by Yuri VLADIMIROV, Academician of the Russian Academy of Medical Sciences, Head of the Department of Medical Biophysics, Faculty of Fundamental Medicine, Lomonosov Moscow State University

The nonthermal phosphorescence of a substance-luminescence (fluorescence) has been known since the 18th century.

However, only in the 20th century, with the development of physics of elementary particles, it received a clear scientific explanation.

It is now used not only for practical purposes (lamps, cathode ray tubes, etc.), but also as an instrument for studies of the matter.

Now we know that absorption of photons (electromagnetic radiation quanta) in living systems often leads to formation of chemically aggressive particles-free radicals.

Reactions with their participation are often associated with energy release in the form of photons.

The Russian scientists made an important contribution to studies of these intricate physicochemical processes.

Electronic transitions in the course of absorption and luminescence (above) in oxidation-reduction reactions (in the middle), in photochemical reactions and hemiluminescence (below). Blue circles-upper filled in electronic orbitals, red circles-lower free orbitals of oxidized forms of molecules (A). AH₂-reduced forms of molecules, A*-molecules in electronic excited state. Dots to the left from letters-unpaired electrons in radicals, figures-reaction numbers.

BIOPHOTONICS

The term "photonics" was suggested in 1961 by Academician (from 1939) Alexander Terenin, physico-chemist, founder of the Soviet school of photochemistry. He defined it as a "sphere of science, studying the totality of interrelated photophysical and photochemical processes which occur during light absorption by a substance". Hence, biophotonics (previously called quantum biophysics) is a science of radiation, absorption, diffusion, and activity of photons in biological systems. It includes studies of the chemiluminescence (luminescence utilizing the energy of chemical reactions) and bioluminescence (light absorption and diffusion by tissues, cells, and biomolecules) phenomena, and other photobiological processes, including photosynthesis and vision.

The basic laws of luminescence of organic molecules became an object of studies of outstanding Russian scientists: Sergei Vavilov, physicist, founder of the Soviet school of physical optics, Academician (from 1932); Alexander Terenin mentioned above, Alexander Krasnovsky, biochemist and biophysicist, Academician (from 1976), Sergei Konev, specialist in photobiology, Academician of the Academy of Sciences of Byelorussia, Vadim Levshin, Dr. Sc. (Phys. & Math.) from P. Lebedev Physical Institute, et al. Of the four laws of luminescence, known today (Stokes law, Cachie's rule, Vavilov's law, and Levshin's rule), the latter two--independence of luminescence quantum release from the wavelength and the law of mirror symmetry of absorption and fluorescence spectra--have been formulated by Russian scientists. In addition, Sergei Vavilov together with the German physicochemist Theodore Foerster proved and explained the effect of the electron stimulation energy* transfer between molecules (1946). Alexander Terenin in 1943 in parallel with Gilbert Lewis, American physicochemist (Foreign Honorary Member of the USSR Academy of Sciences from 1942) interpreted phosphorescence** as a result of the molecule transition into the basic state from the triplet state (two electrons with parallel spins***, in contrast to common molecules, where the spins of all electron pairs are antiparallel, i.e. are directed to opposite sides).

Returning to biophotonics, let us note that photon interactions with molecules in human body lead to modification of the direction of movement of the former (light diffusion) or to their absorption; then the latter transfer into an electron stimulated state. The main method for studies of such molecules is measurement of luminescence characteristics (photon emission during their return to the initial state). The spectra of stimulation and emission, quantum release, time of fluorescence quenching, and other fluorescence parameters, in their turn, provide important information about the mechanism, energy and kinetics of intra - and intermolecular redistribution of energy in the system after photon absorption.

* According to the principles of quantum mechanics, atoms and molecules are stable in only some stationary states, to which certain energy values correspond. The lowest energy state is called basic, other ones-stimulated. When an atom passes from one stationary condition to another, the structure of electron coating is modified.--Ed.

