Protein Purification Flashcards
Protein purification
The isolation of a single protein through the physical separation and complete removal of all other proteins have been removed.
Compromise sometimes met between retaining as much as possible of the protein of interest while removing as mug as possible of extraneous proteins.
Why purify
Substrates may be specific for more than one protein- hence, if there is contamination substrate may go to both enzyme types and impact on the metabolic pathway
Determination of amino acid sequence
Determination of protein structure
Methods of protein purification
- size (MW)
- charge- IEP, pH dependant
- binding affinity for other molecules
Should use more than one technique as they rarely have absolutely unique size or charge
Column chromatography
- solid but porous material (matrix) with appropriate properties is supported inns column (cylinder) to make up the stationary phase
- buffered solution (mobile phase called eluate) flows through the matrix.
- fluid exiting base of column (effluent) constantly replaced by fresh fluid applied at top.
- protein mixture to be separated layer on top of column.
- proteins travel through column at different rates depending on their physical properties which determine the degree to which they interact with the matrix
- fluid exiting from base of column collected in fractions
- individual fractions monitored for total protein and for specific protein of interest
Size exclusion chromatography
Separation based on differences in size MW of proteins
Matrix contains pores of a particular size range
- larger proteins too large to enter pores (excluded), so take more direct route through column (effectively see smaller volume) so emerge more quickly
- smaller proteins can enter pores, so have access to larger volume of fluid; take longer route through column - emerge after a long time
- separation optimised by choosing matrix with appropriate pore size range.
Ion exchange chromatography
Exploits differences in the sign and magnitude of the net charge on proteins at a particular pH
- an anion exchanger binds anions by virtue of cationic (+ charged) groups covalently attached to its matrix.
- a cation exchanger binds cations by virtue of anionic groups covalently attached to its matrix
Strength of binding of each protein to matrix depends on: magnitude of charge on protein, concentration of free salt ions in buffer
Separation can be optimised by choice of matrix, varying pH, varying [salt]
Affinity chromatography
Separation depends on specific binding affinity of a protein for another molecule (ligand)
Column contains matrix cross- linked to that specific ligand- proteins that bind specifically to the ligand will be retained by the matrix
- proteins that don’t bind pass through
Protein of interest is then specifically eluted with solution of free ligand (competition) or by manipulation pH or ionic strength to disrupt ligand- macromolecule interaction
Assessment of protein purity
Use a separation technique with high resolving power to separate all proteins present in the sample
Visualise all proteins with a non- specific proteins rain
Protein is pure if only one protein species appears in sample
Analytical techniques depend on differences in physical characteristics of different proteins such as size, charge,’IEP
SDS PAGE
Electrophoresis separates molecules depending on their behaviour in an electric field- separation is potentially based on charge,’size and shape.
- special case that separates proteins based solely on size: form cross linked polyacrylamide gel (matrix) from polymerise acrylamide monomers, gel acts as a sieve, slowing down molecular movement
- treat protein sample with SDS
- SDS binds to protein backbone evenly along length
- large neg charge from SDS swamps any charge originally present on native protein (natural charge now irrelevant)
- all proteins now carry massive negative charge (proportional to size) but some “charge density” so subjected to same force in electric field
- electrostatic repulsion forces tend to liberalise all protein molecules.
- protein molecules with covalent disulphides linkages are unable to linearise fully, so reducing agent added (beta- mercaptoethanol or dithiothreitol)
- all molecules now have same extended linear shape and same large charge density- molecular size is now the only variable
Isoelectric focusing (IEF)
Separation of proteins based on IEP
mixture of ampholytes (small organic molecules containing both acidic and basic groups) used to set up a pH gradient in a gel matrix, under the influence of an electric field
Protein mixture then applied to gel and power reconnected
Proteins move to position where pH equals their IEP: at this point there net charge is zero and they no longer move, so molecular migration stops
Two dimensional gel electrophoresis (2D page)
Separation by IEF (1st dimension) then SDS- page (2nd dimension at 90 degrees)
Provides higher resolving power (better separation of proteins)
Provides protein mixture “fingerprint”
When coupled with more sensitive stain, illustrates true complexity of biological samples (sheer number of proteins present)
Specific activity as a measure of protein purity
The specific active of an enzyme preparation expresses the enzyme activity in terms of the total amount of protein present.
- specific activity has units of units (U) enzyme activity/ mg protein ie. U.(mg protein) ^-1 or micromole substrate transformed/ min/ mg protein
A measure of the purity of the enzyme preparation:
- Lower the levels of contaminating proteins present, the purer the enzyme is and the higher its specific activity
- specific activity of an enzyme preparation reaches a maximal value when the enzyme has been purified to homogeneity
Turnover numbers of enzymes
Specific activity is useful for comparison of purity, but not very tangible, hard to visualise at the molecular level, provided the MW of the enzyme is known, the specific activity of an enzyme can be used to calculate the:
Turnover number: Number of substrate molecules tra softened to produce by each molecule of enzyme per second (Kcat)
- easy to visualise at molecular level
- must be based on reaction rates (enzyme activities) measured when the enzyme is pure, working at maximal rate, Vmax
- cannot calculate rate enhancement ratio for every enzyme
- enhancement ratio is not reflected in turnover number, does not account for rate of in catalyses reaction
- turnover numbers don’t enable us to reliably compare the catalytic efficiencies of performances of different enzymes, but they illustrate how quickly the catalyses reaction occurs at the enzymes active site
Better measure of catalytic performance
- also consider the concentrations of their substrates at which they can speed up their reactions appreciably:
- if two enzymes have the same Vmax but one can achieve that Vmax at a lower substrate concentration than the other, then is is a more catalytically efficient enzyme
- the Km of an enzyme for its substrate conveniently accounts for this factor
- Km is the (S) at which the reaction rate itself is half maximal.
- the Km for an enzyme tends to be Silat to the cellular concentration of its substrate
- an enzyme that acts on a substrate present at very low levels in the cell usually has a lower Km than an enzyme that acts on a more abundant substrate
- the km is physiologically relevant kinetic parameter to use
Specificity constant better indicator of catalytic efficiency
- best indicator of enzyme efficiency is the ration Kcat/Km - specificity constant
Turnover number reflects Vmax, Km reflects affinity of E for S (higher affinity = lower Km = higher ratio)
