Bio 98 (Luecke/Ribbe)
Midterm Review!
Tutors:Priyanka SaxenaOH: Mondays, 9:00-11:00amSH [email protected]
Tutors:Justin KoOH: Wednesdays, 3:00-5:00pmSH [email protected]
First off…Evaluation Sheet!
Overview of Review Session
PowerPoint/Packets will be posted afterwards No need to take copious notes!
Will be having normal OH next week
Format: Quick reviews, Q/A, working through problems (pI, pH)
This review DOES NOT cover all the material presented in lecture and is only meant as a guide.
Water
Density Ice cubes Lakes
Structure Hydrogen bonding Dipole
Ice
Water as a solvent
Like dissolves like Polar, charged molecules Ex: NaCl
Water separates from: Hydrophobic molecules Ex: Oil Why?
Biochemical Forces
Covalent (Strong)
Non-covalent (weak, but more important biologically) Hydrogen bonding Ionic interactions Van der Waals forces Hydrophobic interactions
The pH scale
pH = -log [H+]
Lower the pH, the more acidic the solution
Strong acids and bases
A strong acid/base COMPLETELY dissociates in water
Example: HCl Strong acid ALL of the HCl put into water will become H+ and
Cl- ions
Weak acid/bases
Weak acid/bases do NOT completely dissociate
Example acetic acid When put into water, some acetic acid will not
dissociate and still be in acetic acid form
In this case: Keq = Ka = [H+][Ac-]/[HAc] pKa = -log (Ka)
Titrations
http://www.chembio.uoguelph.ca/educmat/chm19104/chemtoons/chemtoons9.htm
Henderson-Hasselbach
pH = pKa + log [Ac-]/[HAc]
Problem 1
Benzoic acid has a pKa of 4.2. How many ml of 0.1M benzoic acid and 0.1M are needed to make 5 liters of 0.1M buffer at a pH of 5.2?
Buffers and physiology
Consider 2 cases
Blood has to be maintained tightly at a pH of 7
If the pH of the blood decreases (meaning there is more H+ than OH-) then the body has to be able to offset the excess H+ to increase the pH back up to 7
So, how does this happen?
Buffers and physiology
The excess protons combine with HCO3- to form H2CO3 which quickly dissociates into water and CO2.
CO2 can then be expelled out
Problem 2
Find the pH of 0.8M acetic acid.
pKa = 4.8 pKa = -log(Ka) so Ka = 1.7x10-5M
Concept: Part of the acetic acid that has been put into the solution will dissociate into equal parts H+ and acetate
Ka = [Ac-][H+]/[Hac]
Problem 3
How many moles of NaOH should be added to a solution with 0.44 moles of formic acid, HCOOH, to prepare a formic acid/formate buffer with a pH of 4.0? (Ka of formic acid = 1.7x10-4) **
**Example from this website:
http://www.files.chem.vt.edu/chem-ed/courses/equil/buffers/prac1.html
Solution 3
1) Using the given pH, find the concentration of H+ ions (pH = -log[H+])
2) We also know that the total moles of HCOOH and HCOO- needs to be 0.44 moles
3) HCOOH(aq) H+(aq) + HCOO-
(aq)
4) Using the formula, write an equation for the Ka
Solution 3, cot’d
5) Plug in value for Ka and [H+] to get a ratio of [HCOO-]/[HCOOH]
6) Rearrange equation to solve for [HCOO-]
7) The number of moles of NaOH that needs to be added is the same as the moles of HCOO-
Amino acids
Total of 20 amino acids Combine in different orders and numbers (based on
mRNA code) to make up PROTEINS
Structure Do not memorize – know key characteristics of ones
mentioned in lecture (proline, isoleucine, methionine, threonine, cysteine)
Amino acids
EVERY amino acid has the following: NH3+ (N-terminal) COOH (C-terminal) H
What is variable is: The SIDE CHAIN! (R group)
Problem 4
What is the pI of Tyrosine?
Peptide bond
Estimating MW
Average mass of the 20 amino acids (used for calculations) = 110Da
Example: estimate the mass of a 300 amino acid protein 300x110=33000 Da * Note: Da is just another term for grams/mol (MW)
1 Da = 1gram/mole
Lambert-Beer Law
A=ecl A= absorbance e = molar extinction coefficient (unique to each
molecule) C= concentration L = path length; usually 1cm
Problem 5
Imagine a protein has the following sequence: A-R-M-Y-M-N-M-W-Y-Y-W-W-W-W-W (Y: tyrosine, W:
tryptophan) The molar coefficient per Trp is 5,500/Mcm and for
Tyr is 1,400/Mcm Find the TOTAL molar extinction coefficient What concentration of the protein would give an
absorbance value of 0.35?
