Extraction science

Question 1

Extraction sciences and sample collection

Answer 1

PH, buffers, and cell homogenisation

Question 2

Separation science

Answer 2

Centrifugation, dialysis, lyophilisation, precipitation

Question 3

Chromatography

Answer 3

Gel filtration, ion exchange chromatography, affinity chromatography

Question 4

Protein separation and purification

Answer 4

Electrophoresis

Question 5

Proteomics and metabolomics analysis

Answer 5

Protein sequencing, genome, transcriptome and metabolome

Question 6

Femtomoles

Answer 6

10-15

Question 7

Attomoles

Answer 7

10-18

Question 8

Cell homogenisation techniques

Answer 8

Non Mechanical and Mechanical

Question 9

Cell homogenisation - non mechanical

Answer 9

Osmotic shock Freeze-thaw Lytic enzymes Lysozyme

Question 10

Cell homogenisation - mechanical

Answer 10

Pestle + mortar, abrasives Ball mills and glass beads Blenders and rotor stators Homogenisers Solid Extrusion Liquid Extrusion Ultra-sonication (>20 kHz)

Question 11

Osmotic shock

Answer 11

Animal soft tissues Some plant cells Small scale only

Question 12

Freeze thaw

Answer 12

Animal soft tissues Some bacteria Time consuming; small scale; closed system- suitable for pathogens with appropriate safety measures; some enzymes are cold-liable

Question 13

Lytic enzymes

Answer 13

Animal cells, Plant cells, Mild and selective; small scale; expensive; enzymes must be removed once lysis is complete

Question 14

Lysozyme

Answer 14

Some bacteria Gram-negative bacteria must be pre-treated with EDTA. Suitable for some organisms resistant to mechanical disruption

Question 15

Pestle and mortar + abrasives

Answer 15

Tough tissues Not suitable for delicate tissues

Question 16

Ball mills + glass beads

Answer 16

Bacteria and fungi May cause organelle damage in eukaryotes

Question 17

Blenders and rotor-stators

Answer 17

Plant and animal tissues Ineffective for microbes

Question 18

Homogenizers (glass & Teflon

Answer 18

Soft delicate tissue e.g. white blood cells, liver Glass may shatter- wear safety glasses during use

Question 19

Solid extrusion (Hughes press)

Answer 19

Tough plant material; bacteria; yeasts Small scale

Question 20

Liquid extrusion (French pressure cell)

Answer 20

Microbial cells Small scale

Question 21

Ultrasonication

Answer 21

Microbial cells Cooling required; small scale; may cause damage to organelles, especially in eukaryotic cells

Question 22

Cell disruption - type dependencies

Answer 22

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

Question 23

Disruption media

Answer 23

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.

Question 24

Proteins and their separation

Answer 24

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.

Question 25

Properties of proteins - MCHDM

Answer 25

Mass - most sensitive and specific of techniques, mass spec Charge – pH Hydrophobic/Hydrophilic Properties Differential Solubility Mobility in Applied Fields

Question 26

Protein isolation

Answer 26

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

Question 27

Amino acid properties

Answer 27

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

Question 28

Measures of charge, pH, pKa and pl

Answer 28

* 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

Question 29

Interpreting pH and pKa

Answer 29

* 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 •

Question 30

Acid base equilibria

Answer 30

Question 31

Hendersen Hasselbach equation

Answer 31

Question 32

Measurement of pH

Answer 32

pH sensitive electrodes Glass combination electrode Electrode incorporates a pH sensitive layer and a reference Ag/AgCl electrode

Question 33

pH measurement

Answer 33

Question 34

Poly-Acid Titration – Phosphoric Acid

Question 35

Isoelectric Point

Answer 35

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

Question 36

Ion Selective Electrodes

Answer 36

Question 37

Controlling pH with Buffers

Answer 37

* 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

Question 38

Buffers – change in pH

Answer 38

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

Question 39

Acetate Buffer

Answer 39

Question 40

Buffer titration curve

Answer 40

Question 41

Blood buffer - carbonic acid

Answer 41

Question 42

Properties of ideal buffer

Answer 42

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

Question 43

Example Buffers

Answer 43

* 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

Question 44

Separation of proteins

Answer 44

* 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

Question 45