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14. Free radicals
Def.: Free Radicals= Particles that have 1/more unpaired electrons Generation:
1. Homolysis:
R1: R2 -> ˙ R1 + ˙ R
-> Needs lots of energy: VIS light, UV, X- and ƴ−rays, ionizing radiation
Easier for bonds with H atom: e.g.: C = C—H or —S—H
RSH -> ˙ RS + ˙ H Dot ( ˙ ) = radical
(unpaired electron)
2. One-electron transfer: = typical case in the living systems
RH – e-^ −> ˙ R + H +
e.g.: RH + Fe 3+^ -> ˙ R + Fe 2+^ + H+
H 2 O 2 + Fe2+^ -> Fe 3+^ + ˙ OH + OH-
-> Does NOT need a lot of energy
3. Metabolism of xenobiotics (different toxic chemicals, insecticides, CHCl3, CCl4, tobacco smoke): hv
CCl4 -> ˙ CCl 3 + ˙ Cl
˙ CCl 3 + RH -> CHCl 3 + ˙ R
4. Physical factors: - Visible and UV light - X-rays - ƴ-rays - microwave energy of large intensity (cell phones – not proven yet), ultrasound
Types:
- Monoradicals
- Biradicals
- Stable
- Unstable (reactive) radicals
- Electoneutral
- Radical-anions
- Radical-cations
Basic reactions:
1. Recombination:
˙ R 1 + ˙ R 2 -> R1—R
2. Disproportionation:
2 R 1 —R 2 —R 3 ˙ -> R 1 —R 2 =R 3 + R 1 —R 2 —R 3
3. Isomerization:
2 R 1 —R 2 —R 3 ˙ -> 2 R 1 —R 2 — ˙ R 3
4. Substitution:
˙ R 1 + R 2 -> R 1 + ˙ R 2
5. Addition:
˙ R 1 + R 2 -> R 1 —R 2 ˙
Reactive oxygen species (ROS): (^3) O 2 triplet (groundstate) oxygen, usually denoted as O (^2) (^1) O 2 singlet oxygen
˙ O2-^ superoxide radical (radical- anion)
˙ OH hydroxyl radical
H2 O2 hydrogen peroxide (not a radical)
˙ O2 H hydroperoxyl radical
ROO˙ peroxyl radical
ROOH hydroperoxide
(ROS) also includes some reactive non-radicals such as:
- Hypochlorous Acid ( HOCl )
- Ozone ( O3 )
Methods for registration and investigation of ROS:
- (^) ESR (Electron Spin Resonance)
- (^) CL (Chemiluminescence) which is based on - (^) Radical recombination - (^) Electron transfer reactions
- (^) UV-VIS Spectrophotometry
15. Biomembrane oxidation
Oxidation of the membrane components, consequences.
- Oxidation of lipids
- Oxidation of proteins -> cataract
- Oxidation of carbohydrates
- Oxidation of nucleic acids
- Vitamin E (α, ƴ-tocopherol)
- Vitamin C (Ascorbic Acid)
- Vitamin A
- Beta-carotene
- Carotenoids
- Xanthophylls
- Еstrogens
- Metallothionein
- Coenzyme Q
- Flavonoids, Polyphenols
- Herbals, Theaflavin, Ginko biloba, etc.