** Photoluminescence differs by duration and is subdivided into fluorescence (life time 10^-9-10^-6 s) and phosphorescence (10^-3-10s).--Ed

*** Spin is a proper moment of the quantity of microparticle movement, its quantum nature and not connected with the movement of the particle as a whole.--Ed.

On the basis of measurements of crooked chemiluminescences (1-2), there is made up a hypothetical diagram of reactions, which result in phosphorescence (3), and by means of computer program (4) calculations of reaction kinetics are carried out.

PROTEIN FLUORESCENCE AND ENERGY MIGRATION

Today fluorescence is a routine method for studies of molecules in a live cell and of the characteristics of adjacent structures. One of the pioneers in this sphere was Alexander Krasnovsky. In the second half of the 1940s, before creation of photomultipliers, he obtained at A. Bach Institute of Biochemistry unique data on the chlorophyll state in plant leaves using a visual fluorometer. He headed studies of protein luminescence in the mid-1950s at the Moscow State University. However, attempts to detect this phenomenon were made in the middle of the 19th century, but the scientists who observed it in fact saw just the luminescence of admixtures in a visible part of the spectrum. It were Virgie Shore and Arthur Pardy (USA), who proved in 1956 fluorescence of proteins proper in the ultraviolet spectrum (though, they did not measure the luminescence spectra). A year later F. Teel and G. Weber in the UK measured the luminescence spectra of aromatic amino acids. At the same time, Sergei Konev and the author of this paper, as members of Alexander Krasnovsky's team, in the USSR measured the first spectra of protein fluorescence. And in 1959 we showed that tyrosine* fluorescence in proteins was limited by release of a small number of quanta due to the formation of hydrogen bonds with the adjacent amino acids and migration (transfer)

* Tyrosine is an amino acid, a component of many proteins and peptides (casein, insulin, etc.); in animal and human body serves as an initial substance for synthesis of thyroid hormones, epinephrine, etc.--Ed.

of electron stimulation energy from tyrosine to tryptophan*, usually characterized, on the contrary, by release of great amounts of quanta. The results of these early studies were summed up in our monograph, issued in 1965. The method is now widely used for studies of structural rearrangements in protein molecules.

It was no less important to understand the mechanism of electron stimulation energy transfer. The interest to it was conditioned by the papers published in the Nature and Science journals in 1941 by Albert Szent-Gyorgyi, Hungarian biochemist (Nobel Prize Winner of 1937, Foreign Corresponding Member of the USSR Academy of Sciences from 1947). He suggested that proteins possessed the characteristics of semiconductors and hence, electron transfer together with the relevant stimulation energy was possible in their molecules. These concepts were criticized in two papers written by Sergei Konev and the author of this article (1957, 1959) and confirmed in experiments, proving the possibility of inductive-resonance mechanism of transfer of energy in proteins. By the present time this method is widely used for evaluating distances between sites of their molecules or the complexes formed by them.

When the molecule is in an electron stimulated state, after realization of respective energy transfer it can enter a chemical reaction. Its primary products are, as a rule, rather reactive chemical compounds, often free radicals (aggressive molecular particles with one or two unpaired

* Tryptophan is an essential amino acid, a component of gamma-globulin, casein, and other proteins.--Ed.

electrons on the outer electron membrane). We should like to mention here, a propos, the chlorophyll photore-duction reaction, initiating photosynthesis, which was discovered in 1954 by Alexander Krasnovsky and named after him. Its primary products are chlorophyll free radicals and electron donor molecules.

Photochemical reactions which take place under conditions of UV (ultraviolet) irradiation of proteins also start from the formation of radicals. We studied the mechanism of these processes in 1961-1965 on aromatic amino acids and proteins irradiated at a liquid nitrogen temperature (--196°C). Common chemical reactions are impossible in their hardened solution, but electrons are transferred between energetic levels, connected with photon absorption or emission. By measuring the absorption, fluorescence, and phosphorescence spectra, it is possible to calculate the energy of a molecule in its stimulated state. One of its electrons can be released into the environment, where it will be captured by frozen solvent molecules. The electron return becomes possible if UV-exposed object is heated, and electron transfer will then result in thermoluminescence.

