BIO1010 Practice Document PDF

BIO1010 | Cellular & Biochemical Basis of life Prokaryotic Cells Prokaryotic cells differ from eukaryotic as they do not have a defined nucleus but they possess a region or area which is filled with genetic material. These cells are generally divided into 2 major groups which are Eubacteria (true ba...

BIO1010 | Cellular & Biochemical Basis of life Prokaryotic Cells Prokaryotic cells differ from eukaryotic as they do not have a defined nucleus but they possess a region or area which is filled with genetic material. These cells are generally divided into 2 major groups which are Eubacteria (true bacteria) and Cyanobacteria. Another group which was recently discovered and added to these groups is the Archaea. Archaea are extremophiles meaning that they are highly adapted to thrive in extreme conditions and thus are able to adapt in all types of habitats. Cell wall and plasma membranes are present within these prokaryotic cells but mitochondria and other membrane bound organelles are absent. The Endosymbiotic theory suggests that different prokaryotes in the early stages of evolution clustered together in order to form other cells which are known as Eukaryotes. The area of clustered genetic material in prokaryotic cells is often called pseudonucleus or nucleoid. The genophore is a circular (sometimes linear) area of DNA organised into a nucleoid which is described as being an irregularly shaped region in the cytoplasm that delimits genetic material. The genophore is equivalent of chromosomes in prokaryotic cells and has nucleic acids but lacks histones which are associated with them. Eukaryotic Cells Cells which are defined as having a true nucleus, as the genetic material they possess is enclosed in a circular structure known as the nucleus which is in turn surrounded by a nuclear envelope (nuclear membrane). Eukaryotic cells can be either animal or plant cells. The major difference between the two is that plant cells usually contain a cell wall around the cell membrane whereas animal eukaryotic cells lack this cell wall. Viruses (or Virions) Viruses are infectious particles that replicate only inside a living cell/host. Viruses can infect all forms of life. They are either bacteriophages which are generally seen in the group Archaea or disease causing entities in eukaryotes. They are usually composed of a head region which houses genetic material in the form of single or double stranded RNA or DNA. This head region is referred to as the protein coat or nuclear capsid. Sometimes a tail and hooks are present in order for the virus to attach itself to the host cell and inject its genetic material within it for replication to start. Typically viruses are 20-300nm across. Viruses are very diverse ranging from the influenza virion to the rotavirus which causes severe diarrhoea in infants. Some viruses can in fact grow quite large. Some examples include the Pandoravirus Salinus and the P.dulcis virus. These giant viruses were recently discovered from sludge living as Amoeba (or other unicellular organisms) parasites. Other examples include the Mimivirus and megavirus chilensis. Viroids Viroids are plant pathogens possessing short RNA which is mostly circular and singly-stranded but also have double-stranded RNA in highly complementary regions. These RNA molecules do not code for any particular protein. Their replication cycle involves the rolling circle mechanism using enzyme RNA polymerase I. They lack a protein coat and are mainly responsible for causing distorted growth and stunting in plants. 1|Page Prions – Protein infectious particles Prions are malfunction proteins usually due to result of mutation that caused some misfolded protein which lacks nucleic acids. They are protease resistant and can mediate transmission of some very serious and harmful diseases. They are largely associated with brain degeneration diseases such as encephalopathies, Alzheimer’s, Creutzfeldt-Jakob, fatal familial insomnia and kuru. These misfolded proteins consist of a single infectious sialo-glycoprotein (includes sialic acid + glycoprotein) which is called PrP. This protein induces spontaneous alpha to beta transformation of peptide which aggregates into amyloid fibrils, killing neurons in the process. Prions do not contain any nucleic acids. Cell Biology Tools A huge range of tools which determine the outcome of an investigation have been developed over the past 50 years. New discoveries often follow new experimental tools as well as novel experimental methods using existing tools. Electron microscopy has enabled us to get a better understanding of structures as well as structural organisation in cells. Other advanced tools include confocal fluorescence microscopy, atomic force microscopy, flow cytometry and DNA analysis tools. Experimental Methods in Cell Biology 1. Microscopy techniques 2. Animal cell culture 3. Cell fractionation and centrifugation 4. Analysis of biomolecules 5. Use of isotopes and traces 6. X-ray diffraction and other methods of investigating macromolecules Microscopy Logarithmic scale: suggests cell sizes and components Units which must be taken into consideration: mm – 10-3m; µm – 10-6m; nm – 10-9m Å (Angstrom unit) – 10-10m Light microscope image – Phase contrast gives a very crisp light microscope image which uses no stains and a contrast is still created using changes in the amplitude of light. Confocal fluorescent light microscope (CFLM) – This type of microscope is able to show with clarity images of the nucleus, chromosomes and actin cytoskeleton filaments. CFLM uses stains in order to create a contrast within the specimen for better identification of structures being studied. The stains used are generally Phalloidin – FITC (f-actin, green colouration) and Hoechst 33342 (nuclear stain, blue colouration). Resolving Power The resolving power (R) of a microscope is defined as the ability of a lens system to distinguish between adjacent points as separate objects. The resolving power of a microscope depends on several factors: 1. Wavelength of light 2. Numerical Aperture 3. Lens defects on aberrations 2|Page Resolving Power and Wavelength Resolving power (R) of a light microscope with white light is 2m. R for violet light is about 1.87m. R for an electron microscope is about 5nm. R and Numerical Aperture (NA) NA is a function of the cone of light that passes through the lens, it is the light collecting ability of the microscope lens. The width of the light cone collected by the objective lens depends on the angular aperture (A). NA is calculated using the following equation: 𝑵𝑨 = 𝒏 × 𝒔𝒊𝒏(∝) Angle alpha is defined as ½ the angle aperture Advantages: Higher NA = greater resolution and brighter image Disadvantage: Larger NA = shorter working distance and depth of field becomes increasingly small. Hence the resolving power of a microscope can be calculated as follows: 𝑹𝒑 = 𝟎. 𝟔𝟏𝛌/𝐍𝐀 = 𝟎. 𝟔𝟏𝛌/𝐧𝐬𝐢𝐧 ∝ But 𝟎. 𝟔𝟏𝛌 ≈ 𝛌/𝟐𝐍𝐀 𝐍𝐀 Theoretical Resolution Limit: 𝟎. 𝟔𝟏 × 𝟒𝟓𝟎𝒏𝒎 𝑹𝒑 = = 𝟏𝟖𝟕𝒏𝒎 𝟏. 𝟓𝟔 × 𝟎. 𝟗𝟒 Limitations to Resolving Power Interference between waves of different phases may affect the image’s brightness leading to a dark image. Diffraction effects will lead to distortion of the edge of the image and a blurring effect on the total image obtained. Lens aberrations also limit resolving power. Spherical aberrations arise generally due to the curvature of the lens. Chromatic aberrations which are coloured fringes at the edge of the image are caused by the dispersion of white light through an aberration. To correct chromatic aberrations, compound lenses are generally used (They can be Achromatic lenses and Plan-Achromatic lenses). 3|Page Limitations of light Microscopy Optical thickness of the specimen (depth of material/medium) affects the quality and image appearance. The intensity of light also affects how the image is viewed as, if the image is too bright the structures being observed will not be visible as the intense white light will wash them out and if the image is too dark the structures within will not be clear enough for observation. The larger the optical thickness the poorer the image appearance. The optical thickness of a specimen may be defined by the following equation: 𝑶𝒑𝒕𝒊𝒄𝒂𝒍 𝑻𝒉𝒊𝒄𝒌𝒏𝒆𝒔𝒔 = (𝒏𝒐 − 𝒏𝒎)𝒙 The term (no – nm)x refers to the difference of refractive indices of the object/specimen medium. ‘x’ is defined as the thickness of the specimen parallel to the direction of light. Increasing Contrast of Image To increase the contrast of the image, specimens have to be stained using particular dyes. Stains help reduce the amplitude of particular wavelengths of light passing through them. However, stains also tend to distort, damage and introduce artefacts to tissues being studied. Different stains obviously can produce different images because each stain has a particular colour and a particular way in which it interacts with the tissue being observed. Wavelength phase changes occur through an unstained specimen (Phase Contrast Microscopy) – (Living sample cells appear with a bright halo whereas dead cells appear dark only when no stains are used) Modification of the light microscope. 1. (A) Bright Field Microscopy: Specimen against a bright background 2. (B) Phase Contrast Microscopy: Thick areas appear dark; edges/ thin areas appear bright as explained above. 3. (C) Nomarski- differential- interface- contrast microscopy (NIC): Optical microscopy illumination technique used to enhance contrast in unstained, transparent samples. 