- Мonounsaturated fats
- Enzymatic
- Superoxide Dismutase (SOD)
- Catalase (CAT)
- Glitathion peroxidase (GSH-P)
Oxidation of pharmaceutical preparations:
- Vitamin E (Tocopherols) -> in nut oils, seeds, vegetable Antioxidant mechanisms: Transfer of phenolic hydrogen, Scavenging of singlet oxygen, Regeneration of tocopherol in the presence of ascorbate
- Vitamin C (Hydrogen donation to lipid radicals, Quenching of singlet oxygen, Removal of molecular oxygen, Regenerate tocopherol radicals, May act as a prooxidant, reduces ferric iron to ferrous iron producing ROS
- SOD: converts superoxide to hydrogen peroxide and non-active triplet oxygen
- CAT: converts hydrogen peroxide to water and non-active triplet oxygen directly without producing hydroxyl radical
- GSH-P: catalyses the reduction of different peroxides by glutathione
III. MEMBRANE TRANSPORT
16. Passive membrane transport
Nature and importance of membrane transport: Def.: Passing process of small molecules and ions through a membrane; process = high selectable Functions:
- Regulation of cell volume
- Import of substances necessary for cell growth and energy
- Maintains ion gradient and transforms energy
Importance:
- Faults in transport are cause of many diseases
- Different kinds of drugs can restore the normal cell functions through effect on membrane transport
Types of transport (classification): Types of membrane transport: according to…
- …Number and flow direction of transported substances
- Uniport: moves 1 molecule in 1 directio
- Symport: moves 2 molecules in same direction
- Antiport: moves 2 molecules in opposite directions
- …Change of charge and transmembrane potential
- Electrogenic: changes charge and transmembrane potential
- Non-electrogenic/ electroneutral: does not change charge and transmembrane potential
- …Molecular mechanism:
- Without carrier
- With (mobile/immobile) carrier Types of cell transport:
- Passive transport
- Cell does not use energy
- Molecules move randomly
- Movement to an area with high concentration -> area with low concentration
- 3 types:
- Diffusion
- Facilitated Diffusion: diffusion with help of transport proteins
- Osmosis: diffusion of water
- Active transport
- Cell uses energy
- Molecules move actively to where they are needed
- Movement to an area with low concentration -> area with high concentration
- 3 types: 1) Protein pumps (Sodium-Potassiom pump) 2) Endocytosis 3)Exocytosis Free diffusion of non-charged particles - first and second Fick’s laws: First Ficks law: concentration gradient does not change over time
Second Ficks law:
=>
-> proportional to electric potential In terms of dimentionless potential Nernst-Planck equation has simpler form:
Hindered diffusion across a porous membrane: -> to describe transport first Fick’s law is used, which is simplified by following restrictions:
- All concentrations stay constant with time
- There is linear dependence of concentration on the distance within the membrane
Fig.: Distribution of substance within pore membrane (concentration profile) Flux through pore membrane=
Collander-Barlung equation:
<- apply obtain -> Collander-Barlung equation P [m/s] = membrane permeability (same as velocity)
18. Transport of water solutions across membranes
Osmosis and filtration:
Def.: Osmosis= The diffusion of a solvent from a dilute solution through a semipermeable membrane to a more concentrated one (diffusion from higher to lower concentrations)
-> hydrostatic pressure stops osmosis in final stage
Van’t Hoff’s law: π = RTc
i = isotonic effect; R = gas constant; T = absolute temperature; c = concentration of solute
Def.: - Osmotic pressure = the hits of molecules on the system’s walls if the solute was in the form of ideal gas at the same volume and temperature
- Hypotonic= lower concentration of solutes; water moves into cell from out of the solution - Hypertonic = higher concentration of solutes; water moves out of cell into solution (e.g. sea water) - Isotonic = solutions with equal concentrations of solute; no movement of water
- Oncotic pressure = special type of osmotic pressure, induced by substances of high molecular weight ( e.g.: biomacromolecules)
Ratio btw experimentally measured and theoretically calculated values defined as reflection
coefficient σ : σ = πexp / πtheor 0 ≤σ≤ 1
Fig.: Osmotic pressure - the real (experimental) graph of its effect on the cell’s volume
Def.: Filtration= Movement of fluid through selectively permeable membrane from an area of higher hydrostatic pressure to an area of lower hydrostatic pressure
Filtration law (actually Poisuelle’s law applied to membrane channels):
or
Filtration coefficient (hydraulic conductivity) =
Kidney dialysis (haemodyalisis): -> purification of blood Same principle
- kidney cannot extract waste from blood
- blood is circulated through a saline solution (to prevent change in ion concentrations)
- waste products diffuse out through membrane into saline solution
19. Facilitated transport
- 2 basic types of transporters
Facilitated diffusion:
Ionophores – mobile carriers (valinomycin) and channel-forming carriers (gramicidin A):
Def.: Ionophores= Substances that seletively catalyse the diffusion of ions through membranes 2 general types:
- mobile ion carriers: carry abound ion directly across membrane
