Bio 98 (Luecke/Ribbe)

Midterm Review!

Tutors:Priyanka SaxenaOH: Mondays, 9:00-11:00amSH 149priyanks@uci.edu

Tutors:Justin KoOH: Wednesdays, 3:00-5:00pmSH 237justinko@uci.edu

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