Physical Chemistry of Proteins/Analytical Methods Flashcards
How do histidine side chains often fulfill important functions?
Its pKa is lose to physiological pH (6 vs. 7.4); where acid/base catalysis occurs in some proteases
How does the ionisation state of the histidine side chain change with pH?
- Imidazole ring is mostly protonated below pH 6; carrying a positive charge equally distributed between both nitrogens.
What do posttranslational modifications of proteins achieve?
It can influence the physicochemical properties of proteins, resulting in potential changes in solubility, stability, bonding etc.
Which hydroxyl groups are commonly phosphorylated in posttranslational modifications and what can this achieve?
- Ser, Thr, Tyr
- Phosphorylation gives -ve charge and increases bulk; encouraging protein-proteins interaction
- E.g. Tyr phosphorylation in tyrosine kinase receptors; key to signalling pathways in the cell
What does glycosylation entail and how does it affect the physio-chemical properties of proteins?
- Attachment of sugar moieties to Ser, Thr or Asn residues
- Can alter solubility ( -OH group makes protein more soluble/stable)
What does hydroxylation entail (and to which AAs) and how does it influence the physio-chemical properties proteins?
- Addition of hydroxyl (OH) group
- To Pro or Lys residues
- Can alter H-bonding
What does methylation entail and how does it influence the physio-chemical properties or proteins?
- Addition of methyl groups (CH3) to N or O atoms of AA side chains
- Added hydrophobic (non-polar) group
- Thus sterically bigger
What does disulfide bond formation entail and what effect does it have on the physio-chemical properties of proteins?
- Covalent bond between two Cys AAs (thiol groups/sulfur)
- Renders protein more stable due to additional covalent linkage
If a protein has many basic side chains, what charge will it possess at physiological pH?
- Protein is positively charged
- Abundance of basic side chains = easily protonated = H+
If a protein has mainly acidic side chains, what charge will it possess at physiological pH?
- Protein is negatively charged
- Easily deprotonated = COO-
What factors influence a protein’s state of ionisation?
- Amino acids
- pH of the solution environment
What is the isoelectric point (IEP)?
The pH at which a particular molecule molecule or surface carries no net electrical charge; does not migrate in an electric field.
What does a protein being at its IEP influence, and what’s the common range for protein IEP?
- Protein is at its least soluble
- But this also means it’s at its most permeable
- Occurs for most proteins at pH 5.5 - 8
How is the influence of IEP on solubility be exploited in an insulin glargine (long-acting) formulation?
- Insulin glargine contains two extra Arg residues at the end of the B-chain
- Arg residues are basic
- Thus raising the IEP and altering its solubility; it is more soluble in the acid conditions used in the formulation (more protonation) but less soluble upon injection (physiological pH)
- Insulin precipitates out and slowly dissolves in blood (due to raised IEP) giving long-lasting action
What can the separation of different proteins be based upon?
- Differences in charge
- Differences in hydrophobicity
- Differences in solubility
- Differences in size
What is the principle of gel electrophoresis?
- Molecules separated according to their size and charge (size-to-charge ratio)
- Proteins and nucleic acids electrophoresed within a matrix or “gel”
- Charged molcules migrate toward either postive or negative pole according to charge in an electric field
What are the different forces of attraction and retardation at play in gel electrophoresis?
Attraction:
- Size of charge
- Size of electric field
Retardation:
- Friction (of the matrix)
- Repulsion in medium
What factors give a protein high mobility in gel electrophoresis?
- Small
- Highly charged
What factors give a protein low mobility in gel electrophoresis?
- Large
- Minimally charged
What are the requirements for the analytes in electrophoresis and why?
- Must be charged (or have a charge induced)
- Usually contain acidic or basic functional groups
- Can’t be at IEP; ionisation dependent on pKa of functional group and pH of electrolyte
What are the benefits of using Native Polyacrylamide Gel Electrophoresis (PAGE) over other electrophoresis?
- Native structure of protein maintained during electrophoresis (3D structure not disrupted)
- Separation according to SIZE and CHARGE
How dose PAGE achieve keeping native protein structure?
- Protein isn’t denatured; acrylamide gel is a size-selective sieve during separation
- Smaller molecules travel more rapidly than larger proteins through the gel in response to an electric field
What does SDS-polyacrylamide gel electrophoresis entail?
- Separation of proteins according to SIZE only
- Native (3D) structure of protein is not maintained; proteins are denatured by heat and the addition of the detergent SDS prior to electrophoresis
How is it ensured that proteins are only separated by size in SDS-polyacylamide gel electrophoresis?
- Via the addition of sodium dodecyl sulfate
- A detergent; emulsifies protein and gives a net negative charge, with different proteins in the same SDS solution given approximately the same charge:mass ratio; predominantly migrating on size
- Hydrophobic tail interacts with oil/lipid membranes, negative hydrophilic head sticks out; making it water soluble too
What are the typical detection methods for proteins post-electrophoresis?
- Coomassie Brilliant blue dye staining (Bradford assay)
- Western blot (protein immunoblot); transfer of gel contents onto a membrane and detection via labelled antibodies
What are the advantages of UV absorption (spectrophotometry)?
- No additional reagents or incubations required
- No protein standard needs to be prepared
- Assay does not consume protein
- Relationship of absorbance to protein concentration is linear
What are the disadvantagse of UV absorption (spectrophotometry)?
- Any non-protein component of the solution that absorbs UV light will interfere with assay
Why are there maxima at 280 nm and 200 nm with protein absorbance UV?
280nm: amino acids with aromatic rings (Trp and Tyr)
200nm: peptide bonds
What other factors can affect the absorbance spectrum and why?
- Secondary, tertiary and quaternary structure all affect absorbance
- Thus factors such as pH, ionic strength etc. affect the spectrum
What colorimetric methods for measuring protein concentration are available and what can be generated as a result?
- Bradford assay (dye based)
- BCA Protein Assay (copper based)
A standard curve with samples of known protein concentrations can be created, and thus the concentration of the unknown protein is determined from the curve.
What does the Bradford assay entail?
- Colorimetric method for measuring protein concentration
- Dye based
- Coomassie Brilliant Blue dye binds to proteins in acidic solution (via electrostatic and van der Waals forces)
- Results in shift of absorption maxima of dye from 465 to 595 nm
What does the BCA Protein Assay entail?
- Colorimetric method for measuring protein concentration
- Copper based
- Reduction of Cu2+ to Cu+ by protein in an alkaline medium with subsequent colorimetric detection of Cu+ cation by bicinchoninic acid
- Intense purple-coloured reaction product results from chelation of two molecules of BCA with one Cu+ ion
- BCA/Cu+ complex exhibits strong linear absorbance at 562nm with increasing protein concentrations
What is the advantage of using the BCA Protein Assay to the Bradford assay?
Peptide backbone (that reduces Cu2+ in alkaline) also contributes to colour formation, helping to minimise variability caused by protein compositional differences.
