence thermal shift assay. The screening assay identified flavopiridol as a compound that increased the melting temperature of A. fumigatus UGM. Further characterization showed that flavopiridol is a non-competitive inhibitor of UGM and docking studies suggest that it binds in the active site. This compound does not inhibit the prokaryotic UGM from Mycobacteria tuberculosis.

Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa d-Arginine Dehydrogenase.

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the kcat/Km values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the kcat value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.

Fluorescent spectral studies of the toxic effect of chlororganic pesticides on the biochemical parameters of synaptosomes

The results of the study of the fluorescence spectra of suspensions of synaptosomes, which have been exposed to a chlororganic pesticide, thiamethoxam, at a concentration of 50 MPC during different time periods, at the excitation/emission wavelengths of 290 ± 5/310–600, 340 ± 5/360–700, and 420 ± 5/450–800 nm are given for the first time. It has been demonstrated that the development of intoxication results in weakening of the fluorescence intensity of tryptophan, NAD(P)·H, derivatives of vitamin B6, and vitamin А and in an increase in the fluorescence of pyridoxic acid, lipofuscin, and flavin and porphyrin complexes. The results of the spectral studies indicate that the toxic effect of the chlororganic pesticide for the functioning of living systems is based on free radical toxicity.