4. (D) Dark field microscopy: Describes microscopy methods, in both light and electron microscopy which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e. where there is no specimen to scatter the beam) is generally dark. 4|Page Light microscopy: Steps in Tissue Preparation 1. Fixing: Killing tissues without damaging them. In order to arrest biological decay and preserve the structure of the material, cells must be rendered inert and possibly strengthened using soft tissue, in order to retain shape. Formaldehyde is commonly used for this and is often dissolved in water, when it is known as Formalin, it causes covalent cross-linkages to be added between proteins, thus strengthening the material as well as rendering the tissue sterile. (Other reagents can be used such as – acetic acid 5% - 10%, various alcohols 90% - 100%, copper sulfate 2% or 5% or 10%, lactic acid 10%, copper acetate 2- 5% and potassium dichromate at 1% or 2% or 5%) Timing of fixation will depend on the thickness of the specimen and the strength of fixative, and may range from as little as 15 minutes for heavy metal fixatives not listed here, alcohol fixation takes up to 12 hours and formalin may require two whole days. After the desired time elapses, the excess fixative should be removed by soaking the specimen in two changes of water. Fixing can introduce distortions or artefacts. 2. Embedding & Sectioning: Specimen placed in a material, usually wax or resin, to be easily dissected or cut up into small pieces for observation (usually done using a microtome). This process involves dehydration and clearing, meaning that first, water has to be removed from the tissue of the specimen by repeated soaking in ethyl or Isopropyl alcohol solutions of progressively stronger strength. Once specimen is dehydrated, it is saturated with alcohol that is not miscible in paraffin wax, so this has to be removed. This process is known as clearing as the sample changes colour from opaque white to transparent. Usually chemicals such as Toluene. 3. De-waxing: Removing wax or resin layers using a sharp object. 4. Staining: Stains are chemicals having chromophores that may be basic, acidic or neutral. Stains require mordants or accentuators to bring about colour. There are progressive, regressive, counter and double staining techniques. 5. Dehydration: Removal of water through application of Isopropyl alcohol. 6. Mounting: The specimen is mounted accordingly onto the slide. Haematoxylin and Eosin (H&E) Designed to show the basophilic and acidophilic structures. Haematoxylin Eosin Haematoxylin which is a basic blue chemical develops staining properties when it is oxidised into haematin. Haematin needs a mordant (metal salt) that brings contact between dye and tissue. (In fact haematin combines with aluminium ions to form hemalum, the complex which is used for actual staining). 5|Page Eosin stains basic or neutral cells, such as cytoplasm components, collagen or muscle fibres. It is an acid, red/pink dye that shows up in basic parts of the cell. Structures that stain readily with eosin are termed eosinophilic. Eosin stains red blood cells intensely red. This staining technique is one of the most commonly used in histology. Tissues stained with H&E show that the cytoplasm stains pink-orange whereas the nuclei are stained darkly, either purple or blue. It is important to note that Eosin is an acidic dye and it only shows up in the basic parts of the cell, i.e cytoplasm. On the other hand, Haematoxylin is a basic dye and tends to show up in the acidic regions of the cell like the nucleus, where there is a concentration of DNA and RNA. Fast green and Safranin Fast green is used to stain cellulose green. In this chemical technique Safranin is used as a counterstain. Safranin is able to counterstain cell nuclei and lignified walls (xylem vessels) in a red colouration. Mallory’s Trichome stain and Haematoxylin This stain is utilised in histology to aid in revealing different macromolecules that make up the cell. It uses the three stains: aniline blue, acid fuchsin and orange G. As a result, this staining technique can reveal collagen, ordinary cytoplasm and red blood cells. Counterstaining with haematoxylin is done so as to further show particular staining. Principles of fluorescent staining Fluorescence occurs when a fluorophore is excited by light of a particular wavelength and hence, its electrons are raised from the ground state to an excited state. There are a number of factors which affect the intensity of fluorescent images:  Wavelengths of excitation and detection  Intensity of excitation light  Quantum efficiency of fluorophore  Saturation of fluorophore (transition to dark states) – photon output in linear range  Resonant energy transfer patterns  Oxidising / reducing agents in medium 6|Page Excitation and Emission Wavelengths There are different excitation maximums/levels and emission wavelengths for a number of fluorescent dyes. Such stains include Green fluorescent protein (GFP), 4’,6 – Diamidino-2-Phenylindole, Dihydrochloride (DAPI) and Fluorescein isothiocyanate (FITC). Fluorescent Microscope Optics In most cases, the sample of interest is labelled with a fluorescent substance known as fluorophore and then illuminated through lens with the higher energy source. The illumination light is absorbed by fluorophores (now attached to the sample) and causes them to emit longer lower energy wavelength light. This fluorescent light can be separated from the surrounding radiation with filters designed for that specific wavelength allowing the viewer to see only that which is fluorescing. The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light from the image. First, the microscope has a filter that only lets through radiation with the specific wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. To become detectable (visible to the human eye) the fluorescence emitted from the sample is separated from the much brighter excitation light in a second filter. This works because the emitted light is of lower energy and has a longer wavelength than the light that is used for illumination. Most of the fluorescence microscopes used in biology today are epi-fluorescence microscopes, meaning that both the excitation and the observation of the fluorescence occur above the sample. Most use a Xenon or Mercury arc-discharge lamp for the more intense light source. For further info visit: http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html N.B – Fluorescent dyes tend to become quenched when they are exposed time and time again to light. Hence, at some point, if the dye was used a number of times, excitation of the fluorophore won’t be possible. As a result, no fluorescence will be observed in the specimen. Natural Fluorescent Compounds Some compounds are naturally fluorescent on exposure to certain wavelengths of light. Example: Chlorophyll, sesquiterpene lactones & 7-membraned ring alkaloids. Some examples of the naturally fluorescing dyes include Azulene, Artemisinin & Colchicine. Fluorescent Compounds Most are synthetic, inorganic or organic compounds. They are commonly used and found in detergents. Fluorophores are used to label components of the cell directly (example: nucleic acids). Fluorophores can also be combined with antibodies for a particular antigenic molecule (protein, carbohydrate, lipids) and thus, they can act as tracers to locate molecules of interest within the cells or tissues under study. Fluorescent dyes usually emit a bright coloured light that gradually decreases with exposure to other light sources – quenching (equivalent of bleaching of the fluorophore). 7|Page  Nuclear Fluorescent Stains Most bind stoichiometrically to nucleic acids (in an amount proportional to the quantity of DNA present). Example: Propidium iodide, Ethidium bromide, Quinacrine dihydrochloride etc They also can be used for quantitative measurements flow-cytometry - cell ploidy and cell cycle visualisation are undertaken. Changes in fluorescent properties of the fluorophore occurs before and after binding to the nucleic acids. Most stains do not pass freely across the cell membrane because they are bulky, and as a result, cells must be fixed and/or permeabilised before staining. Cell membrane must be stretched by example exposing to water which makes cell expand and allows chemicals to enter through pores. However, some stains are so small that they are able to pass freely across membranes. An example of this exception are benzimides – Hoechst 33342 & Hoechst 33258 – penetrate cell membrane without permeabilization. Examples of Fluorescent Stains 1 Propidium Iodide and Ethidium Bromide: Intercalate between the purine and pyrimidine bases in double stranded nucleic acids. They Bind to both RNA (double stranded) and DNA. The former must be removed if only DNA is to be measured. They are excited by UV of blue light to give red or orange fluorescence. Propidium Iodide and Cell Cycle Analysis Cell cycle analysis is a method that employs flow cytometry (laser- based, biophysical technology employed in cell counting, sorting, bio marker A) Untransfected B) AllStars Negative Control siRNA C) CDC2 siRNA detection & protein engineering) to distinguish cells in different phases of the cell cycle. Before analysis, the cells are permeabilised and treated with a fluorescent dye that stains the DNA quantitatively, usually PI or Propidium iodide. The fluorescence intensity of the stained cells at certain wavelengths will therefore correlate with the amount of DNA they contain. As the DNA content of the cells duplicates during the S phase of the cell cycle, the relative amount of cells in the G0 phase and G1 phase (before S phase), in the S phase and in the G2 phase and M phase (after S phase) can be determined, as the fluorescence of the cells in the G2/M phase will be twice as high as that of the cells in the G0/G1 phase. N.B: Cell cycle anomalies can be symptoms for various kinds of cell damage, example DNA damage, which cause cell to interrupt cell cycle at certain checkpoints to prevent carcinogenesis (transformation into cancer cell). Other possible reasons for anomalies include lack of nutrients, for example after serum deprivation. 8|Page Examples of Fluorescent Stains 2 1. Acridine Orange and Quinacrine dihydrochloride: Dyes which are chemical derivatives of acridine. Intercalates between bases of double stranded RNA & DNA or by external ionic bindings and stains (yellow-green). Acridine orange will also enter acidic compartments such as lysosomes and become protonated and sequestered. In these low pH conditions, the dye will emit orange light when excited by blue light. Thus, Acridine orange can be used to identify engulfed apoptotic cells, because it will fluoresce upon engulfment. Simultaneous DNA & RNA measurement. 