Extraction science Flashcards
To introduce the experimental techniques and principles of extraction and separation science for preparation and characterisation of biological samples. Prof Shaw
Extraction sciences and sample collection
PH, buffers, and cell homogenisation
Separation science
Centrifugation, dialysis, lyophilisation, precipitation
Chromatography
Gel filtration, ion exchange chromatography, affinity chromatography
Protein separation and purification
Electrophoresis
Proteomics and metabolomics analysis
Protein sequencing, genome, transcriptome and metabolome
Femtomoles
10-15
Attomoles
10-18
Cell homogenisation techniques
Non Mechanical and Mechanical
Cell homogenisation - non mechanical
Osmotic shock Freeze-thaw Lytic enzymes Lysozyme
Cell homogenisation - mechanical
Pestle + mortar, abrasives Ball mills and glass beads Blenders and rotor stators Homogenisers Solid Extrusion Liquid Extrusion Ultra-sonication (>20 kHz)
Osmotic shock
Animal soft tissues Some plant cells Small scale only
Freeze thaw
Animal soft tissues Some bacteria Time consuming; small scale; closed system- suitable for pathogens with appropriate safety measures; some enzymes are cold-liable
Lytic enzymes
Animal cells, Plant cells, Mild and selective; small scale; expensive; enzymes must be removed once lysis is complete
Lysozyme
Some bacteria Gram-negative bacteria must be pre-treated with EDTA. Suitable for some organisms resistant to mechanical disruption
Pestle and mortar + abrasives
Tough tissues Not suitable for delicate tissues
Ball mills + glass beads
Bacteria and fungi May cause organelle damage in eukaryotes
Blenders and rotor-stators
Plant and animal tissues Ineffective for microbes
Homogenizers (glass & Teflon
Soft delicate tissue e.g. white blood cells, liver Glass may shatter- wear safety glasses during use
Solid extrusion (Hughes press)
Tough plant material; bacteria; yeasts Small scale
Liquid extrusion (French pressure cell)
Microbial cells Small scale
Ultrasonication
Microbial cells Cooling required; small scale; may cause damage to organelles, especially in eukaryotic cells
Cell disruption - type dependencies
Animal cells No cell wall. Plasma membrane and cytoskeleton weak. Can be easily disrupted by using Waring Blender Plant cells Cellulose cell wall and additional lignin or waxes Can be disrupted by blenders or Press-solid extrusion or enzymes eg pectinases and cellulase Bacterial Cells Gm+ and Gm- bacteria- cell wall. Peptidoglycan Can be disrupted by sonication or using the enzyme lysozyme Fungal Cells Filamentous Fungi and Yeasts Robust- 90% polysaccharide (Chitin, mannan + protein microfibrils) Can be disrupted by grinding with glass beads, acidic washed sand or celite
Disruption media
Buffer to replace intracellular buffer system. Usually ~pH 7.0 Inorganic salts. KCl or NaCl. Usually below 100mM Sucrose. Used to prevent osmotic lysis of organelles eg. Mitochondria or lysosomes. Also used to stabilise proteins. Mg2+- integrity of membranes – counteracting negative charge of membrane phospholipids. Also ATP in complex with Mg2+ EDTA (ethylene diamine tetra acetic acid). To chelate heavy metal contaminants that would otherwise inactive cysteine amino acids which are often important for protein stability and activity. Removes Ca2+ which can activate some proteases and nucleases. Protease inhibitors.eg. PMSF (phenylmethylsulfonyl fluoride- serine protease inhibitor). Intracellular proteases are released from lysosomes on cell breakage (lysosome acid pH so at neutral pH less active). Reducing agents. eg 2-mercaptoethanol, dithiothreitol (Clelands reagent). Prevent oxidation of proteins especially with free cysteine thiol groups which are needed for activity. They may be oxidised to disulfide bridges or sulfenic and sulfinic acid which can inactivate the protein. Detergents cause dissociation of proteins and lipoproteins from the cell membrane. SDS (sodium dodecyl sulfate) will denature proteins and is used for SDS-PAGE. Some non-ionic detergents such as Triton X100 are used for membrane protein isolation since they do not denature protein.
Proteins and their separation
An understanding of the structure and function of proteins is important to understand since they make up a large proportion of the living cell. They are important for 1) Enzymes to carry out all of the reactions going on in the cell; 2) Structural proteins make up connective tissue, muscle and bones; 3) The Immunoglobulins (IgG, IgM, IgA, IgE) are part of the humeral immune response; 4) Proteins form complexes with RNA in ribosomes and DNA in nucleosomes; 5) Proteins interact with lipids in the cell membrane and act as transport systems into the cell; 6) Proteins interact with sugars in glycoproteins and most proteins in higher cells are glycosylated.
