lecture 1 biochemistry (week 1)

Question 1

why are proteins purified?

Answer 1

structural analysis

sequence analysis

therapeutic preparations

Question 2

what features determine how proteins can be separated?

Answer 2

differences in size/shape

surface change

hydrophobicity

ligand affinity

methods give differing levels of yield and resolution

Question 3

what must be done before starting a protein purification

Answer 3

collect info about characteristics and properties of most important impurities

molecular weight

isoelectric point, hydrophobicity

presence of carbs

free sulfhydryl groups (disulphide bridges -> reduction by addition of beta-mercaptoethanol)

stability - temp, pH, organic solvents, oxygen, heavy metal, mechanical shear

proteolytic degradation

minimise purification steps (4 steps is average)

Question 4

how does extraction from the source material occur?

Answer 4

homogenisation

sonication

freeze thawing

cell debris removed by centrifugation

protease inhibitors (eg, PMSF, Pepstatin A, Aprotinin) included to prevent deg. and extract is kept on ice

Question 5

what is homogenisation?

Answer 5

process whereby tissue is disintegrated into subcellular particles - nuclei, mitochondria

resulting mixture = homogenate

Question 6

what is sonication?

Answer 6

process by which sound waves are used for the lysis of the cell

Question 7

what is freeze thawing?

Answer 7

process of using freezing cycles and thawing to obtain the desired bioproduct

Question 8

what is in the crude cell lysate?

Answer 8

lipids - removed by centrifugation - lipids will float, removed by adsorption

nucleic acids (cause high viscosity) - removal by precipitation, addition of nucleases

Question 9

what techniques are used during the initial fractionation of the clear lysate?

Answer 9

ultrafiltration

precipitation - means to concentrate the solution, removal of most of the bulk proteins and other contaminants (proteases, membrane fragments)

fractional precipitation - bulk protiens are preciptated and removed with residual particulate matter by centrifugation, protein of interest is precipitated afterwrds from the supernatant, or protein is purified from the supernatant by column chromatography

Question 10

describe the process of ultrafiltration

Answer 10

cut-off limits for separation from 1000 Da to 300kDa –> removal of salts, concentration of protein solution (gentle, fast, inexpensive)

Question 11

what is precipitation

Answer 11

precipitation - means to concentrate the solution, removal of most of the bulk proteins and other contaminants (proteases, membrane fragments)

Question 12

describe fractional precipitation

Answer 12

bulk proteins are precipitated removed with residual particulate matter by centrifugation

protein of interest is precipitated afterwards from the supernatant

or protein is purified from the supernatant by column chromatography

Question 13

why are most proteins soluble?

Answer 13

there are charged groups on their surface that are solvated by water

decreasing interaction with water decreases the solubility of the protein precipitation

Question 14

how can the interaction with water of a protein be reduced?

Answer 14

antichaotropic salts (increase of hydrophobic effect in solution, salt competes with the protein for interaction with water, aggregation) e.g. (NH4)2SO4 ammonium sulphate

organic solvents (replacement of water, decrease in solubility)

organic polymers (e.g. polyethylene glycol PEG MW 6,000 or 20,000 neutral, viscous, inflammable, not poisonous, inexpensive)

varying the pH (lowest solubility at isoelectric point)

varying the temperature

Question 15

what are the principles of chromatography?

Answer 15

the molecule of interest partitions between 2 phases:

mobile phase (solution or solvent)

stationary phase (solid resin)

Question 16

where is the resin usually packed?

Answer 16

into a column, and the mobile phase is passed through it

properties of the protein determines how well it binds to resin

Question 17

what are the fundamentals of column chromatography?

Answer 17

different distribution of a protein between a stationary and a mobile phase

upright column made of plastic, glass or stainless steal filled with column material –> chromatographic medium (adsorption medium)

minus-column: column retains most of the proteins, but the wanted protein

plus-column: column retains the wanted protein, most of the other proteins end up in the flow through

Question 18

what is the analyte?

Answer 18

substance to be analysed

Question 19

what is the mobile phase?

Answer 19

consists of the sample being separated and the solvent that moves the sample through the column

Question 20

what is the stationary phase?

Answer 20

substance, which is fixed in place for the chromatography procedure –> particle size (3-100um) and pore size (10-100nm) are characteristics of stationary phases

Question 21

what are functional groups?

Answer 21

determine the properties of the column, attached to the material via hydroxyl groups

Question 22

what is the eluate (effluent)?

Answer 22

mobile phase leaving the column –> which removes the sample components

Question 23

what is the flow rate?

Answer 23

the volume of fluid, which passes through a given surface per unit of time

Question 24

what is the bed volume?

Answer 24

synonymous with column volume for a packed column

Question 25

what is the bed height?

Answer 25

height of the column material in a packed column

Question 26

what is the retention time (tR)?

Answer 26

the characteristic time it takes for a particular analyte to pass through the system

Question 27

what is the retention volume (VR)?

Answer 27

characteristic volume, which is required for a substance for its elution

Question 28

what is the exclusion volumen (V0)?

Answer 28

volume of the mobile phase on the column

Question 29

what is the isocratic separation?

Answer 29

the composition of the solvent i the mobile phase remains constant during the separation

Question 30

what is the gradient separation?

Answer 30

during elution the composition of the solvent changes stepwise or continuously

Question 31

what is present in the material in the stationary phase?

Answer 31

neutral, hydrophobic inert (no binding) functional groups generate different protein - binding properties chemical and physical stability(derivatisation, regeneration, sterilisation, high flow rate, high pressure) various, but constant pore sizes

Question 32

what are the gel forming materials?

Answer 32

2% agarose, Sephacryl S-500 (Poly-N,N‘-bisacrylamide Dextrane), Sephacryl S-500

Question 33

describe the monitoring of fractionation? (specific)

Answer 33

measurement of biological activity - enzymatic assay (dependent on pH, buffer temp, and conc. of substrates and co factors) use of antibodies in immunoblotting

Question 34

describe the monitoring of fractionation? (unspecific)

Answer 34

uv absorption --> 280nm (try, tyr) 205nm (peptide bond) biuret assay, lowry-folin-ciocalteau, bichinchonin acid assay, bradford assay

Question 35

what are the column chromatography methods?

Answer 35

gel filtration ion exchange chromatography chromatofocussing hydrophobic interaction chromatography reversed phase chromatography affinity chromatography affinity chromatography via immobilised metal ion covalent chromatography

Question 36

what are the protein features used for purification?

Answer 36

charged groups - asp, glu, lys, arg, his --> ion exchange chromatography and chromatofocussing metal chelating groups - his, trp, cys --> metal chelate chromatography size and shape --> gel chromatography thiol groups - cys --> covalent chromatography hydrophobic patch - phe, trp, ile, leu, val, etc --> hydrophobic interaction chromatography binding site - biospecific affinity chromatography