2. Benzimides (nucleus-blue)- Hoechst 33342 and Hoechst 33258: They fluoresce when excited by U.V light at around 350nm and both emit blue/cyan fluorescent light around an emission maximum at 461nm. The fluorescence intensity of Hoechst dyes also increases with the pH of the solvent. At high concentrations the colour of fluorescence will appear red. Hoechst dyes are soluble in water and in organic solvents. They bind to the minor groove of double-stranded DNA with a preference for sequences rich in adenine and thymine. Hoechst dyes are cell-permeable and can bind to DNA in live or fixed cells. Therefore these stains are often called supravital, which means that cells survive a treatment with these compounds. Cells that express specific ATP-binding cassette transporter proteins can also actively transport these stains out of their cytoplasm. 3. Fluorescein isothiocyanate (FITC). This is the fluorophore of choice in immunohistochemistry. FITC is a derivative of fluorescein used in wide ranging application including flow cytometry. FITC is the original fluorescein molecule functionalised with an isothiocyanate reactive group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure. This derivative is reactive towards nucleophiles including amine and sulfhydryl groups on proteins. FITC fluoresces green when excited. The fluorophore conjugates with phalloidin (attaches to actin) and also Annexin V. FITC like most fluorochromes, is prone to photobleaching. Immunocytochemistry or Immunohistochemistry This refers to the process of detecting antigens (example: proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumours. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand distribution and localisation of biomarkers and differentially expressed proteins in different parts of a biological tissue. Visualising an antibody – antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme such as peroxidase that can catalyse a colour-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine. Immunohistochemistry can be direct or indirect (Target antigen detection methods). Both involve marker- linked antibodies. The direct method is a one-step staining method and involves a labelled antibody reacting directly with the antigen in the tissue sections. This technique utilises only one antibody and therefore it is simple and rapid, in contrast to indirect approaches. Ergo, this stratagem is utilised with lesser frequency than its multi-phase counterpart. 9|Page The indirect method involves an unlabelled primary antibody (first layer) that binds to the target antigen in the tissue and a labelled secondary antibody (second layer) that reacts with the primary antibody. As mentioned above, the secondary antibody must be raised against the immunobody of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody, if the secondary antibody is conjugated to the fluorescent or enzyme reporter. The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labelled) secondary antibodies need to be generated. Other advantages include that, microtubules distribute themselves using TEM of cultured epithelial cells. Also fluorescent antibodies on tubulin help to aid recognition. It is important to note that the primary antibody will take the longest time to bind to the antigen. The secondary antibodies take less time to bind because the molecule has increased in size due to the primary antibody and hence it is easier to target and locate. Multi-Fluorescent Probe Microscopy Three fluorescent probes are used to stain three different cell structures in mitosis with different colours – the spindle microtubules; the centromeres; and the DNA. Annexin V – FITC staining During apoptosis, phosphatidylserine (PS), is translocated to the outer leaflet of plasma membrane using scramblase – bidirectional (flippases – from exoplasmic to cytoplasmic and floppases – reverse). PS serves as an apoptosis marker for macrophage recognition and engulfment. PS-binding protein - Annexin V - is a Ca2+ dependent phospholipid-binding protein. It can bind other phospholipids, but has highest affinity for PS. Annexin V can be conjugated to fluorescein isothiocyanate (FITC). Apoptosis vs Necrosis Stages of cell death. A & B: cell apoptotic early stage with PS transferred to outside, membrane still intact – thus no PI staining C & D: cell necrotic, with some PS externalised, however membrane structure compromised – thus PI stain accumulating inside cell 10 | P a g e FISH – Fluorescent in situ Hybridisation Technique used to detect and localise the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out when the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counselling, medicine and species identification. FISH can also be used to detect and localise specific RNA targets (mRNA, IncRNA and miRNA), in cells, circulating tumour cells and tissue samples. A small probe is prepared to get a purified section of genome specific to target gene by nick translation. For Nick Translation use tagging technique DNAase and digoxigenin or biotin to tag probe. Denature chromatin (formamide at 42degreesC) and add tagged probe. Probe binds that part of chromosome with which it shows a high degree of sequence complimentarily. Seek out probe with antibody tagged to fluorophore. Nick translation is a tagging technique in molecular biology in which enzyme DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labelled analogues. Avidin is a tetrameric biotin-binding protein. Advanced Microscopy Techniques Confocal Scanning Fluorescent Microscopy (CSFM) Houses a fine laser beam which scans through several layers of tissue. It virtually cuts sections of variable thickness. Reveals differences in structure at various levels. Capable of building a 3D construction image showing maximum detail of the specimen (cell contents). Images of the cell sections can be taken at 0.5micrometre intervals and a virtual reconstruction of the whole specimen can be constructed from these various sections. They are stained with fluorescent dyes so that structures are easily identified (example Phalloidin FITC for the actin cytoskeleton and with Hoechst stain for the DNA/RNA). Laser illuminates a small pinhole on plate A. Image focused at a single point in the 3D specimen. Emitted fluorescence from this focal point focused at a second (confocal) pinhole on plate B. Conventional and Confocal meaning that not every light coming from the specimen is focused, some is filtered out as only the selected wavelength passes. Micrographs of intact gastrula-stage Drosophila embryo (FITC probe for actin filaments). (A) The conventional, unprocessed image is blurred by the presence of fluorescent structures above and below the plane of focus. (B) In the confocal image, this outof-focus information is removed, resulting in a crisp optical section of the cells in the embryo. 11 | P a g e Following figure is a 3D reconstruction from CSFM Images. Pollen grains have a complex sculptured cell wall containing fluorescent compounds. CSFM images at different depths through the grains recombined to give a 3D view. Electron Microscopy While lenses in light microscopy are made of glass or quartz, lenses in electron microscopy are magnetic coils. LM uses light with an average wavelength of 0.53 m (530nm) while EM uses an electron beam with an average wavelength of 0.004nm at an accelerating voltage of 100KV. In electron microscopy the specimen is placed in a vacuum. Specimen must be dead, ultrathin and specifically stained (generally with heavy metal stains). The electron microscope has a limit of resolution which theoretically can be calculated to a resolving power of 0.002 nanometres but in actual fact it is about 0.1 nanometres. Transmission electron micrograph of a thin layer of gold shows the individual files of atoms in the crystal as bright spots. The distance between adjacent files of gold atoms is about 0.2 nm. Common EM fixative and stains include glutaraldehyde and osmium tetroxide (OsO4). Glutaraldehyde cross- links molecules ad forms covalent bonds between them. OsO4 is reduced by many organic compounds to form cross-linked complexes. This fixative and stain is especially useful for fixing cell membranes as it reacts with the unsaturated c=c double bond in many fatty acids in the bi-layer, hence staining them. Uranyl acetate and Lead citrate are also commonly used. EM has the capability of examining ultrathin sections of about 50-70 nm thick. These thin sections give sharply focused images at high magnification. The electron microscope uses electrostatic and electromagnetic lenses to control the electron beam and focus it to form an image. These electron optical lenses are analogous to the glass lenses of a light optical microscope. Electron microscopes are used to investigate the ultrastructure of ta wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals and crystals. Heavy metal shadowing of bacteria, viruses, isolated molecules and macromolecular assemblies is another high-resolution method for observing the ultrastructure of biological specimens. The prepared specimen is metal shadowed whilst strengthening with a carbon film at an angle resulting in a replica of the surface structure. The specimen is dissolved away by a solvent. Then the replica is put on a carbon grid for study under the electron microscope. 12 | P a g e Immunogold labelling or staining is a technique used in electron microscopy. Colloidal gold particles are most often attached to secondary antibodies which are in turn attached to primary antibodies designed to bind a specific protein or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast ‘dark spots’. The figure on the left shows a thin section yeast mitotic spindle with microtubules. Antibodies against four proteins are used to localise the position within the spindle pole body of yeast. Antibodies are linked to collided gold parts. Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained with an electron dense material, leaving the actual specimen untouched, and thus visible. This contrasts with ‘positive staining’, in which the actual specimen is stained. (Thus this technique is used to highlight the surface features on individual particles) Positive staining is almost the opposite of negative staining. This technique is carried out by staining the specimen under study using heavy metal stains so that a contrast is created between the background and the actual specimen. However, this contrast is inadequate for sharp imaging of structure and sections, it must be counterstained using heavy metal salts which bind to components at the section surface imparting enhanced electron density and sharp, contrasted electron images. Scanning Electron Microscopy (SEM) Scanning electron microscope produces images by probing the specimen with a focused beam of electrons that is scanned across a rectangular area of the specimen by electromagnetic coil lenses. Quantity of electrons scattered or emitted from surface of specimen is measured by a detector. An image is then built up on a video screen. Generally the image of an SEM is about an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimetres in size and has a great depth of field, and so it produces images that are good representations of the 3D shape of the sample. The SEM though, produces a highly detailed 3D image of biological specimens. Transmission Electron Microscopy (TEM) 13 | P a g e Transmission electron microscope uses a high voltage electron beam to create an image. An important mode of TEM utilisation is electron diffraction. The advantages of electron diffraction over X-ray crystallography are that the specimen need not be a single crystal or even a polycrystalline powder and also that the Fourier transform reconstruction of the object’s magnified structure occurs physically, thus avoids the need for solving the phase problem faced by X-ray crystallographers. The major disadvantage is that the TEM requires extremely thin sections of the specimens, typically of about 100 nanometres. Biological specimens have to be chemically fixed, dehydrated and embedded in a polymer resin to stabilise them sufficiently, to allow ultrathin sectioning. These sections may in turn require ‘staining’ with heavy metal ion stains so as to achieve the required contrast image. Scanning & Transmission Electron Microscopy (STEM) Scanning transmission electron microscope rasters a focused incident probe across a specimen that (as with TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterwards in the TEM. The STEMs use of SEM-like beam rastering simplifies annular dark-field imaging, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion. Often TEM can be equipped with the scanning option and then it can function both as TEM and STEM. Cryoelectronic Microscopy In this technique, rapid freezing to form vitreous ice is the key. A very thin (about 100 nm) film of an aqueous suspension of virus or purified macromolecular complex is prepared on a microscope grid. The specimen is then rapidly frozen by plunging it into a coolant. A special sample holder is used to keep this hydrated specimen at –160°C in the vacuum of the microscope, where it can be viewed directly without fixation, staining, or drying. Unlike negative staining, in which what we see is the envelope of stain exclusion around the particle, hydrated cryoelectron microscopy produces an image from the macromolecular structure itself. However, to extract the maximum amount of structural information, special image-processing techniques must be used. 14 | P a g e Atomic Force Microscopy (AFM) Also known as scanning force microscopy (SFM) is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometre, more than 1000 times better than the optical diffraction limit. This type of microscopy is able to examine details of the specimen at an atomic level. A fine laser beam directed on a silicon cantilever etched with a fine tip point. This point brushes/scans the surface of the specimen and sends the data to a detector. Part of the cantilever is coated with gold and thus it acts as a mirror reflecting the laser beam on a photodiode which in turn is connected to the detector. The cantilever is placed on a holding strip with a fine micrometre stage for adjustment. The stage on which the cantilever is attached is vibrated by what is known as a piezo crystal. A picture is built from the vibrations detected by the probe on the specimen surface. This microscope detects the smallest of vibrations, thus it must be operated away from urban areas and during the night. AFM can be operated in two modes: either Tapping mode (most suitable for biological samples) or Scanning mode (most suitable for nonbiological samples). Pulse Chase Autoradiography (Light Microscope) The chambers labelled A, B, C, and D represent either different compartments in the cell (detected by autoradiography or by cell- fractionation experiments) or different chemical compounds (detected by chromatography or other chemical methods). This is a method used in biochemistry and molecular biology for examining a cellular process occurring over time by successively exposing the cells to a labelled compound (pulse) and then to the same compound in an unlabelled form (chase). Radioactivity is a commonly used label. This process is used for tracing a pathway of formation of cell products such as proteins and other metabolites inside the cell. Pulse: Involves addition of a measured dose of a radioactive tracer which acts for a short time Chase: Addition of excess unlabelled chase compound pushes the pulse (usually an unreactive material used) Photographs are taken at different intervals recording the movement of the chemical under study. Very often the radioactive tracer used is 3H Thymidine (Tritiated thymidine) incorporated into DNA (but not RNA). Thus metabolites are CHASED by unlabelled thymidine and DETECTED by photographic means. Other heavy isotope tracers include 14C, 32P or 35S. It is important that the radiation used must not be too penetrating as the area of effect has to be small. Labelled tissue or cultured cells on glass slide are covered with a glycerine and AgBr photographic emulsion. (AgBr light/ radioactivity Ag). Unused AgBr is fixed from further development. Developed Ag is eventually photographed as a dark precipitate. Similar experiments were important to establish intra-cellular pathway for newly synthesized secretory proteins. 15 | P a g e Cell Culture Techniques Cell culture techniques (CCT) are used in various studies in biology such as:  Progression of a process with time  Movement of RNA and protein synthesis  Cell cycle changes  Energy metabolism  Intracellular activity & relation between organelles  Translocation of hormone receptor complexes  DNA replication and transcription  Signal transduction processes Cells from certain tissue cultures outside body. 2 types of these cells: Monolayer (adherent) or Suspension Culture (non-adherent). Specialised media are used to contain & sustain the culture thus, a well-balanced supply of salts & supplements is maintained. Culture conditions must also be highly controlled (temperature, CO2, O2, pH [7.2 or close], nutrients [respiration and build-up of new cells] and waste products [use of buffering agents]). Best Sources of Cells  Foetal or neo-natal tissues contain a lot of viable cells generally for short lived cultures.  Umbilical cord or cord blood contains stem cells which are excellent for culturing.  Haematopoietic tissues RBCs that give rise to all other blood cells thus, invaluable source of stem cells.  Tumour tissue is also a good source of cells for long lived cultures Culturing 1. Isolating Cells of a uniform type. Different cells held in the extracellular matrix must be sorted so as to isolate the needed cells. Cells of explant are generally dispersed enzymatically (e.g Trypsin, collagenase, pronase) by the use of chelating agents (e.g EDTA, trisodiumcitrate). The cells derived from the explant are a mixture, therefore, they have to be sorted using a number of techniques such as centrifugation, differential cell adhesion and antibody assisted/fluorescence activating cell sorting. Differential cell adhesion: Mechanism by which heterotypic cells in mixed aggregates sort out into isotypic territories. DAH theory – Viscoelastic liquids, and as such possess measurable tissue surface tensions. These surface tensions have been determined for a variety of tissues, including embryonic tissues & cell lines. The surface tensions correspond to the mutual sorting behaviour: the tissue type with the higher surface tension will occupy an internal position relative to a tissue with a lower surface tension (if these tissues can interact with each other through their adhesion machinery). Quantitative differences in homo and heterotypic adhesion are supposed to be sufficient to account for the phenomenon without the need to postulate cell type specific adhesion systems: fairly generally accepted, although some tissue specific cell adhesion molecules are known to exist. Fluorescence activated cell sorting: (or flow cytometry) is a laser- based, biophysical technology employed in cell counting, sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus (monitoring using fluorescence). It allows simultaneous multiparametric analysis of the physical and chemical characteristic of up to thousands of particles per second. Groups of cells passing through the stream contain a given charge 16 | P a g e  which if negative, produces fluorescence whereas if positive, indicates non-fluorescence. Droplets of the stream are deflected by an electric field according to their charge. Hence, cells of interest can be selected within the sample. 2. Establishing a cell line A cell line can be maintained over several generations by being repeatedly sub-cultured. A primary culture is often short lived whereas a malignant culture is long lived (example: Cancer cells). Cell cultures can be of two types; either Adherent or Suspension cultures. Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix (such as collagen and laminin) components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture, which involves growing cells in a 3D environment as opposed to 2D culture dishes. Adherent Cell Culture Suspension Cell Culture From adherent and solid tissues; cells secrete matrix From blood forming tissue; no matrix secreted Put out a number of processes to mark out territory and mark May form clumps which are easily broken down by syringing where offspring cells are deposited Appropriate for cells adapted to suspension culture and few Appropriate for most cell types, including primary cultures other non-adhesive cell lines (e.g. hematopoietic) Easier to passage, but requires daily cell counts and viability Requires periodic passaging, but allows easy visual inspection determination to follow growth patterns; culture can be diluted under inverted microscope to stimulate growth Cells are dissociated enzymatically (e.g., TrypLE™ Express, Does not require enzymatic or mechanical dissociation trypsin) or mechanically Growth is limited by surface area, which may limit product Growth is limited by concentration of cells in the medium, yields which allows easy scale-up Can be maintained in culture vessels that are not tissue-culture Requires tissue-culture treated vessel treated, but requires agitation (i.e., shaking or stirring) for adequate gas exchange Used for cytology, harvesting products continuously, and many Used for bulk protein production, batch harvesting, and many research applications research applications Culture Conditions The culture must be kept in a completely sterile environment. All materials, equipment, and air must be sterile, therefore a laminar flow cabinet with filter sterile air is used. Cell cultures must also be kept in areas away from microbes and/or chemicals which could lead to contamination of the culture itself. In fact, many scientists working in culturing cells have a problem known as cross-contamination, in which cell lines are contaminated by other cells. Physical & nutritional conditions present in organism are controlled & reproduced. Osmolality is monitored; hence, salt concentration must be controlled so as not to allow any imbalance which could lead to osmosis. 17 | P a g e The medium of a cell culture must supply energy and requirements for normal cell function i.e. cell growth and multiplication. Hence, the medium must possess; bio-chemicals or precursors, basal media, buffering system, accessory factors and supplements. Xenopus laevis and Cell Biology Also known as the African clawed toad, this organism is used in many biological studies such as for fertilisation & cancer studies. The advantages of culturing such an organism for research is that they are easy to maintain food and temperature needs, resistant to disease, males can be stimulated to mate, female frog induced to lay eggs by injecting (HCG) – human chronic gonadotrophin and also the eggs reach maturity in only a year’s time. In the figure on the right, Pelvic amplexus male (smaller) grasps the female (larger). Hybrid Cells Somatic cell hybrids are culture lines that contain the entire complement of the mouse genome and a few human chromosomes. These culture lines are developed by mixing human and mouse cells in the presence of the Sendai virus. The virus facilitates the fusing of the two cell types to a form a hybrid cell. For a reason that is not entirely known, most, but not all, human chromosomes are lost from the hybrid cell lines. Usually only a few human chromosomes are retained. Because the human and mouse chromosomes can be distinguished by chromosome staining techniques, it can be determined which human cells are retained with a specific cell line. Hence these hybrid cells are used to extensively study the human genome. Cell fractioning techniques Fractioning of sub-cellular parts To separate and purify sub-cellular particles mainly:  Organelles or components  Biological membranes (plasma, mitochondrial or plastid membranes)  Large organelles (nucleus) are isolated quickly as pure components (due to size)  Smaller organelles are more difficult to isolate as pure components  Other membranes are isolated to near purity  Some cells are easier to work with than other such as erythrocytes, since they lack many organelles as the cell is obviously specialised for carrying haemoglobin. Functional organelles and macromolecules are generally separated using gentle fractioning. Breaking Cells – Homogenates or Extracts Cells are broken up in many ways: subjected to osmotic shock or ultrasonic vibration, forced through small orifice, or ground up in blender. These procedures break many of the membranes of a cell (including plasma membrane & endoplasmic reticulum) into fragments that immediately reseal to form small closed vesicles. Artefacts can result (NB. Endoplasmic reticulum forms microsomes). If carefully carried out, however, the disruption procedures leave organelles such as nuclei, mitochondria, Golgi apparatus, lysosomes and peroxisomes largely intact. The suspension of cells is thereby reduced to a thick slurry (homogenate or extract) that contains variety of membrane-enclosed organelles, each with distinctive size, charge & density. Homogenisation carried out in isotonic solution (0.25M sucrose). Provided that homogenisation medium has been carefully chosen (by trial and error for each organelle), the various components (including microsomes) retain most of their original biochemical properties. 18 | P a g e Homogenate Fractionation All fractions are initially impure – contaminants can be removed by:  Re-suspension and repeating centrifugation  Use of different centrifugation speeds and  By altering density of suspension solution The different components of the homogenate must then be separated. Carefully applied procedure gives intact organelles of distinctive size, charge and density while retaining their original biochemical properties. Such cell fractionations are possible with the help of an instrument known as preparative ultracentrifuge that rotates cell extracts of broken cells at high speeds. The treatment separates cell components by size and density: in general, the largest units experience the largest centrifugal force and move the most rapidly. At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speeds, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first the small closed vesicles and then the ribosomes can be collected. All of these fractions are impure, but many of the contaminants can be removed by re-suspending the pellet and repeating the centrifugation procedure several times. Ultracentrifuge  Sample tubes mounted on a metal frictionless rotor.  Armoured chamber, rotor and motor  Vacuum – reduces friction & prevents rotor heating  Refrigeration system maintains sample at 4°C.  High rotation speeds - 80,000 rpms  Enormous centrifugal forces as high as 500,000 x g Centrifuge rotors Fixed angle particles have  Short distance to travel before pelleting  Easier to disturb pellet on withdrawing supernatant Swinging bucket have:  Longer distance of travel  Allows better separation e.g. in density gradient centrifugation  Easier to withdraw supernatant without disturbing pellet Centrifugation is the first step in most fractionations, but it separates only components that differ greatly in size. A finer degree of separation can be achieved by layering the homogenate in a thin band on top of a dilute salt solution that fills a centrifuge tube. When centrifuged, the various components in the mixture move as a series of distinct bands through the salt solution, each at a different rate, in a process called velocity sedimentation. For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles) finds itself on top of a lighter one (example, salt solution). This is achieved by augmenting the solution in the tube with a shallow gradient of sucrose prepared by a special mixing device. The resulting density gradient – with the dense end at the bottom of the tube – keeps each region of the salt solution denser than any solution above it, and it thereby prevents convective mixing from distorting the separation. Centrifugation techniques are mainly based on separating particles in their shape or size. 2 basic techniques:  Differential Centrifugation: (or preparative ultra-centrifugation) cell membrane is disrupted and components separated centrifugation at different speeds  Density Gradient Centrifugation uses a density gradient for separation. 19 | P a g e Principles of Centrifugation Sediment force (SF): SF = Mass x Centrifugal field SF = m r ω2 where m is the mass of particle, r is the radius of centrifuge and ω is the angular velocity of the rotor (i.e. Rotations per sec) Relative Centrifugal Force (RCF): RCF = r ω2/g = (2πN)2/g RCF = 1.118 x 10-5 x (N2) x r – However this is not a final equation due to compensation of a number of counter forces. Preparative Ultracentrifugation Simplest separation technique suitable for parts that differ greatly in size. Size of particles sedimented depends on the speed and time of the actual centrifugation. Repeated centrifugation at progressively higher speeds (Multistep process). Pellets at each speed, which are sedimented at the bottom of the centrifuge tube, contain particles of similar size. Typical values for centrifugation steps are:  Low speed = 1000xg for 10 minutes  Medium speed = 20,000xg for 20 min.  