Though the final result of the complex scheme of reactions associated with absorption of UV radiation quantum turns out to be protein inactivation, their first products are free radicals of amino acids-tyrosine, tryptophan, and cystein (which was shown by Ofelia Azizova (Institute of Biophysics of the USSR Academy of Sciences) in 1963-1969, and Dmitry Roshchupkin (Biology and Soil Department, MSU), later on both doctors of biological sciences.

Despite the fact that this system is quite simple (the object is an amino acid molecule, the exposure is UV irradiation), we see here all main phenomena studied in biophotonics and the science on radicals in much more intricate situations. These are photophysical processes (light absorption by molecule --> fluorescence --> transition to triplet state (electron spin reversion) --> phosphorescence, as well as photochemical (photoionization --> chemiluminescence) processes.

FREE RADICALS

As we have already mentioned, the radicals are chemically very aggressive, as they strive to get back the missing electron by taking it away from some molecule or, vice versa, to get rid of a "spare" electron or to combine with another free radical, thus forming a molecule. Now, let us make a short digression to history.

The formation of free radicals in a chemical reaction was first demonstrated by Moses Homberg, a German scientist, in 1897. Later it was found that the radicals served as intermediate products of chain reactions in biological systems under conditions of exposure to ionizing radiation. Analyzing the similarity of manifestations of its effects and ageing on animals, Denham Harman, American scientist, suggested the involvement of free radicals in ageing (1956) and development of diseases of midde-aged people, including atherosclerosis (1957). Later Academician Nikolai Emanuel (Institute of Chemical Physics, now named after N. Semyonov) in his classical works revealed the role of free radicals in chain reactions of organic compounds (including lipids) oxidation. And in 1968 American scientists Joe McCord and Irwin Fridovich found out that a previously known plasma protein, erythrocuprein, is able to eliminate superoxide radicals (oxygen molecules with unpaired electron).

Diagram of heliumneon laser light impact on a live cell as a result of photodynamic effect of the endogenous sensitizer (porphyrin); 1-4-consecutive stages of the process.

It is quite obvious today that free radicals play an important role in our organism. To begin with, a common oxygen molecule has two unpaired electrons and is, so to say, a double radical; that is why oxygen is very active chemically. Its structural formula can be written so: • OO •, a dot denoting an unpaired electron by the atom to which it belongs. By binding one electron, oxygen molecule transforms into a superoxide (• 0₂), by binding two electrons in water solution it transforms into hydrogen peroxide (HPj), and binding three electrons it becomes a hydroxyl radical (• OH). All these products are called active oxygen species (AOS). Under some conditions they act as regulators of cell processes, under other conditions they inflict severe damage to cells, leading to their death.

One of the natural radicals is nitrogen monoxide (NO). In human body it is formed by enzymes--NO synthases, regulates many intracellular processes, for example, serves as an important mediator of vascular wall relaxation. Its deficit leads to disorders in local blood flow and hypertension (arterial pressure elevation). Its excess is also undesirable, as NO metabolites (active nitrogen species) are rather toxic.

Spontaneous oxidation of organic molecules by oxygen usually takes place by the mechanism of chain reaction in which free radicals are involved. Lipids (for example, polyunsaturated fatty acids) and live cell biomembranes are oxidized by the same mechanism. The products of chain reaction are lipid peroxides, and the process is usually called lipid peroxidation.

A series of studies we carried out at the Department of Biophysics of Biomedical Faculty of N. Pirogov 2nd

Moscow Medical Institute (today Russian State Medical University named after N. Pirogov) in 1972-1983 showed: lipid peroxidation leads to degradation of the membranous bimolecular lipid layer, to increase of membrane permeability for ions, and reduction of its electric strength, as a result of which the membranes are destroyed under the effect of electric field generated by themselves. That eventually leads to cell death (necrosis) or triggers the mechanism of their programmed death (apoptosis); the latter fact was proved mainly by experiments of Kazuhiro Nomura, a Japanese specialist, in 1999-2000, and Valerian Kagan, an American professor, in 2002-2004.

