Hemoglobin & Oxygen

Question 1

what protein structures are considered secondary?

what interactions induce formation of secondary protein structure?

Answer 1

• alpha helices & beta sheets
• hydrophobic interactions

Question 2

define tertiary protein structure. what is a key capacity of tertiary structures?

Answer 2

• 3 dimensional folding of polypeptides
• tertiary structures are able to bind other molecules

Question 3

define quaternary structure

Answer 3

a protein structure consisting of multiple independent polypeptides

Question 4

hemoglobin is

• what type of protein?
• made of what subunits/bonds?

Answer 4

• globular protein - aka, water soluble
• made of 4 subunits
  
  • 2 alpha (141 AA) , 2 beta (146 AA)
    
    • a1b1 & a2b2 bonds:
      
      • strong bonds
    • a1b2 & a2b1 bonds:
      
      • weaker than the above bonds, but still strong: H-bonds, hydrophobic bonds, salt bridges

Question 5

what are the strongest protein denaturants (and at what molar concentration)?

Answer 5

1. 9M urea - strongest
2. 6M Guanidine HCl (Gdncl)

Question 6

describe the affinity/effects of protein, transitional metals, and organometallic compounds to oxygen. where does Hb fit into this?

Answer 6

• protein = low affinity (esp in side chains)
• transitional metals = high affinity, but generate free radicals
• organometallic compounds (Fe^2+/​³⁺) = less toxic than transition metals, but free Fe³⁺ still reactive

hemoglobin provides a solution, as the 4 Fe+ containing heme groups are not “free” and thus not risks for reactivity.

Question 7

contrast the affinity of Mb & Hb for O2. what is the reason for this & why is it important?

Answer 7

• myoglobin
  
  • higher affinity
  • hyperbolic curve
  • requires lower O₂ partial pressure to release it to environment
• hemoglobin
  
  • lower affinity
  • sigmoidal curve
    
    • due to 4-subunits working in cooperation
  • more readily releases O2 to the environment

Question 8

define holoprotein & apoprotein

Answer 8

Holoprotein: a protein with attached prosthetic group

Apoprotein: without the prosthetic group

Question 10

describe the structure of heme & the characteristics of the atoms that form it.

Answer 10

• heme = Fe²⁺ (ferrous iron) bonded to a surrounding porphyrin ring
  
  • the porpyrin ring = carbon chain + 4 N groups & methyl, vinyl and propionic acid (- charge) side chains

there is one heme group per globin chain

Question 11

Fe²⁺ is bonded to what stuctures in hemoglobin?

Answer 11

• 6 bonds total
  
  • 4 to the porphyrin ring
  • 1 to the proximal histidine - pulls Fe²⁺ slightly out othe plane of the ring
  • 1 to O₂ when hemoglobin is oxygenated
    
    • this induces a conformation change that pulls Fe²⁺ BACK into the & realigns the His/F-helix

Question 12

explain the T & R states of Hb & what they allow

Answer 12

• T and R states are responsible for the “cooperativity” and sigmoidal curve characteristic of Hb
• T vs R
  
  • T (tense) state
    
    • low affinity for O2
    • more stable (more interactions)
  • R (relaxed) state
    
    • high afifnity for O2
    • more flexible (fewer interactions)

Question 13

what triggers the T–>R conformation change & what bonds are broken?

Answer 13

• triggered by O2 binding
• involves breaking of ion pairs between the a1-B2 interface

Question 15

what are the agents that decrease the affinity of Hb for O2?

Answer 15

1. H+ ions (low pH)
2. 2,3-BPG
3. CO2 binding to Hb chain

Question 16

2,3- BPG

• where is if found/where does it come from?
• size/charge?
• how does it effect Hb?

Answer 16

• found in erythrocytes
• is a glycolytic intermediate
• - charge/small
• is a negative heterotropic regulator of Hb
  
  • binds to the + charges between B-subunits
  • stabilizes the T-state –> decreases Hb’s affnity for O2
    
    • upon oxytenation, spot for BPG dissapears

Question 17

draw out the O2 saturation curve of Hb vs Hb + BPG (pO2 plotted against O2 saturation)

Answer 17

Question 18

draw the O2 saturation curve of Hb at various pH levels (on a graph of pO2 vs % O2 saturation)

Answer 18

lower pH/high H+ = less O2 sound

Question 19

how does the pH of blood change as it crosses capillaries and why is this the case?

Answer 19

• when tissues consume O2 from lungs, it is converted into CO2.
• that CO2 is converted to –> H₂CO₃ –> then H⁺ + HCO₃.
• this H+ boots the O2 of incoming Hb.
• that’s why Hb in blood passing oxygen poor tissues is acidic

Question 20

how does CO₂ interact with Hb? what does it form?

Answer 20

• CO2-Hb = carbamate of hemoglobin
  
  • binds to the N-terminal groups of Hb
  • 15% of CO₂ in the blood is carried to lungs bound to Hb
  • CO₂ stabilizes the T-state (deoxy)

Question 21

discuss the interaction of carbon monoxide (CO) with Hb

Answer 21

• CO = highly toxic
  
  • Hb has a much higher affinity (~250 fold) for CO than O2
    
    • CO blocks Mb/Hb - O₂ interactions
    • CO blocks mitochondrial cytochromes in ox-phos
  • poisoning cause cause –> nausea/dizziness/confusion/disorientation/visual disturbances

Question 24

what is methemoglobin & its characteristics? what is its clinical relevance?

Answer 24

• a variation of Hb containing ferric (Fe³⁺) iron instead of ferrous iron (Fe²⁺)
  
  • Fe³⁺ cannot bind O_2.
  • it actually increases the affinity of normal Hb for O2, thus slowing O2 unloading onto tissues
  • beyond if our Hb is > 1-2 % metHb variant, we are under oxygenated and can become cyanotic (blue skin)

Question 25

what is *metHb reductase*? how does it work & what does it require?

Answer 25

* a **cytochrome b5** enzyme that reducesmethemobloin (ferric form) back to normal Hb (ferrous form) * NADH dependent * NADH reduces cyt b5 to reduced form * cyt b5 reduces Hb-Fe³⁺

Question 26

discuss the cause and pathology of sickle cell anemia

Answer 26

* cause: point mutation leading to a change in a single aa residue (allele from both parents) * leads to abrnormal erythrocytes * are long, thin sickle shaped **insoluble polymers** that aggregate into **tubular fibers** * Hb on these erythrocytes (called **HbS**) is **deoxygenated**