Properties of proteins - MCHDM
Mass - most sensitive and specific of techniques, mass spec
Charge – pH
Hydrophobic/Hydrophilic Properties
Differential Solubility
Mobility in Applied Fields
Protein isolation
Study of protein structure and function requires them to have a preserved quaternary structure They cannot be denatured To isolate proteins from a cell and keep under conditions close to the at of the cell; pH, ionic strength, viscosity, solvation, temperature Protect against degradation by proteases Preservation of their oxidation state - over-oxidised Separation and purification based on their molecular properties
Amino acid properties
Non polar + hydrophobic:
Alanine, isoleucine, leucine, methionine, phenyalanine, proline, tryptophan, valine
Charged + polar:
Arginine, histidine, lysine, aspartic acid, glutamic acid
Polar (hydrophilic) + uncharged
Asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine
Charged + polar
Arginine, histidine, lysine, aspartic acid, glutamic acid
Measures of charge, pH, pKa and pl
- pH is more complex than just a measure of the hydrogen ion concentration, especially in complex fluids
- The formal definition of pH is:
pH=-Log(a_(H_3O^+) )
- Log is the Base 10 logarithm
- a_(H_3O^+)is the activity of the hydrated hydrogen ion
- Activity = g[H_3O^+]
- gis the activity coefficient takes values 0 – 1
- Activity reflects the non-idealityof a liquid such as the cytoplasm
Interpreting pH and pKa
- Lower pKa the higher Ka(because of the minus sign) hence
- STRONGER ACID
- pKa of an acid is the pH at which it is exactly half dissociated
- pH > pK_aacid is fully dissociated
- pH < pK_aacid is predominantly HA
- Strong Acid HCl,Ka= 107
- Weak Acid MeCOOH,Ka= 1.74 × 10-5
•
Acid base equilibria
Hendersen Hasselbach equation
Measurement of pH
pH sensitive electrodes
Glass combination electrode
Electrode incorporates a pH sensitive layer and a reference Ag/AgCl electrode
pH measurement
Poly-Acid Titration – Phosphoric Acid
Isoelectric Point
Isoelectric Point (pI)
- The Isoelectric Point is the pH at which the molecule contains no net electrical charge
- The protein may still be charged attracting a solvation shell
- The protein may still be polar
- The net charge – equal numbers of positive
and negative charges
• Incorporation into lipid bilayers may be favoured/required
Ion Selective Electrodes
Controlling pH with Buffers
- Used to control pH of a solution
- Usually a mixture of weak acid and conjugate base
- Defined as- one which resists a change in H+ concentration (pH) on addition of acid or alkali.
- In whole cells the cellular constituents act as buffers. eg proteins which have a large number of weakly acidic and basic groups as amino acid side chains.
- Extent of pH change is determined as buffer capacity. This can be measured experimentally by a titration curve. The mid-point of the plateau of the sigmoidal curve is the pKa where there is best buffering capacity
Buffers – change in pH
Add 10 mL of NaOH to 1L of water. What is the pH change?
pH of water is 7, Kw = [OH-] [H3O+]
[OH-]= 0.01moles in 1.01 L = 0.0099 M
So [H3O+] = 10-14/(0.0099) = 1.01 × 10-12
= pH 11.99
A pH change of 5 by adding only 10 mL of NaOH to 1L
Acetate Buffer
Buffer titration curve
Blood buffer - carbonic acid
Properties of ideal buffer
Impermeable to biological membranes
Biological stability and lack of interference with metabolic and biological processes
Lack of significant absorption of ultraviolet or visible UV light
Lack of formation of insoluble complexes with cations
Minimal effect of ionic composition or salt concentration
Limited pH change in response to temperature
Example Buffers
- Citric and Phosphate form insoluble complexes with divalent cations. Phosphate can act as a substrate, activator or inhibitor of certain enzymes.
- TRIS often toxic to biological systems and can penetrate membranes decoupling electron transport systems in whole cells or isolated organelles.
- Markedly affected by temperature with a tenfold increase in H+ concentration from 4oC to 37oC.
- Zwitterionic buffers eg HEPES
- Interfere with protein determination eg. Lowry assay.
- Overcome many of the problems associated with other buffers since structure based on amino acids so more ‘Biological’.
BUFFERS WILL WORK BEST AT PH VALUES ONE UNIT EITHER SIDE PKA
Separation of proteins
- Mass
- Centrifugation
- Passage through membranes - dialysis
- Affinity
- Hydrophobic/Hydrophilic Properties columns • Differential Solubility extraction buffers
- Charge and Mobility in Applied Fields • Buffer control
- Separation on gels
- Electrophoresis
• Single Cell Analysis