High speed = 80,000xg for 1 hour  Very high speed = 150,000xg for 3 hours Typical pellet components are:  Low speed = whole cells, nuclei, cytoskeleton  Medium speed = mitochondria, lysosomes,  High speed = microsomes, small vesicles  Very high speed = ribosomes, viruses, macromolecules Density Gradient Centrifugation In the absence of a density gradient, separated bands of solute in the centrifuge are gravitationally unstable. This is an important technique for purifying similar but intact cells, proteins and nucleic acids. Concentrated, dense solute band overlying less dense solvent leads to mixing by convection – nullifies separation. Overcome by creating DENSITY GRADIENT in tube. There are two types of Density gradient centrifugation:  Velocity/Rate Zonal centrifugation (Sucrose or Ficoll density gradient centrifugation) (explained above)  Isopycnic or Equilibrium Centrifugation (Caesium chloride density gradient centrifugation) The isopycnic centrifugation refers to centrifugation of equal density particles. In this process the ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape. In this case the sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or caesium chloride. Each cell component begins to move down the 20 | P a g e gradient, but it eventually reaches a position where the density of the solution is equal to its own density. At this point the component floats and can move no farther. A series of distinct bands is thereby produced in the centrifuge tube, with the bands closest to the bottom of the tube containing the compounds of highest buoyant density. This process is so sensitive, that it can easily separate macromolecules that have incorporated heavy isotopes from the same macromolecules that contain lighter, and more common isotopes. It can also be used to separate proteins and nucleic acids. Separation and Identification of Macromolecules  Chromatography (Large molecule separation)  Polyacrylamide gel electrophoresis or PAGE; (Separation of DNA or its fragments)  Western Blots (analytical technique to separate and detect proteins in homogenate)  Northern Blots (RNA nucleic acid separation/hybridisation)  Southern Blots (DNA separation and hybridisation) Thin Layer Chromatography or TLC Composed of a stationary phase made up of an immobilised thin layer, often made of glass or aluminium, and the mobile phase which is a single solvent or solvent mixture which moves the sample (acts as a carrier). The solvent must be allowed to saturate before placing the sample for TLC hence, the lid of the TLC container must be closed so as to allow the air inside to be saturated by the solvent. Solvent moves up the TLC plate or chromatogram by simple capillarity. From the distance of each sample, that moved from the bottom of the TLC plate, to as close as possible to the solvent line, the Rf value can be calculated; 𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒎𝒐𝒗𝒆𝒅 𝒃𝒚 𝒔𝒑𝒐𝒕 Rf = 𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒎𝒐𝒗𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒇𝒓𝒐𝒏𝒕 The retention factor values calculated are important for the identification of each particular sample. Column Chromatography Column chromatography is used to separate a mixture in solution (example proteins). A glass column is packed with a porous solid matrix, often silica gel is used. The different proteins are retarded to different extents by their interaction with the matrix, and they can be collected separately as they flow out of the bottom of the column. Obviously, different molecules, will interact differently with the matrix and hence, move at different rates. The sample collected at the bottom of the column at the end of the chromatography is referred to as the eluent. This can be collected into separate tubes. 21 | P a g e Depending on the choice of matrix, proteins can be separated according to:  Charge basis: ion-exchange chromatography (therefore separated on +/- charged matrices)  Different hydrophobicity: Hydrophobic chromatography (hydrophobic protein retarded by hydrophobic side chains of matrix)  size exclusion: gel-filtration chromatography (small proteins linger in pores of matrix beads; large proteins move on in spaces between matrix beads)  ability to bind to small molecules or to other macromolecules: affinity chromatography (matrices attract molecules of interest) Ion-exchange Chromatography relies on charge – charge interactions. Insoluble matrix carries ionic charges (retard movement of molecules of opposite charge). Protein column Chromatography proteins with the lowest attraction for the ion-exchange resin pass directly through the column and are collected in the earliest fractions eluted from the bottom of the column Affinity chromatography and SDS-PAGE The elution position of the still-impure protein determined by its enzymatic activity. The active fractions pooled and purified on an affinity column that contains an immobilized substrate of the enzyme. SDS PAGE Electrophoresis Different proteins can have similar sizes, shapes, masses and overall charges, most separation techniques such as SDS polyacrylamide-gel electrophoresis or ion-exchange chromatography cannot typically display all the proteins in a cell or even in an organelle. In contrast, 2D GE, which combines two different separation procedures, can resolve up to 2000 proteins – the total number of different proteins in a simple bacterium – in the form of a 2D map. In the first step, the proteins are separated by their intrinsic charges. 22 | P a g e The sample is dissolved in a small volume of a solution containing a non-ionic detergent, together with beta- mercaptoethanol (powerful reducing agent) and the denaturing reagent urea. This solution solubilises, denatures and dissociates all the polypeptide chains but leaves their intrinsic charge unchanged. The polypeptide chains are then separated in a pH gradient by a procedure called isoelectric focusing, which takes advantage of the variation in the net charge on a protein molecule with the pH of its surrounding solution. At low pH or high [H+] most proteins have a net positive charge  carboxylic acid group (–COOH) of proteins is uncharged  nitrogen-containing basic groups are fully charged (–NH3+) At high pH or low [H+] most proteins have a net negative charge  carboxylic acid groups are negatively charged (–COO–)  basic groups tend to be uncharged (–NH2) Every protein has a characteristic isoelectric point, the pH at which the protein has no net charge and therefore does not migrate in an electric field. In isoelectric focusing, protein moves to a position in the gradient that corresponds to its isoelectric point and remains there. In the second step, the narrow gel containing the separated proteins is again subjected to electrophoresis but in a direction that is at a right angle to the direction used in the first step. This time SDS is added, and the proteins separate according to their size. The original narrow gel is soaked in SDS and then placed on one edge of an SDS PAGE slab, through which each polypeptide chain migrates to form a discrete spot. This is a second dimension of the 2D PAGE. The only proteins left unresolved are those that have both identical sizes and identical isoelectric points, a relatively rare situation. Even trace amounts of each polypeptide chain can be detected on the gel by various staining procedures – or by autoradiography if the protein sample was initially labelled with a radioisotope. This technique has such great resolving power that it can distinguish between two proteins that differ in only a single charged amino acid. 23 | P a g e  It is important to note that Mercaptoethanol opens up proteins by disrupting their disulphide bridges. The polypeptide chains form SDS-protein complex. If carbohydrates are present they may hinder results as they might attach to a protein slowing down its migration process (amount of protein should always be kept equal so as to ensure validity and accuracy in results). N.B – Generally in 2D PAGE E.coli is used. Also each spot, as described above, is a different polypeptide. Compounds of Life Molecules and Ions Cell biochemistry depend on:  The molecules or ions it contains  Their interaction with other molecules/ions Molecules are covalently bonded clusters of atoms. Ions are charged particles that interact differently from molecules. Formation of molecules and ions depends on:  electron number  whether the electrons are shared or donated  their distribution in electron shells Polar and Non-Polar Structures Atoms of different elements joined by a single covalent bond attract shared electrons to different degrees. Example compared to C, N and O attract electrons strongly, whilst H attracts weakly. Polar structures arise when there is unequal sharing of electrons: a +ve charge is concentrated against one end and a –ve charge is concentrated at other end. As a result, polar covalent bonds arise (Example N-H and O-H but not N-N, O- O, C-H as they are attracted equally). Polar & Non-polar Covalent bonds Permanent Dipoles are created that allow molecules to interact through electrical forces. Compare the electron distributions in polar water molecule (H2O) and non-polar oxygen molecule (O2). Compare (+, partial positive charge; –, partial negative charge). Van der Waals Forces Applies for forces between atoms on different molecules as they come close together. One must consider space filled models - solid envelope representing extent of electron cloud. Strong repulsive forces PREVENT closer approach of another non-bonded atom. This is called the van der Waals radius for an atom. As nuclei of two atoms approach each other, they initially show a weak bonding interaction due to their fluctuating electric charges. However, the same atoms will strongly repel each other if they are brought too close together. The balance of these van der Waals attractive and repulsive forces occurs at the indicated energy minimum. 24 | P a g e Water Properties of water have determined how life evolved. This is the most abundant substance in cells comprising about 70% of the cell by weight. Most biochemical reactions occur in a medium of water and hence, most biochemicals are soluble in water. Some of which are partially insoluble and partially soluble. Insoluble substances are meant to act as boundaries to prevent access of water and other dissolved substances (Not meant to act as barriers). Water has two covalent bonds which are highly polar (O is strongly attractive to electrons, H is weakly attractive to electrons) and therefore water carries a permanent dipole. This particular characteristic gives rise to a special type of bonding, hydrogen bonding. Hydrogen bonding is highly directional and although they are weaker than covalent bonds, the combined effect produces a huge net stability to molecules. Four Types of Noncovalent Interactions  Ionic bonds: purely electrostatic  Hydrogen bonds: attraction between the electropositive hydrogen on one molecule and electronegative atom on another molecule.  