Influence of low intensity laser radiation on different biological systems Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Chemie der Philipps-Universität Marburg vorgelegt von Olga Tsivunchyk aus Grodno Marburg/Lahn 2003 Vom Fachbereich Chemie der Philipps-Universität Marburg als Dissertation am 15.1.2004 angenommen Erstgutachter: Prof. Dr. H. Bäßler Zweitgutachter: Prof. Dr. M. Hofrichter Tag der Disputation am 16.1.2004 2 Sincere Gratitude My work would be not done without support of these people and here I convey all my cordial gratitude to them. I am truly thankful to Prof. Dr. H. Bäßler for the given possibility to finish my work under his leadership; for used laser equipment and working place; for advising and help in preparing of discussion; for his understanding and taken responsibility at my Ph.D. work. I do consider my work under his supervision as a great honour for me and appreciate it very much to Prof. Dr. M. Hofrichter from Zittau for his agreement to take co-supervision at my work; for his advising concerning experiments and writing of thesis structure; for kind hospitality visiting Zittau to Prof. Dr. D. Gemsa for the given opportunity to work in his working team; for his leadership during my DAAD study in Philipps-University of Marburg and all help for it to Prof. Dr. L.-O. Essen for the help and advises in preparing of discussion topic and presentation of my thesis to Prof. Dr. P. Galland for the help and consulting in preparation of discussion topic to Dr. E. von Löw for the great help in laboratory work; for very nice and pleasant atmosphere in joint co-operation; for his help in literature study and friendly support to Dipl. Biol. R. Kottke for teaching and help in practical work in microbiological laboratory to Dr. T. Khomich for time of training in her working group in Institute of biochemistry; for advices in planning of experiments to Dr. K. Mandrik for teaching and help in work with plant tissues 3 to Irina Osakovich for her help in biochemical laboratory and work with experimental animals to Dmitry Zverev for his help in physical laboratory, with data processing and for our friendship to Dr. Y. Romanovsky for his help in work with laser equipment during experiments performance to Prof. Dr. A. Rubinov for his help in preparing of discussion topic to Prof. Dr. R. Frey from Brandenburg Technical University Cottbus, for his interest towards my work and help in continuation in Marburg to Claudia and Andreas Karber for their help in cases of technical PC-problems to my parents and family for possibility to study further more and make researches, especially to my father, who had shown me the world of science to my dear husband Andrei and our son Eugen for their great patience and understanding of my work; for lovely support and home feeling being far away from each other to my best friend Dr. R. Haas for the given power to finish this work; for his help overcoming difficulties and problems; for best and worst time that we have spent in fun and work together. 4 Index page Zusammenfassung 8 1 Introduction 9 1.1 Background 9 1.2 Lasers 9 1.2.1 General description 9 1.2.2 Quantum Properties of Light 11 1.2.3 Stimulated Emission 11 1.2.4 Characteristics of Laser Light 12 1.2.5 Kinds of lasers 13 1.3 Biological antioxidant system 14 1.3.1 General overview 14 1.3.2 Biological antioxidant system 15 2 Literature review: Physiological and biochemical effects of laser light 16 2.1 Effects of LILI in general 16 2.2 Influence of LILI on cell membrane 17 2.3 The mechanisms of photo- and biological LILI activation 18 2.4 Photoactivation of enzymes 20 2.5 Biostimulation 21 2.6 Biological responses on LILI and their application 24 2.7 Summary 25 3 Materials and methods 26 3.1 Lactic dehydrogenase activity determination 26 3.2 Succinate-dehydrogenase (succinic dehydrogenase system) activity detection 27 3.3 Glucose-6-phosphatase activity detection 28 3.4 Biuret – Method 29 3.5 Alpha-amylase activity detection 29 3.6 Determination of superoxide dismutase (SOD) activity 30 3.7 Electrophoresis of proteins 31 3.8 Detection of glutathione peroxidase activity in red cells 32 3.9 Detection of catalase activity 33 3.10 Detection of malonate dialdehyde (MDA) in red cells 34 5 page 3.11 Detection of Mg2+- ATPase activity in red blood cells 35 3.12 Method for the estimation of phosphate 36 3.13 Detection of Ca2+-ATPase activity 37 3.14 Detection of MnP activity 38 3.15 (Triphenyltetrazoliumchloride) TTC-test 39 3.16 Detection of glutathione reductase activity 40 3.17 Method of Lowry 40 4 Experiments 41 4.1 Investigations of the influence of low intensive laser irradiation (LILI) on the antioxidant system of animals 41 4.2 Low intensity laser irradiation (LILI) as a modulator of the antioxidant system of animals 42 4.3 Investigations of changing activity Ca2+-ATPase and Mg2+-ATPase of erythrocytes-membranes after