M10 L7-L9

​Lecture 7- Lecture 9

By: Varsha Katoju​

​Lecture 7 High Yield Topics

​- Ammonium Sulfate Precipitation

-Chromatography​

• Ion Exchange

• Gel filtration ​

• Affinity​

​-Gel Electrophoresis

Multiple Choice

In Ammonium Sulfate Precipitation, low concentrations of salt often ________ the solubility of a proteins; this is known as _______ ___. (L7; Slide 6)

• decrease; salting out

• increase; salting out

• increase; salting in

• decrease; salting in

• Remember proteins have different positive and negative charges and salt is NaCl or Na+ and Cl-

• In solution salt will becomes attracted to these charges and affects the protein differently based on salt concentrations​

• We can use this technique to remove undesired proteins or isolate desired proteins (Slide 7)

• Why (NH₄)₂SO₄? Because it is extremely soluble meaning increased interaction with water!

How does salt affect proteins? ​

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Chromatography​

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Ion Exchange​

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​-Negative charged resin (Cation exchanger) or positively charged resin (Anion exchanger)

-Typically proteins with smaller charges elute first and then proteins with highest charges

-Analogy: Biggest magnets will stick the hardest to opposing charge so we leave them for last!​

​-Separate molecules based on size and shape

-Resin matrix has pores with a defined size range

-Small molecules get stuck while the large ones pass more freely eluting out!

-Analogy: Opposite of a coin machine

Size Exclusion​

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​-Proteins bind due to affinity with ligands on resin

-Unwanted proteins elute out of solution

-Then ligand solution is added and protein of interest is eluted out of solution

Analogy: ​Broken brita filter

Affinity​

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Multiple Choice

SDS or sodium doceyl sulfate coats proteins and overwhelms the with negative charge. Therefore, during gel electrophoresis separation becomes dependent on _____. Heavier proteins are close/far from the positive charge

• Velocity;

  closer

• Mass;

  farther

• Interactions; farther

• Charge;

  closer

​Lecture 8 High Yield Topics

• ​Oxygen Binding to Heme (Slide 8, 10)

• Myoglobin​ Oxygen Storage (Slide 6)

• Hemoglobin Oxygen Binding/Transport​ (Slides 15, 16)

• Hemoglobin transport Oxygen (Slide 19,20)

• Hemoglobin transport CO₂ (Slide 21)

• 2,3-Bisphosphoglycerate (BPG) (Slide 22)

• Sickle-cell amenia (Slide 26)

• Globin genes​ (Slide 31, 32)

Multiple Choice

Ture/False: Oxygen binds to Myoglobin irreversibly

• True

• False

Heme​

• We have a heme group in Myoglobin (Mb) and Hemoglobin (Hb). Fe^{2 +} s bound to imidazole group (octahedral coordinated) and O2 is held on the other side also by a histidine

• O₂ bind at an angle to the Fe²⁺ while CO does not. A bent paper straw is weaker than a straight one

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Multiple Select

Hemoglobin is allosteric protein. How is the affinity of Hb affected by area of high oxygen concentration like our lungs? Select all that apply.

• As oxygen binds to deoxy-hemmoglobin, Hb's affinity for O₂ decreases

• As O2 is released from oxy-hemoglobin Hb's affinity for O2 decreases

• As O₂ is released from oxy-hemmoglobin, Hb's affinity for O₂ increases

• As O2 binds to deoxy-hemoglobin Hb's affinity for O₂ increases

Co2-binding to deoxy-Hb​

• CO₂ binds to the amino-terminal end of each globin stabilizing the T-state (deoxy-Hb) releasing H+ to kidney's and carrying CO2 to lungs​

• Hb transports 40% of H+ and about 15-20% of CO₂ formed in tissues​; CO₂ can also be transported as dissolvable HCO₃-

• Lungs have partial pressure so here the oxy-Hb conformation is favored (we want to oxygenate the Hb) so we let go of the CO2 which we breath out!!!​

​Watch video on 2,3-Bisphosphoglycerate

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https://www.youtube.com/watch?v=JUe64NwOejo

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​Sickle Cell Anemia

• Amino acid mutation from Glu-6 to Val-6

• Causes a "sticky" hydrophobic ​ends which grabs other HbS molecules creating crystallization

• These sickled cells have difficulty traveling through capillaries​

• ​Extra fact***Causes sharp, stabbing, and intense pain anywhere is your body

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Multiple Choice

Fetal hemoglobin can bind oxygen at lower partial pressures than the mother hemoglobin can. This means that it has a lower or higher affinity for O₂

• Higher

• Lower

Lecture 9 High Yield Topics​

• Metal Cofactors (Slide 8) - MEMORIZE THIS TABLE!

• Enzymatic reaction (Slide 13)

• Reaction rates (Slide 14, 15)

• Example mechanism (Slide 19) -MEMORIZE THESE DEFINITIONS!

• Transition state (Slide 21)​

Multiple Choice

Enzymes lower the activation energy barrier of a reaction rate and affect the reaction equilibria.

• True

• False

K'_eq, k, and Δ​G‡

• K'_eq is the equilibrium constant [P]/[S]

• k is the rate constant and measure how fast a reaction occurs (velocity of reaction in material that reacts per unit time)​

• Know the relationship between the rate constant [k] and the free energy of activation [​Δ​G‡]

  • The rate constant is larger when the free energy of activation decreases AKA speedier reaction when we lower activation energy barrier​

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Multiple Choice

Transition state analogs are good inhibitors of enzymes and are used in drug design. Suppose that you create a drug that mimics the transition state of an enzyme, how will this affect the forward and backwards rates of the reaction. (Slide 21)

• Forward rate would stay the same. Backward rate would increase by a factor of 2

• Forward rate would decrease by a factor of 2. Backward rate would increase by a factor of 2

• Forward rate would decrease by a factor of 2. Backward rate would decrease by a factor of 2

• Forward rate would decrease by a factor of 2. Backward rate would decrease by a factor of 3

• None of the above

​YAY we are almost done! This week you will finish all the content for Exam 1 L1-L13!

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Start studying now so you do not fall behind...

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P.S. Plan to come to lecture next week!