Isoelectric point
What is it? The pH at which the protein or amino acid has no
NET charge (neutral) When pH > pI = negative charge When pH < pI = positive charge
pI: no titratable side chain The pI is the average of the two pKa’s on either
side of zwitterion
When solving pI problems: always start with the fully protonated form of the amino acid/protein! (NH3+, COOH, and side chain)
Example: Tryptophan
NH3+, COOH NH3+, COO- NH2, COO-
pI: titratable side chan Example: Arginine
Fully protonated form = NH3+, COOH, NH2+ (side chain)
NH3+, COOH, NH2+ NH3+, COO-, NH2+ NH2, COO-, NH2+ NH2, COO-, NH2
pI = (9.04 + 12.48)/2 = 10.76
Problem 6: pI of a protein
Val-Pro-Ala-Trp-Cys-Gln
Steps: 1) Look at amino acid residue on N and C terminal 2) Look for any amino acid residues that have a
titratable side chain (using table 3-1)
pI of protein
1) pKa of amino group on Val = 9.62
2) pKa of carboxy group on Gln = 2.17
3) Any side chains? 1) Cys (pKa of R group = 8.18)
Start with protonated form of everything:
NH3+, SH, COOH NH3+, SH, COO- NH3+, S-, COO- NH2, S-, COO-
pI = 2.17 + 8.18/2 = 5.175
Problem 7
Find the NET CHARGE of the same sequence at a pH of 8.18
Val-Pro-Ala-Trp-Cys-Gln
Make a chart (if time, draw a quick picture)
If pH > pKa : amino acid will be deprotonated
If pH < pKa : amino acid will NOT be deprotonated
If pH = pKa: special case
Answer : Net charge = -0.5
Val Pro Ala Trp Cys Gln
Side chain?
No No No No Yes No
pKa 9.62 8.18 2.17
pH 8.18 8.18 8.18
Change?
NH3+
NH3+
SH S-
COOH
COO-
Charge
+1 Avg = -O.5
-1
Protein purification
Need to separate your protein of interest from everything else inside of a cell!
Why? Research Pharmaceuticals Therapies Sequencing (HGP!)
4 key steps
1) Homogenize: Prepare CFE
2) Centrifuge
3) Ammonium sulfate precipitation: solubilities
4) Column chromatography (3 types)1) Ion exchange
2) Gel
3) Affinity
Chromatography
Ion exchange Cation: column binds POSITIVE peptides/amino
acids Anion: colum binds NEGATIVE peptides/amino acids
Gel SMALL beads elute/come out of column LAST (get
caught in beads)
Affinity Ligand interaction, etc.
Monitoring purification
SDS PAGE (electrophoresis)
SDS is negatively charged –binds to fragments and makes them negatively charged
Can now flow towards POSITIVE (bottom) of gel
Quantifies proteins by molecular weight
Sequencing a Protein
You have a folded protein, which you now need to linearize to study each amino acid & peptide sequence
Things to do: Break disulfide bridges Make the protein smaller (easier to work
with/sequence) Separate, sequence
5 key steps
1) Break disulfide bonds (DTT/2-betamercaptoethanol), then block using iodoacetic acid
2) Cleave proteins (using proteases)
3) Separate using HPLC
4) Sequence: Edman degradation or mass spec
5) Align correct sequence
Protein ID
Identifying an unknown protein
2-d gel electrophoresis (MW and pI)
Peptide mass fingerprinting
Protein: Myoglobin,Hemoglobin,
and Enzyme
Protein Structure
Primary Structure Sequence of AAs
Secondary Structure Alpha and beta
conformation
Tertiary Structure 3D structure. Protein
Quarternary Structure Multiple proteins
coming together Homo-oglimer vs.
Hetero-oglimer Ex: Hemoglobin
3D structure of Polypeptide and its Restriction
Alpha carbon limited to phi and psi angle
Partial double bond restricts movement
Result is planar conformation
The Ramachandran PlotPossible movement of phi and psi angle denoted.
Alpha and Beta Sheet
3.6 residues per turn
5.4 Angstrom height per turn
R group always pointed down
i-> i-4 H linkage
Helical Wheel DiagramUseful for deciding hydrophobicity/hydrophilicity
Alpha and Beta Sheet
R groups alternate up and down
Parallel or Anti-parallel
C=O and N-H groups switch “left” and “right”
Protein Conformation
Most stable in its final folded shape Lowest energy
Linear form of protein is not stable Hydrophobic
residues exposed Disrupt water by
decreasing entropy
Chaperone Protein
Provides a chance for mis-folded protein to take its final conformation Provides right
kind of environment for this to happen Acidic or basic.
Different from intracellular environment
Oxygen-binding curve for Myoglobin
Hyperbola
[O2]0.5 Concentration
of O2 where half of Mb is saturated with Oxygen molecules.
Oxygen-binding curve for Hemoglobin
“S” Shaped
Sigmoidal curve is suited better for transport. Off loads more
O2 to tissues
T state and R state for Hb
Allosteric Quaternary
structure change Information relayed
to other subunits
R= relaxed state High affinity for O2
T= tensed state Lower affinity for
O2
Concerted vs. Sequential Model
Concerted Sequential
Transitioned in a “concerted” manner.
Equilibrium shifted to the R state.
Transitioned one by one. One subunit affecting the
other.
Bohr Effect
Body’s way of adapting to the demand
Affinity of Hb for O2 changes with the change in blood pH level Lower the pH,
lower the affinity
The Effect of BPG on Hb
Body’s way of adapting to the demand
More BPG produced with falling pO2 lvl (ie: high altitude)
Same affect on Hb as pH change Lowers the
affinity Increase of P50
Enzyme-- the Biological Catalyst
No Enzyme Enzyme Added
Enzymatic Kinetics
Variable K1,k2(Kcat), K-1, Km
Michaelis-Menten Equation
Hyperbolic Curve
Vo= rate at which the product is produced by the enzyme
Vmax when [S]= inifnite
Km= vmax/2
Enzyme Models
Michaelis-Menten Equation
Lineweaver-Burk Plot
Lineweaver-Burk Plot
Enzyme Inhibition
Enzyme Inhibition
Enzyme Inhibition
Enzyme Regulation
Negative Feedback
Homoallostery vs Heteroallostery
Heteroallostery
Heteroallostery: Example