Class notes
Full Course Notes for Introduction to Biochemistry (BIOC2580)
- Institution
- University Of Guelph (U Of G )
These notes include all important lecture information as well as examples and definitions of key terms from each unit.
[Show more]
Seller
Follow
Content preview
FULL COURSE NOTES BIOC 2580Henderson Hassle back Equation
- If pH is one unit > pKa, the group is fully deprotonated
- If pH = to pKa, the group is 50% deprotonated and 50% protonated
- If pH is one unit < the pKa, the group is fully protonated
- If pH is less than one unit away from pKa, a calculation needed
- Average N-terminal has pKa of 9.5
- Average C-terminal has pKa of 2.5
- Average side chain has a pKa of 12.5
- Degree of deprotonation
Amino Acid analysis
- Partition /thin layer chromatography (Amino acids exchange between phases, based on polarity)
o Stationary phase: Particles of solid are chosen with a specific property
o Mobile phase: Liquid solvent or buffer flows past the particles and is non polar
o Polar amino acids: spend more of their time hydrogen bonded to silica and move slowly (last)
o Non-polar amino acids: N spend more time in solvent and move fast (first)
o Very polar amino acids have low Rf, non-polar amino acids have high Rf
- Column chromatography
o Sample mix ABC applied at top
o Buffer added carries sample mix through column to collection tubes
o Contents of collection tubes analysed, results plotted
o Volume of buffer needed to move a compound through the column is the elution volume. Compounds
can be identified by their characteristic elution volume
- Ion exchange chromatography
o Separates on the basis of charge, uses charges resins
o Cation exchangers have negative groups which bind positive molecules
o Anion exchangers have positive groups which bind negative molecules
o Elution by changing the pH to alter amino acid so it no longer binds
- How amino acids are detected
o They can be detected by adding ninhydrin which reacts with primary and secondary amine
o Ninhydrin reacts with amino groups to give a purple colour, and is used to detect and measure the
quantity of amino acids on (for example) TLC plates.
o Gives intense purple colour or yellow colour for proline
o Colour intensity is proportional to quantity of amino acid and can be measured
o Alternative is fluoresceine giving yellow fluorescence under UV light
- Metal affinity chromatography
o Clusters of His in a protein bind tightly to Ni or Co
o Column is made up of chelating resin containing Ni
o The His-tagged protein is eluted by adding imidazole to the buffer
o Imidazole out competes His-tag and protein no longer binds to the column
- Separating proteins on the basis of size
o Gel filtration or molecular exclusion chromatography allows separation of proteins on the basis of size
, o Protein molecules can enter the pores if they fit
o Larger proteins are excluded from the pores
- Two dimensional gels
Allow separation of proteins with similar isoelectric points that differ in molecular weight.
- Column chromatography using gel filtration
o Small molecules enter pores and are delayed in their movement down the column
o Intermediate size molecules can only enter some pores and are delayed less
o Very large molecules are excluded from gel and stay in the buffer flowing around the beads… pass
through column quickly
- Electrophoresis
o Separation based on movement of charged molecules in an ELECTRIC FILED
o Positive goes to negative, Negative goes to positive
- SDS PAGE
o the protein is pre-treated with the detergent SDS
o SDS causes protein molecules to extend, and give uniform charge per unit size
o When SDS is used, native protein charge is swamped out and overall negative charge is used to move the
protein through the gel (Towards positive electrode)
o Separation is based on SIZE
o Small proteins fit through the pores and move fast
- Isoelectric focussing
o Separation based on isoelectric point of proteins (the pH a which the net charge on the protein is 0)
o pH where net charge of protein is zero
o At high pH, protein is deprotonated, moves towards the + electrode
o As it passes through gradient of decreasing pH, becomes protonated and negative charge decreases
o Protein stops moving when net charge is 0
- Finding Molar mass of proteins using gel filtration
o Measure elution volume of proteins of known mass
o Elution volume: volume of buffer needed to move a protein from top to bottom of column
o Elution volume is a linear function of log molar mass (negative slope) (x)
o Then measure elution volume of unknown protein and project back to the log mass axis (y)
o Y= mx + b
o Find slope using (Y2-Y1) / (X2 -X1)
Basis of reactivity and hydrolysis
- Peptide bonds of protein are hydrolysed
o Acid hydrolysis
o Base hydrolysis
o With digestive enzymes: proteases
o After hydrolysis, amino acids can be analyzed using chromatography, tells you how much of each amino
acid is present
o Hydrolysis destroys the rest of the polypeptide chain
- Nucleophile: initiate biochemical reactions, nucleus lover, an atom with a lone pair of electrons available to share
o Seek out other groups that are electron deficient (electrophiles)
o Nucleophilic substitution or displacement: incoming nucleophile attacks target atom to displace its
leaving group
o If it has double bond, it only has to lose one of them
o Nucleophilic displacement: Hydrolysis is attack by H2O as nucleophile on the e- deficient C of the peptide
bond
, Determining amino acid sequence
- Sangers method
o can only determine the N-terminal amino acid, ONCE, we can’t repeat it, since hydrolysis destroys the
rest of polypeptide chain
o we can identify the first amino acid in a protein (The N-terminus) by tagging it with fluorodintirobenzene
(bright yellow)
o Flurodinitrobenzene reacts with the amino group of the N-terminal amino acid of a peptide/protein,
which can then be identified after hydrolysis based on the yellow colour of the labelled amino acid.