Today the role of lipid peroxidation in the development of the major diseases of middle-aged people is amply described. These processes contribute to the development of cardiovascular diseases associated with atherosclerosis, neurodegenerative disorders (Alzheimer's, Parkinson's diseases, etc.), chronic inflammatory diseases such as rheumatoid arthritis, ocular diseases (cataract and retinal degeneration), diabetes, and many others.

WHEN RADICALS GENERATE PHOTONS

Chemiluminescence can be regarded as a process opposite to photochemical. During the latter the light is absorbed and triggers chemical reactions, while during the former these reactions lead to formation of products in electron-stimulated state and to light emission.

Alexander Gurvich, a Russian scientist, cytologist, was the first to discover fluorescence of live objects in

In case of adding to solutions or live cells, there takes place a reaction of chain oxidation of lipids, fluorescent dye-stuff C-525, quantum release of radiation (φ) and, accordingly, intensity of chemiluminescence increases more than thousands of times!

UV spectrum--it was in the 1920s. Fluorescence of these objects was recorded due to its stimulation of division of other cells, and for this reason it was called mitogenetic. Later on other specialists confirmed the existence of UV radiation of live cells, for example, in 1934 Sergei Rodionov and Gleb Frank (Academician from 1966) through registration of individual UV photons by a quartz gas-discharge tube with a metal photocathode.

Development of photoelectron multipliers abruptly improved the quality of measurements of radiation of plant and animal cells and tissues. Numerous biological objects emitting fluorescence called superweak were studied by these devices (the author of this paper and Fyodor Litvin, later on Dr. Sc. (Biol.), 1959). The nature of this fluorescence was disclosed due to studies carried out by the founder of the Department of Biophysics at the Moscow State University Boris Tarusov, Dr. Sc. (Biol.), and his team (1961) and our studies (1964-1965). It was shown that the main source of fluorescence in live cells was the reaction between polyunsaturated fatty acids' free radicals in cell membranes and the lipoproteins.

It is known that oxidation of organic compounds by air oxygen runs as a chain reaction, in which free radicals of these compounds are involved. Research carried out at the Laboratory headed by Viktor Shlyapintokh, Dr. Se. (Chem.) (N. Semyonov Institute of Chemical Physics, RAS), revealed that these reactions were associated with slight luminescence, its mechanism (as was later found) being very close to that of superweak fluorescence of live cells and tissues, revealed in our studies. Detailed studies of chemiluminescence mechanism in oxidation of organic substances by molecular oxygen were carried out by Rostislav Vasilyev, Dr. Sc. (Chem.), and his team at the Institute of Chemical Physics in 1963-1965. They showed that the luminescence was caused by reaction between two peroxyl radicals participating in the chain reaction. And in 1971 we established that superweak fluorescence of cells and tissues, associated with the lipid oxidation chain reaction, was also caused by interaction of such radicals.

As it is impossible to measure the concentration of free radicals by direct chemical methods because of extremely high reactivity of the radicals and it is extremely difficult to detect them by electron paramagnetic resonance* (particularly in biological objects) because of low concentration, no wonder that the chemiluminescent method very soon became the basic one for studies of the radicals responsible for lipid peroxidation.

Discovery of superweak fluorescence of human cells and tissues (including plasma and blood cell fluorescence) initiated numerous studies. This phenomenon was then used as a new method of clinical laboratory analysis, enabling physicians correctly diagnose a disease and monitor the patient's state in the course of treatment. Studies carried out at N. Pirogov Second Moscow Medical Institute in 1974-1990 showed that human and animal plasma and blood cell fluorescence increased in inflammatory diseases and dropped under conditions of oxygen deficit in tissues (hypoxia), indicating a serious warning.

Unfortunately, this method has two flaws. First, in many cases the fluorescence is caused not only by one reaction (interaction between peroxyl radicals), but also by other ones, poorly studied. Secondly, its intensity is

* See: V. Popov, "A Pioneer in Paramagnetic Resonance", Science in Russia, No. 6, 2008.--Ed.