Van der Waals attractions: fluctuating electron clouds produce a flickering dipole - a very weak interaction between atoms  Hydrophobic force: when a non-polar surface is pushed OUT of the hydrogen bonded water network. Hydrophilic and Hydrophobic Hydrophilic: Charged molecules or contain polar bonds, form H-bonds with water and thus dissolve readily Hydrophobic: Uncharged molecules and usually have long carbon chains are deemed hydrophobic. N.B – It is important to note that there are in total 4 types of non-covalent bonding interactions; Ionic bonds, H-bonds, VdW forces and hydrophobic forces. Four Families of Biomolecules 1. Sugars: Polysaccharides and oligosaccharides 2. Fatty acids: Fats, lipids and membranes 3. Amino acids: Proteins 4. Nucleotides: Nucleic acids (nitrogen bases (purines & pyrimidines) Adenine, guanine, thymine, cytosine and uracil in RNA) Proteins and Amino acids Proteins are highly complex organic molecules found in all living cells (most abundant). Proteins are the most functionally diverse molecules in all living systems. Every life process depends on proteins. Huge variety of proteins: enzymes, hormones, oxygen & electron carriers, energy converters, motor & skeletal proteins etc. Important Proteins in cells Regular array of two types of proteins in insect flight muscles – actin and myosin. DNA is surrounded by several types of proteins called histones. This is done so that DNA is able to keep its shape, as histones protect, package DNA and also regulate expression. 25 | P a g e Amino acids Amino acids are building blocks of proteins. Over 300 different amino acids have been described over the years, which occur in all biological systems, especially plants. Only 20 of these amino acids are commonly found as constituents of animal proteins. Food, obviously, contains a high amount of these variable proteins, some of which are essential and others are regarded as non-essential proteins. Proteins are absorbed upon digestion and carried in the bloodstream. They are mainly used for growth, maintenance and repair. (Cellular Catabolism: a process in which amino acids are broken down into smaller fragments). Non-essential and Essential Amino Acids essential amino acids non-essential amino acids arginine alanine Amino acids can be placed in the category of either essential or histidine asparagine non-essential. Essential amino acids are those that are isoleucine aspartate ‘essential’ in our diet. In other words, they cannot be created leucine cysteine lysine glutamate through our own metabolism and therefore, they must be methionine glutamine obtained from the foods we eat. Fortunately, protein- phenylalanine glycine containing foods contain varying degrees of essential amino threonine proline acids. During times of starvation, our body relies on its own tryptophan serine valine tyrosine protein stores, such as pre-albumin, albumin and ultimately protein from sources that it normally shouldn’t have to utilise (e.g. muscle, tissue, etc). On the other hand non-essential amino acids are those which can be produced from other amino acids and substances in the diet and metabolism. During times of need, the metabolism can shift into producing amino acids that it requires for synthesising proteins essential to our survival. Structure of Amino acids  An amino group (NH2) - basic  Carboxyl group (COOH) - acidic  Distinctive chain or R group The 2 functional groups (COOH & R) are attached to the alpha carbon. Effect of pH on Amino acids  At a low pH the amino group is protonated: NH3+  At a high pH the carboxyl group loses a proton: COO-  However, at physiological pH, 7.35-7.45, both groups are charged, hence the term zwitterion (Dipolar ion). In a zwitterion the ionic groups of opposite charge have to balance out perfectly. 26 | P a g e Stereoisomerism Every amino acid except glycine, shows optical activity through rotation of plane-polarised light. 2 optically active isomers; dextrorotatory d (+) and levorotatory l (-). Both isomers occur in nature but, the l (-) form is more common. Isomerism occurs due to chirality of alpha carbon. Chirality refers to the carbon in the molecule is surrounded by four different functional groups. Chirality is not limited to a single carbon, in fact amino acids with more than one chiral centre exist. Asymmetry is such that the two forms are not superimposable, hence they are referred to as enantiomers. Priority and Handedness By convention the groups are assigned the following priority; where the first is the most important and the last is the least: 1. NH3+ 2. COO- 3. R-group 4. Hydrogen As one moves from one group to another, chirality or handedness emerges. Amino Acids with 2 Chiral Centres An amino acid may have more than one chiral centre. This arises when the R functional group in itself has more than one functional group attached to it, example Threonine. Types of Amino acids The 20 amino acids commonly found in animals are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. The nature of the side group determines the role of the protein molecule. R-groups or side chains determine the physical characteristics of the amino acids and eventually the whole protein once a sequence of amino acids is joined together by polypeptide chains. Aliphatic groups are insoluble in water and are deemed hydrophobic. These hydrophobic parts are generally located deep within the protein so as to decrease the chance of contact with water. R-groups within the amino acids give rise to Relative Hydrophobicity. N.B – Hydrophobicity scales are values that define relative hydrophobicity of amino acid residues. The more positive the value, the more hydrophobic are the amino acids located in that region of the protein. These scales are commonly used to predict the transmembrane alpha-helices of membrane proteins. When consecutively measuring amino acids of a protein, changes in value indicate attraction of specific protein regions toward the hydrophobic region inside lipid bilayer. 27 | P a g e There are in total seven different R-groups: 1. Secondary amino group 2. Aromatic group 3. Aliphatic hydroxyl group – medium hydrophobicity 4. Acidic hydroxyl group – low hydrophobicity 5. Basic 6. Amide 7. Sulfur Hence, the degree of hydrophobicity differs with the different side groups present in each molecule. Amino acid with non-polar side chains Each of these amino acids has a non-polar side chain attached to its molecule. These side chains do not bind to give off protons and they also do not participate in Ionic or Hydrogen bonding. These R groups tend to cluster on the interior of the protein. They are usually aliphatic side chains and can be associate with oily or lipid like therefore hydrophobic interactions.  Glycine (H)  Phenylalanine (CH2Phenyl)  Alanine (CH3)  Tryptophan (phenyl and N-ring)  Valine (CH(CH3)2)  Methionine (CH2CH2SCH3)  Leucine (CH2CH(CH3)2)  ***Proline (imino ring CH2CH2CH2)  Isoleucine (HC(CH3)CH2CH3)  Cysteine (terminal S-group) Location of the non-polar side chains determines the eventual function and location of the protein. In aqueous solutions, the side chains of non-polar amino acids cluster in the interior of folded proteins and help to give it the 3-D structure as well as affect its location in such membranes. In lipid environment of membranes, non-polar side groups interact with the lipid and help localise it as an intrinsic protein. Types of Amino Acids Continued Proline An alpha-amino acid, one of the twenty DNA-encoded amino acids. It is not an essential amino acid, which means that the human body can synthesise it. It is unique among the 20 protein-forming amino acids in that the amine nitrogen is bound to not one but two alkyl groups, thus making it a secondary amine. Proline is also unique in the sense that, the ring structure that it is able to form with the amino group is in fact also referred to as an imino group (Capable of disrupting alpha helices, by forming a kink). Cysteine Cysteine is also an alpha-amino acid. It is a semi-essential amino acid, meaning that it can be biosynthesised in humans. The thiol side chain in cysteine often participates in enzymatic reactions, serving as a nucleophile. Thiol is susceptible to oxidisation to give the disulphide derivative cystine (dimer), which serves an important structural role in many proteins. Disulphide bonds are possible through this sulfhydryl (thiol) group of cysteine. 