o The amino acid at the N-terminus of a protein has a free amino group, At high pH, the group
deprotonates to become: NH2-, a nucleophile
o Reacts with fluorodintirobenzene , HF is a good leaving group
- Edman degradation method
o the two step process keeps the reaction cycle in phase so only one amino acid is released per
cycle
o exclusion of H2O prevents hydrolysis of the remaining peptide bonds
o reactions can be repeated
o Step 1: (basic phase) coupling requires base: reaction must be complete before the next cyclization step
can take place (labels the N-terminal AA with PITC)
▪ Important because The N-terminal amino group becomes deprotonated under alkaline
conditions making it nucleophilic and allowing it to react with the electrophilic PITC reagent.
o Step 2: (acidic phase) reaction must be complete before the next coupling step can take place (cleaves
the first peptide bond). Coupling and cyclization cannot occur simutaneously
Selective hydrolysis
- Trypsin
o Cuts peptide bonds to the right of Lys and Arg (L or K)… but not if next to proline
▪ H-bonding groups of trypsin peptide backbone to enzyme , Selected side chain fits in a
long narrow pocket in trypsin, Negative charge at bottom of pocket attracts positive Lys
or Arg. AA that are too large will not fit
- Chymotrypsin
o Cuts peptide bonds to the right of Phe, Tyr and Trp (F, Y or W)… but not if next to proline
▪ Selected side chain fits in a nonpolar pocket in the chymotrypsin, Pocket is large, to fit
aromatic amino acids, benzene ring makes good contact with pocket… small AA may
allow H2O into non polar pocket
o Proximity and orientation: The binding of a large amino acid in the binding pocket of chymotrypsin
positions the peptide bond to be broken close to the catalytic unit.
o General acid/base catalysis: His-57 donates or accepts protons during catalysis.
o Hydrophobic effect: The binding pocket of chymotrypsin is lined with non-polar amino acids.
o Van der Waals forces: The binding pocket of chymotrypsin is the right size to fit a large amino acid.
o Lowering the energy of activation: Chymotrypsin breaks the peptide hydrolysis reaction into two easier
steps.
o Complementary to the transition state: The oxyanion hole binds to the tetrahedral carboxyanion.
- Cyanogen bromide
o Cuts to the right of Met (on the S)
o Peptide chain is broken on carboxylate side and Met is converted to homoserine, Hse
- Overlap method
o Two samples of the original polypeptide are each cut separately using two hydrolysis methods, each
targeting different sites (trypsin, chymotrypsin)
o Sequences from one set of oligopeptides are lined up to overlap with oligopeptides from the other set,
to deduce how they were originally joined
, - Mass Spectrometry
o Cleave one peptide bond per molecule, in random manner
o Flip the order to make it go from N-C
Secondary structure
- regular repetitive patterns such as helix, in short sections of the polypeptide chain
- The polypeptide chain forms a backbone which appears to be linked by C-C and C-N single bonds
- Single bonded structures are flexible due to bond rotation
- Groups connected by single bonds can rotate about bond axis
- Chain flexibility arises from bond rotation not bond bending
- Linus Pauling
o The peptide bond has double bond character
o The peptide bond has two resonance forms, one with a double bond
o Pauling compared lengths of C-N bonds to correlate bond length with bond order
- Alpha helix
o Peptide bonds form rigid planes connecting tetrahedral alpha-C atoms
o Restricted bond rotation limits freedom of motion, so that only a few regular structures can form
o in a helical shape, every a-C bond down the peptide chain turns in same direction (e.g.clockwise)
o in an extended shape, the a-C bonds turn in alternate directions down the peptide chain
o Distance between each helix turn is 5.4 Å
o Number of hydrogen bonds = n -5 +1
o Oxygen on amino 1 will always match up with Hydrogen on amino 5
o Ala, Arg, Gln, Glu, His, Leu, Lys, Met, (Phe) tend to form a-helix
- Beta strand/sheet
o Strands in the SAME direction make a parallel B-sheet
▪ H-bonds connect strand to strand
o Strands in OPPOSITE directions make anti-parallel B-sheet
▪ H-bonds align better in antiparallel mode
o Maximum space available for bulky or awkward shaped side chains
o Distance between each strand is 7 Å
o Trp, Tyr, (Phe), Val, Ile, Thr, Cys need room, prefer b-sheet structure
- Secondary Structure breakers
o GPNDS: Gly, Pro, Asn, Asp ,Ser
o 2 breakers in a group of 4 amino acids interrupts the secondary structure
o Forms a turn or flexible loop
Tertiary structure
- is the overall pattern of folding of the whole polypeptide chain, fiborous proteins
- The simplest possible tertiary structure is continuous secondary structure
- Most proteins are globular: this requires the polypeptide to fold back on itself (causing breaks)
- Allows for flexible loops and turns where polypeptide can change direction to allow folding
- The hydrophobic effect
o is a major force driving protein folding
o Non-polar Aas group together to minimize contact with H2O (hydrophobic effect).. in core
o Polar Aas form outer layer, interact well with surrounding H2O (good H-bonding) or with ions in solution
o Close contacts attract by weak van de Waals forces… Aka London dispersive forces
- A sequence with mostly groups of a-helix-forming AAs will fold into an a-helix bundle
o Non-polar AAs every 3 or 4 places in the helix make a non-polar patch or stripe e.g. -PPNPPNNP-, which
fold to inside of bundle
o AAs that prefer b-sheet are present, but scattered
- B-sheet-forming amino acids in majority fold into antiparallel B-sheet
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller emilygiles-duhamel. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for CA$10.61. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
78462 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now