Transformation of a vector of electrons-cytochrome c (CytC) into the peroxidase enzyme takes place in cell mitochondria, if protein combines with cardiolipin (1-3). In the presence of hydrogen peroxide there is formed the so-called compound 1 (4), and in the process of its interaction with lipids (LH)-their radicals (5), there begins a chain reaction of lipid oxidation, resulting in launching an apoptosis. In the experiment, instead of lipids, is usedluminol, formation of its radicals is accompanied by bright chemiluminescence. Superoxide radicals (6) participate in this reaction.

very low and hence, rather much material is needed (for example, blood for plasma analysis must to be collected from the vein, which is rather unpleasant). The solution of the problem was found due to special compounds, called chemiluminescence activators. They are divided into chemical and physical ones (the latter do not participate in chemical reactions; their effect is based on amplification of the quantum emission of chemiluminescence). During almost thirty years Viktor Sharov, Cand. Sc. (Biol.), from the Department of Biophysics of the Russian State Medical University searched for an effective physical stimulator. Substances--quinolysine coumarines--were found, repeatedly amplifying the chemiluminescence intensity. Coumarine C-525 was found to be the best: it increased the fluorescence intensity during chain oxidation of lipids more than 1,500 times (!) without affecting the chemical reactions in the system and not entering into reactions itself. Obviously, with activators of this kind, insignificant amounts of biological material are sufficient (just several microliters of blood or several milligrams of tissue).

As soon as it was found that superweak fluorescence of animal cells was caused by lipid peroxidation reactions in biomembranes, measurements of such chemiluminescence was used for studies of the mechanism of these reactions. The main method for measurements, which we used together with Pyotr Gutenev in 1973 (at that time a student at N. Pirogov Second Moscow State Medical Institute, now Cand. Sc. (Phys. & Math.)), consisted in registration of the time course of luminescence (its kinetics), mathematical simulation of the phenomenon, and comparison of the estimated data with experimentally obtained curves. However, this approach was fully realized only in 2002, when together with Dmitry Izmailov, Cand. Sc. (Biol.), Lomonosov Moscow State University, we developed convenient software for the same purpose. Eventually, the totality of experimental data is described by five chemical reactions; their velocity constants are selected. We widely use this method at our laboratory for studies of many other processes associated with chemiluminescence.

WHEN PHOTONS GENERATE RADICALS

It has been shown in recent years that the biological effect of intense light (laser or light-emitting diodes) is associated with the formation or release of free radicals. In 1994, based on the data accumulated by that time we formulated a hypothesis according to which the stimulatory effect of low-intensity laser radiation was based on three photochemical reactions: 1--photodynamic effect on cell membranes, associated with the formation of lipid radicals during illumination of cells in the presence of endogenous sensitizers--porphyrines (pigments highly prevalent in living nature); 2--photochemical stimulation of antioxidant enzyme--superoxide dismutase; and 3--photochemical cleavage of nitrosyl complexes of haem* proteins with release of NO (a radical).

Presumably, the mechanism of the second of these processes is more intricate than we initially thought, and includes stimulation of the said enzyme biosynthesis after primary formation of radicals by mechanisms of the first or third reaction. But the involvement of both in the bios-timulatory effects of low-intensity laser radiation and high intensity light-emitting diodes has been confirmed

* Haem--a nonprotein part (prosthetic group) of haemoglobin and cytochromes.--Ed.

by numerous studies carried out at the Department of Medical Biophysics of the Russian State Medical University by Gennady Klebanov, Dr. Sc. (Med.), Anatoly Osipov, Dr. Sc. (Biol.), and their colleagues.