28 | P a g e Serine, Threonine and Tyrosine These amino acids have a net charge of zero at neutral pH although all of them possess a hydroxyl group which by virtue postulates them as polar amino acids. The polar hydroxyl group can form hydrogen bonds with other molecules as well as polar hydroxyl side chains, which may serve as a site of attachment for other groups (e.g. phosphates or oligosaccharide chains in glycoproteins) Aspargine and Glutamine Aspargine is one of the 20 most common natural amino acids on Earth. It has carboxamide as the side-chain’s functional group. It is a non essential amino acids. A reaction between aspargine and reducing sugars or reactive carbonyls produces acrylamide in food when heated to a sufficient temperature. These products occur in baked goods such as French fries, potato chips and toasted bread. Glutamine is also one of the 20 most common amino acids encoded by the standard genetic code. It is not recognised as an essential amino acid but, may become conditionally essential in certain situations, including intensive exercise or certain gastrointestinal disorders. Its side chain is an amide formed by replacing the side-chain hydroxyl of glutamic acid with an amine functional group, making it the amide of glutamic acid. These molecules are both neutral but polar molecules. Aspartate and Glutamate Aspartic acid is an alpha amino acid. The carboxylate anion, salt or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 22 proteinogenic amino acids. Aspartate is pervasive in biosynthesis. As with all amino acids, the presence of acid protons depends on the residue’s local chemical environment and the pH of solution. Glutamic acid is one of the 20-22 proteinogenic amino acids. It is a non- essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates. In neuroscience, glutamate is an impotant neurotransmitter that plays a key role in long-term potentiation and is important for learning and memory. Both of these amino acids have a carboxylate group attached to their side chain making them acidic and thus, prone to give off protons. Amino acids in aqeous solution contain weakly acidic α-carboxyl group and α-amino groups. They may also contain an ionisable group attached as a side chain to the molecule. These attachments to the molecule aid it to act in solution as a buffer. At physiological pH: Rate of formation of a zwitterion depends on the dissociation constant Ka or K1. 29 | P a g e  (𝑹𝑪𝑶𝑶−)(𝑯+) Therefore, Ka or K1 of amino acid = (𝑹𝑪𝑶𝑶𝑯) The reverse of Ka is the A (Association constant) i.e. the reciprocal of the equation above. From the Ka of the amino acid one can derive the Henderson-Hasselbach equation: [𝑹𝑪𝑶𝑶− ] Log Ka = log + log [H+] (x -1) [𝑹𝑪𝑶𝑶𝑯] [𝑹𝑪𝑶𝑶− ] by definition -log Ka = -log [𝑹𝑪𝑶𝑶𝑯] – log [H+] pH = -log[H+] & pka = -logKa [𝑹𝑪𝑶𝑶− ] pKa = -log [𝑹𝑪𝑶𝑶𝑯] + pH (since pk1 is the same as pKa) [𝑨− ] pH = pKa + log [𝑯𝑨] Henderson & Hasselbach EQN Hence, at dynamic equilibrium pH = pKa + 1 Other pK values:  pK2 refers to the pH at which 50% of the base is dissociated. Other values of pK 3 exist.  All amino acids have their unique isoelectric points  Amino acids cannot be stored in the body, as in excess, they are deaminated.  Protein: storage polymer for amino acids.  Amino acids are also used to form hormones and neurotransmitters. Proteins Continued The field of study that deals with proteins is referred to as proteomics. Variety of proteins:  Enzymes: Key actors in synthesis and metabolism  Polypeptide hormones: Regulate metabolic processes  Oligopeptides: Opioid pentapeptides and antimicrobials  Motor proteins: in muscles are responsible for shortening of muscle cells (Contraction)  Cell Support System and Cytoskeletal Proteins  Bone: Scaffold of protein collagen fibres with hydroxyapatite (calcium and phosphate) salts deposited  Albumin: Essential protein in the blood serum  Haemoglobin: RBC protein responsible for transportation of O2 and CO2  Immunoglobins & nanobodies: responsible for immunity responses and to fight infectious organisms ⧿ (Nanobodies: more effectively perform their function since they are small) Proteins as Macromolecules Proteins comprise approximately 50% of the cellular dry weight of a cell. Proteins are large molecules with high molecular weight ranging from 10,000 Daltons (50 – 100 amino acids) to 1,000,000 Daltons. Hundreds of protein molecules have been isolated in pure, homogenous form; many have been crystallized. All contain C, H, and O and nearly all contain S as well. Some also incorporate P and Fe. 30 | P a g e Peptide bond formation Peptide bond forms when a carboxyl carbon atom of amino acid binds covalently to amino nitrogen atom of another amino acid. This is a condensation reaction or dehydration synthesis with release of H2O molecule. Within a protein, all (except terminal) carboxyl and amino groups are combined in peptide linkage. It is not available for chemical reaction, BUT still important for H-bond formation. Each aa is linked via peptide link (amide bond). C–N bond is called a partial double bond. This gives rise to cis-trans isomerism where the trans-isomer is more stable than the cis-form due to relative vicinity (Repulsion of like charges) experienced. Stearic limitations on Bond Angles The peptide bond is planar – being a partial double bond, it is rigid and hence, it does not permit rotation. The most common conformation is a psi angle of -60° and a phi angle of -60°. (Ramachandran plot) A Ramachandran plot or [φ,ψ] plot is a way to visualise backbone dihedral angles ψ against φ of amino acid residues in protein structure. The figure to the left illustrates the definition of all the backbone dihedral angles mentioned above. The omega angle at the peptide bond is normally 180°, since the partial – double bond character keeps the peptide planar. A Ramachandran plot can be used in two somewhat different ways. One is to show in theory which values, or conformations, of the psi and phi angles are possible for amino acid residue in a protein. A second, is to show the empirical distribution of data points observed in a single structure in usage for structure validation, or else in a database of many structures. Either case is usually shown against outlines for the theoretically favoured regions. Peptide Bond Stability The peptide bond is quite stable as it may last up to a 1000 years, if not subjected to extreme conditions which may disrupt it. Prolonged exposure to strong acids and bases at high temperatures hydrolyses the bonds. Equilibrium lies on the hydrolysis side rather than the synthesis due to the requirement of energy. Protein Structural Components A protein consists of a polypeptide backbone with attached side chains. Each protein differs in its amino acid sequence and number. Proteins are always read from left to right (N-terminus to C terminus), as happens always in biological mechanisms. The sequence of amino acids makes the protein distinct. This distinct sequence is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific 3D structure that determines its activity and function (Tertiary structure). A polypeptide is a single linear polymer chain derived from condensation of amino acids. Individual amino acid residue are bonded together by peptide bonds and adjacent amino acid residues. Polypeptides have polarity in their molecules. The sequence of amino acid residues in a protein is defined by sequence of a gene, which is encoded in genetic code. In general, the genetic code specifies 20 standard amino acids; however, in some organisms, genetic code can include selenocysteine and in certain archaea pyrrolysine. Shortly after or even during synthesis, residues in a protein are often chemically modified by post- translational modification, which alters the physical and chemical properties, folding, stability, activity and ultimately the function. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes. 31 | P a g e Properties of polypeptides Regular and repeating parts – Main chain or backbone and distinctive side chains. Stabilised by disulphide bonds and also hydrogen bonding. The main chain has high potential for hydrogen bond formation. Hydrogen bonds are in fact found every fourth residue in alpha-helix or between adjacent residues in adjacent parallel chains. Protein 2° Structure Proteins do not assume a random 3D structure. Spontaneous 3D folding occurs but randomness is a totally different aspect. Amino acids arrange themselves according to the physical interactions between one amino acid and another. Most common structures are the alpha – helix and Beta – pleated sheet. Polypeptide chains with polar amino acids usually fold in such a way as to expose their polar groups on the outside and hide their hydrophobic groups which are generally tightly protected on the inside. Bonds in 2° Protein folding Three types of non-covalent bonds with which the secondary structure of a protein is held together. Generally the predominant bonding is hydrogen bonding but Van der Waals forces or other electrostatic forces are present. Characteristics of α – Helix A common secondary structure of proteins and is a right-handed coiled or spiral conformation (helix), in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier (Hydrogen bonding). The name 413-helix is also used, denoting a hydrogen bond between every carbonyl oxygen and the alpha-amino nitrogen of the fourth residue toward the C-terminus, and 13 atoms being involved in the ring formed by the hydrogen bond. Among types of local structure in proteins, the alpha-helix is the most regular and the most predictable sequence, as weak as the most prevalent. The amino acids in an alpha helix are arranged in a right-handed helical structure where each amino acid residue corresponds to a 100° turn in the helix, meaning that, 3.6 amino acids or residues per turn having a pitch (the vertical distance between one consecutive turn of the helix) of 0.54nm and a translation of 0.15nm along the helical axis. All the peptides except the first one, in an alpha helix, are stabilised by hydrogen bonding. The side chains of the residues or amino acids, as seen in the diagrams above, extend outwards of the spiral or cylindrical shape. N.B: Bovine insulin is an example of intramolecular and intermolecular disulphide bridges. Disruption of the Alpha Helix  Proline with its imino group inserts a kink in the chain and hence, disrupts the smooth helical structure.  Charged amino acids (such as glutamate, aspartate, histidine, lysine & arginine) disrupt the helix especially in large numbers.  Amino acids with bulky side groups also interfere with alpha helix formation. 32 | P a g e Characteristic of the Beta pleated sheet The beta sheet is the second form of regular secondary structure in proteins. It is less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least 2 or 3 backbone H-bonds, forming a generally twisted, pleated sheet. A beta strand is a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an almost fully extended conformation. The higher-level association of beta sheets has been implicated in formation of the protein aggregates and fibrils observed in many human diseases, notably the amyloidosis such as Alzheimer’s disease. Hyd