- channel-forming carriers: provide channel through membrane for passage of ions down their electrochemical gradients
Valinomycin – mobile carrier :
- carrier for K+
- hydrophobic periphery, hydrophilic core, 1 ion coordinatively bonded
- circular molecule, made up of 3 repeats of sequences
Mechanism:
IV. ELECTRICAL PROPERTIES OF CELLS AND TISSUES
21. Membrane potentials
Diffusion, equilibrium (Nernst) and Donnan potentials – conditions and mechanisms of generation, dependencies on concentration and time:
3 ways to move electrical charge in a system:
- move electrons
- move ions 3 processes involved: - diffusion caused by concentration differences - drift caused by electrical potential differences - active ransport: Na-K pump
“holes” in semiconductor devices
Electrodiffusion equation of Nernst-Plank:
c – ion concentration; φ - electrical potential; F - Faraday’s constant; D – diffusion coefficient; u - ion mobility; J – ion flux; z – charge of ion
Ηενδερσσον’σ εθυατιον: Νερνστ ευατιον:
Δονναν εθυιλιβριυμ χονδιτιον: Δονναν ποτεντιαλ:
-> Proportional to protein concentration in intracellular liquid and charge n of protein anion
22. Resting potential
Origin of the resting potential – theories: Assymmetrical ionic composition on the two sides of biomembranes
- [K+]i ~ (20 —40) [K+] (^) e
- [Na+]e ~ (10 —20) [Na+] (^) i
- [Cl+]e ~ (5 —10) [Cl+] (^) i The resting potential is actually transmembrane potential of cell membrane ( negative value of -60 /-90 mV) Theories:
- “potassium” theory of the resting potential Bernstein – 1902:
- resting potential is a Nernst (membrane) equilibrium potential caused by concentration difference of potassium ions (only) in the intra- and extracellular phases
Equations of Hodgkin-Huxley for the ionic currents – 3
= maximal values of the
membrane conductivities (when all channels are open) n,m, h= functions of potential and time; have meaning of probabilities (0 < n,m,h < 1)
Structure of the ion channels:
- Highly selective
- Bidirectional
_2 major categories:
- Voltage-gated channels_ : Conformation state depends on difference in ionic charges on 2 sides of the membrane 2) Ligand-gated channels : Conformation state depends on binding of a specific molecule (ligand)
Different channel types:
Effect of toxins and drugs on ion channel properties: -> local anaestetics
24. Surface electric charge of cells. Electrophoresis
Def.: surface charge = electric charge present at interface
- Processes leading to surface charging: a) dissociation of surface chemical groups b) adsorption of ions to the surface
- Practically all surfaces immersed in water are electrically charged
- Biomembranes are negatively charged Respectively, living cells are also negatively charged
Origin of the surface electric charge of cells: Major sources of biomembrane surface charge
- Lipid polar groups
- Side chains of protein aminoacid residues
Def.: surface charge density = σ 0 = number of charges/unit area
Electric double layer – electric charge and potential distribution in the vicinity of charged membranes: Components:
- Surface charge created by ionized surface groups
- Stern layer created by adsorbed counter-ions
- Diffuse electrical layer created by ions in solution
Ion distribution in diffuse layer determined by 2 opposing forces:
- electrostatic interactions, 2) thermal motion (entropic force)
Free energies of ion in bulk and near surface are equal:
mobility decreases
Measurement:
d – distance, which the particle travels for time t, t – time, S – area, κ – electroconductivity [ Ω -1m-1]
Types of electrophoresis
- Free electrophoresis (microelectrophoresis): boundaries btw fractions are smeared due to convection and diffusion
- Electrophoresis with carrier: Convection eliminated and different fractions are separated in well defined strips
- Analytical electrophoresis
- Preparative electrophoresis Frequently used carriers:
- Cellulose (electrophoresis on paper)
- Polymer gels (gel electrophoresis)
- Polyacrylamide
- Agarose
- Starch SDS gel electrophoresis: SDS(=sodium dodecyl sulfate)= surfactant (large – charge) -> binds to protein molecules -> forms micelles (practically same charge)
- electrophoretic mobility depends on protein molecular weight only -> nearest (highest) strips correspond to largest protein molecules
Preparative paper electrophoresis:
Drug electrophoresis: Principle:
- Electrophoretic introduction of small doses of medicines into epidermis
- Act either
- locally (e.g.: for treatment of joints) or
- spread gradually in organism via blood and lymphatic systems
25. Passive electrical properties of cells and tissues
Electrical conductivity of cells and tissues for direct (DC) and alternating (AC) current:
- Low voltages and currents -> no health risks
- High voltages and currents -> health hazard (electrical shock, burning, electrocution)
Relation btw electric conductivity and Ohm’s law:
Fig.: I(t) dependence for tissues
Types of polarization of dielectrics and heterogeneous systems.
- Elecronic polarization
- Atomic (ionic) polarization
- Dipole (orientational) polarization
- Electric Dipole (pair of + and – charge of equal magnitude, small distance btw)
- Dipole moment (product of + charge magnitude and distance btw charges) P=qd [C×m]
- Macrostructure polarization
- Surface polarization
- Electrode (electrolyte) polarization