However, as we have already mentioned, the radicals can also act as destroyers. For a long time it was assumed that they just nonspecifically damaged cell structures, the cells "were ill" and sometimes died from it. It became clear in recent years that the main mechanism of the effect of radicals was more specific: they triggered apoptosis (programmed cell death). The sequence of the events leading to it is well studied and includes six stages: 1--the cell is exposed to factors inducing apoptosis; 2-a cardiolipin phospholipid complex with cytochrome c (protein, a component of the respiratory chain) forms in mitochondria; 3--lipid peroxidation in the inner mitochondrial membrane, catalyzed by this complex; 4--formation of large pores (or cracks) in the outer membrane of the same organelles and release of cytochrome c; 5--cytochrome c complex with other proteins triggers a cascade of reactions with participation of caspases*; and 6--cell structures are destroyed and absorbed by phagocytes. The key component of the cascade is lipid peroxidation in the inner mitochondrial membrane, catalyzed by the cytochrome c--cardiolipin complex.

Studies of the structure and characteristics of this complex were carried out recently at the laboratory headed by the biochemist Valerian Kagan in Pittsburgh (USA) and by our team in collaboration with scientists from the Moscow State University, Russian State Medical University, and A. Shubnikov Institute of Crystallography, RAS. It was found that a spherical nanoparticle of 11 nm in diameter, consisting of a cytochrome c molecule, was enveloped by a dense monolayer of cardiolipin molecules. The particle is hydrophobic and incorporates in the lipid layer of mitochondrial membranes. The access to the haem is facilitated and the cytochrome c molecule acquires a new characteristic--it can catalyze the radical formation process in organic molecules, including lipids. The mechanism of reactions stimulated by this complex has been studied by the kinetic chemiluminescent method, combining the recording of luminescence kinetics, associated with the reactions of radicals, and mathematical simulation of the kinetics of contemplated reactions.

APOPTOSIS, ANTIOXIDANTS, LASER EXPOSURE

Antioxidants are compounds preventing the formation of radicals and hence, development of chain oxidation reactions. However, radicals are also formed during the peroxidase** catalytic reaction, and they can trigger the

* Caspases are intracellular proteolytic enzymes; they cleave the peptide bond between amino acids in proteins.--Ed.

** Peroxidases are enzymes catalyzing in live cells oxidation of various substances with the formation of hydrogen peroxide.--Ed.

same chain processes. How antioxidants will act in such a case? Recently Yevgeny Deyev from our laboratory thoroughly tested many antioxidants, including tocopherol, flavonoids, and other compounds, for the formation of luminol (chemical indicator capable of chemiluminescence) and lipid radicals in reactions catalyzed by the cytochrome c--cardiolipin complex. As it turned out all these substances suppressed the formation of radicals in these reactions at about the same concentrations which were used for suppression of lipid peroxidation reactions.

As for theoretical significance, it is in fact a revolution. Previously biophysicists (including the author of this paper) thought that antioxidants acted because they broke lipid oxidation chains, and it was found that they could also inhibit the peroxidase work, including the cytochrome c--cardiolipin complex, thus preventing the development of apoptosis.

As for practice, it is a promising observation, as in many cases apoptosis is an undesirable event (it leads to aggravation of the situation after infarction and stroke), while sometimes it is highly desirable (cancer cells survive in the body as they are not subjected to apoptosis despite the signals they receive). Perhaps, the use of antioxidants is a means for regulation of apoptosis?

And one more important observation. Nitrogen monoxide (NO) easily forms complexes with haemin compounds, for example, with haemoglobin and cytochrome c. Grigory Borisenko, Cand. Sc. (Biol.), and Anatoly Osipov, Dr. Sc. (Biol.), Department of Biophysics, Russian State Medical University, showed that those complexes were photosensitive and degraded with the formation of free NO under the effect of laser exposure. Herman Stepanov, a postgraduate student from the Russian State Medical University, and Anatoly Osipov found that the peroxidase activity of cytochrome c--cardiolipin complex was suppressed by NO and restored under the effect of laser exposure. These results suggest that apoptosis would presumably be suppressed or stimulated by NO or laser exposure. To regulate apoptosis means to be able to control complications of cardiovascular diseases and cancer. But this is an object of further research.