Manual for laboratory classes in biological physics. Коллектив авторов

ns Special practical.

      The works presented here do not require complex and expensive equipment and can easily be reproduced in any university laboratory.

      At the end of each Chapter are questions of self-control that will help more firmly and deeply understand the processes and phenomena observed in laboratory work.

      SAFETY REQUIREMENTS DURING BIOPHYSICS WORKSHOPS

      Before carrying out any laboratory work, students are required to undergo safety training. The students will also have to sign the control sheets that indicate completion of the training.

      During labs, instruments that meet standard requirements and have proper technical specifications and normative technical documentation could be used. The equipment is checked directly by the instructor before the laboratory.

      To prevent electrical hazard in this workshop, follow these safety practices:

      1. To prevent the hazard of electric shock, all devices must be grounded according to their documentation.

      2. The wires used in the classroom must not be damaged.

      3. The plug should not be pulled by holding the cord, the body of the plug should be held instead.

      4. If there are any malfunctions in a device, the device has to be switched off and work has to be stopped immediately. Work can be resumed if and only if the device was fixed by a qualified personel.

      5. Students have the right to start the work only after the instructor allows them to work independently, after the equipment and laboratory procedures are checked by the instructor.

      6. Only class-1 lasers, which output radiation harmless to human eyes and skin, are going to be used for this workshop. To prevent retinal damage, the beam should not be observed without protective filters and the light should not be shined directly into the eye.

      7. Upon completion, all of the devices must be switched off.

      Chapter 1

      THERMODYNAMICS

      The temperature range on the Earth from -80 °C to +85 °C significantly exceeds the borders within which the active life is possible. Temperature determines the activity level and distribution of animals. In an open ocean, the temperature of the surface water layers is -2 – +30 °C. Vital processes are only possible at 0-40 °C. In an inactive state, animals tolerate not only negative temperatures, but freezing. For example, small nematodes tissue cultures (epithelial, muscles, etc.), protozoa get frozen when placed in liquid air (-197 °C) and comes to life if to warm them slowly. Some animals inhabit hot springs, like a few bacteria and algae that can reproduce at +70 °C .

      Temperature, as a measure of the velocity of molecular movement, determines the rate of chemical reactions, and is considered to be one of the factors limiting growth and metabolism. Animals that change their body temperature in accordance with the change in environmental temperature are called poikilothermic (changeable, labile). In this case, temperatures of bode and surroundings are not necessarily equal. Body temperature can be higher, especially, at the active state. Thus, fish of 40g have the muscle temperature higher by 0,44 °C.

      Animals able to regulate their temperature are homoeothermic (birds and mammals). This is due to the thermoinduction, protectional behavior, thermoisolation changes, circulation and other factors changing heat transfer. Periods of hibernation or lethargy, is accompanied by the decrease in body temperature. And the physiological thermostat switches to a lower temperature. Sensor mechanisms demonstrate the change in temperature, causing corresponding feedback reactions. Relatively few animals – heterothermic animals – can partially regulate body temperature that is limited by body regions or by environmental conditions. There are a lot of such animals among insects, for example, bees or ants, but most of arthropods are typical poykilothermic organisms. Temperature of any active cell must be higher than the temperature of the environment, as during oxidation processes and glycolysis heat is released. body temperature depends on several factors affecting the thermal balance in a contrary manner. The heat source can be metabotic thermogenesis (endothermy) or environment, mostly, solar energy (ectothermy). Heat transfer occurs by radiation, convection, heat conduction and water evaporation. Blood circulation from insite to outside of the body promotes heat losing, while thermo isolation obstacles it.

      Thermal conductivity of water is 0,0014 kcal/(cm · c · degrees), it is lower than in metals but higher than in other liquids (for example, ethanol – 0,00042 kcal/(cm · c · degrees)). Specific heat ··capacity of water – 1 kcal/g·degrees, ethanol – 0,09 kcal/g·degrees, most of animal tissues – 0,07-0,09 kcal/g·degrees. The coefficient of temperature conductivity is equal to the coefficient of thermal conductivity divided by the product of specific weigh and specific heat capacity. Low temperature conductivity leads to slow cooling or heating of tissues and limitation in heat distribution within the organism. Fat is a good isolator for animals. Animals with a big tissue mass warm and cool themselves slowly, heat transfer is conducted by circulating liquids. Water evaporation cools any surface, the evaporation of 1 kg of water at 20 °C cost 585 kcal. Most of terrestrial animals use this to avoid body overheating.

      Biokinetics studies the rate of biological processes and their dependence from concentrations of substances which participate in biochemical conversions, and also the dependence from external conditions, especially, from temperature. Such dependence is comprehensible if to take into account that any chemical conversion occurs if chaotically moving molecules collide. As temperature rises, the mean free path of the molecules increases, and thus, the likelihood of their collision also increases. So, the relative number of molecules able to participate in the reaction, or active molecules, increases with the rise of temperature, and the rate of the reaction also increases.

      The parameter indicating how many times the number of active molecules and the rate increased at the temperature rise by 10 °C is called temperature coefficient Q₁₀.

Q₁₀= Vт₂ / Vт₁ (1.1),

      where Vт₁ – reaction rate at the initial temperature, Vт₂ – the rate increased at the temperature rise by 10 °C

      There is a ratio between t coefficient and excessive energy that molecules should possess so that their collision could cause a chemical reaction. (so-called Activation Energy)

Е = 0,46 Т₁ · Т₂ · lgQ₁₀ (1.2)

      where Е – activation energy, kcal/mol, Т₁ и Т₂ – temperatures with the difference of 10 °C , i.е. Т₂ = Т₁ + 10°, lgQ₁₀– decimal log of Q₁₀.

      It is clear that with the rise of temperature by 10 °C, the number of molecules with the energy exceeding the critical value will double, although the increase in kinetic energy proportional the absolute temperature will be much lower. In biological range, the temperature of Q₁₀ values for most metabolic reactions lays in the interval between 2 and 2,5. Some complex changes in rates of physiological processes for example, circadian rhythms, are relatively independent from temperature, and Q₁₀ for oxygen consumption of some poykilothermic animals is between 1 and 2. If to compare oxygen consumption at rest and at the active state, we can determine the characteristic exchange levels at different temperature conditions.

      In CАrdium mollusks Q₁₀ values for active state and rest are equal to 1,84 and 1,20 respectively. For most of invertebrates Q₁₀ values are small. Temperature parameters of enzymatic reactions can lower with the decrease in substrate concentration to the limiting level, thus the measurements of temperatures coefficient have no sense. So, in the case of complex reactions with parallel and consecutive stages, with the contrary influence on Q₁₀ it is impossible to perform an elementary analysis of temperatures coefficient. Even though, in biological research, studies of adaptation and acclimatization of animals to different environmental conditions the study and determination of Q₁₀ coefficient are widely used.

      Laboratory work № 1

      Determination of temperature coefficient and calculation of the activation energy of the frog’s heart

      Objective: Determination of temperature coefficient and calculation of the activation energy