bio 98 (luecke/ribbe) midterm review! tutors: priyanka saxena oh: mondays, 9:00-11:00am sh 149...
TRANSCRIPT
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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]
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First off…Evaluation Sheet!
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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.
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Water
Density Ice cubes Lakes
Structure Hydrogen bonding Dipole
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Ice
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Water as a solvent
Like dissolves like Polar, charged molecules Ex: NaCl
Water separates from: Hydrophobic molecules Ex: Oil Why?
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Biochemical Forces
Covalent (Strong)
Non-covalent (weak, but more important biologically) Hydrogen bonding Ionic interactions Van der Waals forces Hydrophobic interactions
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The pH scale
pH = -log [H+]
Lower the pH, the more acidic the solution
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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
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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)
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Titrations
http://www.chembio.uoguelph.ca/educmat/chm19104/chemtoons/chemtoons9.htm
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Henderson-Hasselbach
pH = pKa + log [Ac-]/[HAc]
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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?
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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?
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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
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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]
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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
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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
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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-
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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)
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Amino acids
EVERY amino acid has the following: NH3+ (N-terminal) COOH (C-terminal) H
What is variable is: The SIDE CHAIN! (R group)
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Problem 4
What is the pI of Tyrosine?
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Peptide bond
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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
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Lambert-Beer Law
A=ecl A= absorbance e = molar extinction coefficient (unique to each
molecule) C= concentration L = path length; usually 1cm
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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?
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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
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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-
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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
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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)
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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
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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)
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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
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Protein purification
Need to separate your protein of interest from everything else inside of a cell!
Why? Research Pharmaceuticals Therapies Sequencing (HGP!)
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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
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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.
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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
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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
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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
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Protein ID
Identifying an unknown protein
2-d gel electrophoresis (MW and pI)
Peptide mass fingerprinting
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Protein: Myoglobin,Hemoglobin,
and Enzyme
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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
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3D structure of Polypeptide and its Restriction
Alpha carbon limited to phi and psi angle
Partial double bond restricts movement
Result is planar conformation
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The Ramachandran PlotPossible movement of phi and psi angle denoted.
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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
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Helical Wheel DiagramUseful for deciding hydrophobicity/hydrophilicity
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Alpha and Beta Sheet
R groups alternate up and down
Parallel or Anti-parallel
C=O and N-H groups switch “left” and “right”
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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
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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
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Oxygen-binding curve for Myoglobin
Hyperbola
[O2]0.5 Concentration
of O2 where half of Mb is saturated with Oxygen molecules.
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Oxygen-binding curve for Hemoglobin
“S” Shaped
Sigmoidal curve is suited better for transport. Off loads more
O2 to tissues
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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
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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.
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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
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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
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Enzyme-- the Biological Catalyst
No Enzyme Enzyme Added
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Enzymatic Kinetics
Variable K1,k2(Kcat), K-1, Km
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Michaelis-Menten Equation
Hyperbolic Curve
Vo= rate at which the product is produced by the enzyme
Vmax when [S]= inifnite
Km= vmax/2
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Enzyme Models
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Michaelis-Menten Equation
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Lineweaver-Burk Plot
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Lineweaver-Burk Plot
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Enzyme Inhibition
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Enzyme Inhibition
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Enzyme Inhibition
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Enzyme Regulation
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Negative Feedback
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Homoallostery vs Heteroallostery
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Heteroallostery
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Heteroallostery: Example