LILI radiation in vitro experiments 43 4.4 Enzymatic response of animals' tissues on LILI in vitro experiments 44 4.5 Investigations of LILI influence of different energy on activity of alpha- amylase in grains 45 4.6 The possibilities to activate alpha-amylase in germinated grains 45 4.7 Changing of the activity of the exoenzyme Manganese peroxidase after laser radiation 46 4.8 Investigation of influence of LILI on biochemical properties of yeast 47 4.9 Investigation of influence of LILI on biochemical properties of bacteria 47 4.10 UV-Vis-Spectra of enzymes 48 5 Results 50 5.1 Investigations of the influence of low intensive laser irradiation (LILI) on the antioxidant system of animals 50 5.2 Low intensity laser irradiation (LILI) as a modulator of animal's antioxidant system 54 5.3 Investigations of changing activity Ca2+-ATPase and Mg2+-ATPase of erythrocytes-membranes after LILI radiation in vitro experiments 59 6 page 5.4 Enzymatic response of animals' tissues on LILI in vitro experiments 62 5.5 Investigations of LILI influence of different energy on activity of alpha- amylase in grains 67 5.6 The possibilities to activate alpha-amylase in germinated grains 70 5.7 Changing of the activity of the exoenzyme Manganese peroxidase after laser radiation 72 5.8 Investigation of influence of LILI on biochemical properties of yeast 75 5.9 Investigation of influence of LILI on biochemical properties of bacteria 76 5.10 Summary of Results 76 6 Discussion 81 7 Conclusions and outlook 87 8 References 88 9 Appendix 102 9.1 List of abbreviations and units 102 9.2 List of light sources 104 9.3 List of materials and equipment 105 9.4 List of chemicals 106 9.5 List of biological systems 108 9.6 UV-Vis Spectra 110 7 Zusammenfassung In der Literatur sind viele Beispiele des Einflusses von Laserbestrahlung mit geringer Energie (LILI) auf biologische Syteme beschreiben. Allerdings sind die Ergebnisse wiedersprüchlich. Ziel dieser Arbeit war es, mit verschiedenen Experimenten den Einfluß von LILI auf verschiedene biologische Systeme und Objekte detailliert zu untersuchen. Es wurden verschiedene Experimente mit folgenden biologischen Systemen und Objekten durchgeführt: * verschiedene Enzyme des Antioxidations-Systems von Tieren (Catalase, Superoxid- Dismutase, Glutathion-Peroxidase, Glutathion-Reductase) * Mg2+- und Ca2+-ATPase aus Membranen von menschlichen Erythrozyten und Erythrozyten von Ratten * Lactat- und Succinat-Dehydrogenase von Ratten aus Leber, Nieren, Gehirn, Muskel, Herz * alpha-Amylase aus trockenen und gekeimten Gerstekörnern * Mangan-Peroxidase aus Lignin-abbauenden Pilzen * Dehydrogenasen von Hefe * Dehydrogenasen von Bakterien. Folgende Laser wurden für die Versuche eingesetzt: YAG-Laser (355 nm und 533 nm), Argon-Laser (458 nm, 488 nm und 520 nm), Helium-Neon-Laser (632 nm) und CO2-Laser (10.6 µm). Nach Laserbestrahlung wurden bei verschiendenen Systemen sowohl Aktivitätserhöhung als auch Aktivitätsminderung beobachtet. Abhängigkeiten von der Bestrahlungszeit und Intensität der Bestrahlung wurden ebenfalls ermittelt. Verschiedene Antworten biologischer Systeme nach Laserbestrahlung hängen von den spezifischen Eigenschaften dieser Systeme ab. Unterschiedliche Reaktionen verschiedener biologischer Systeme wurden nach Bestrahlung mit verschiedenen Lasern detektiert. 8 1 Introduction 1.1 Background Scientific experiments at the theme of the Dissertation were started since 1999. At the beginning the theme of Dissertation was formulated as: "Investigations of the influence of low intensity laser irradiation (LILI) on the Antioxidant system of animals". Those investigations were done with the purposes to find out the effects of LILI of some kinds of lasers on the process of lipid peroxidation (LPO) and the activity of antioxidant system in some organs of animals and, at the first, in erythrocytes and in blood plasma. In literature sources the effects of LILI on antioxidant system (AOS) of organism with the most important components such as reduced glutathione (GSH) and the enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GP) mostly have been described for He-Ne-laser irradiation. Since they are contradictory [1], the function investigations of the transport proteins of the erythrocytes' membranes which are responsible to keep the native structure of erythrocytes and ion balance in these cells had caused especial interest. The contradictory obtained results had made a basis to carry out additional researches concerning laser light influence on other enzymatic systems of animals, plants, mushrooms, yeast and bacteria. 1.2 Lasers 1.2.1 General description The light from lasers differs from ordinary light in several important aspects. Ordinary light from a light bulb travels randomly in all directions (unless the bulb is equipped with an integral reflector that directs the light). The light is thus incoherent.