Why do we need oxygen- binding proteins?
- Cells require O2 for production of energy
- O2 only sparingly soluble in blood, thus needs a transport and storage system
What is Hb for and what is Mb for?
Haemoglobin= O2 transport (CO2 transport)
Myoglobin (Mb)= O2 store (in muscles & tissues)
What is Haemoglobin (HbA)
- Protein which makes blood red
- Composed of four protein chains- 2 alpha and 2 beta, each have a ring- like haem group containing an iron atom (4 haem)
- Oxygen binds reversibly to these iron atoms and is transported through blood
What is Haem?
Fe2+ & protoporphyrin IX
- Tightly bound, non-polypeptide unit essential for biological activity
- Non-covalently bound in a hydrophobic crevice
- Fe2+ can coordinate with 6 ligands (4 nitroges from prophyrin ring, nitrogen from proximal His, oxygen atom)
How does Hb and Mb differ?
Hb has a very similar 3D structure to Mb but differ at 83% of amino acid residues
Very diff primary structure can specify similar 3D structures
What type of protein is Hb
allosteric protein
Oxygen binding alters tertiary and quaternary structure
Binding of O2 to one subunit affects interactions with other subunits
Facts about Oxygen binding to haemoglobin & myoglobin
Hb= OB=cooperative, OA= pH and CO2 dependent, regulated by bisphosphoglycerate
Mb= OB= non-coop, OA= non dependent, no regulation, higher affinity for O2 (good storage)
What type of binding curve does Hb have?
sigmoidal because its cooperative
Binding one O2 makes binding the next easier
What happens to Hb conformation when one O2 binds?
- Alters conformation
- Proximal His F8 is pulled in shifts helix F, EF and FG corners
- Altered shape transmitted to subunit surfaces and some interchain salt bridges rupture
- The aB pairs slide and rotate relative to one another
- These structural changes increase the affinity of the remaining subunits for O2
How does Hb shape affect O2 binding?
- Hb uses motion and structural changes to regulate its action
- O2 binding at the four haem sits in Hb= not simultaneous
- When first O2 binds= small changes to structure of adjacent protein chain
- Neighbouring chains nudged into diff shape= O2 can bind easier
- Progressively gets easier and easier
What happens as Hb travels around the body
- Lungs= O2 lots, easily binds to first subunit and rest follow
- Blood circulates, oxygen levels drop & CO2 increase
- Hb releases bound O2
- When first O2 drops off, changes shape prompting the remaining O2 to drop off
- Hb picks up largest possible load in lungs and drops it where and when it is needed
- A CO2 molecule is able to bind to the amino termini of each Hb protein chain in the T- state
What does Haem do?
- Enables Hb to transport other molecules (e.g. NO & CO)
- NO affects the walls of blood vessels, causing vasodilation, reduces blood pressure
-NO binds to specific cysteine residues in Hb and also to Fe in haem groups - CO= toxic, better at binding to haem than O2 (60 deaths per year, nausea, dizziness and confusion)
When does Hb have a lower affinity for O2?- Bohr effect
- pH is lower
- CO2 conc is higher
What is the Bohr effect?
- Increase in H+ (lower pH) decreases affinity of Hb for O2
- Increased CO2 in blood or increased lactic acid will lower pH
- Provides increased release of O2 to tissues respiring rapidly and muscle releasing lactic acid
Why do H+ ions affect O2 binding?
- pH affects the protonation state of amino acid residues
- When H+ conc is high, additional residues are protonated- especially histidine residues
- Additional positively charges residues can form new salt bridges- these stabilise the T-state and decrease affinity for O2
What is BPG?
2,3,- bisphosphglycerate
Where is BPG found?
high concentratons in RBC
- Regulates O2 affinity
-decreases affinity of Hb for O2
- BPG levels are increased at high altitude and under hypoxic conditions
- Means Hb releases more O2 to tissues at high altitude and when hypoxic
How does BPG stabilise T- state?
- Binds in the space between beta chains
- Negative charges on BPG interact with amino acid residues lining the space
- As H+, CO2 and BPG interact with Hb at different sites their effects can be additive
- More CO2= more H+= increased side chain protonation= more salt- bridges= more stable de-oxy T state
What are some features of HbF?
- Higher oxygen affinity
- Dominates for the last 2 trimesters but by 1 yers old almost entirely HbA
- Two alpha and 2 gamma subunits
- Gamma give increased affinity for oxygen
- BPG does not bind and so no effect (due to no beta chain)
- So under same conditions when Hb releases oxgen, HbF can still capture it
What are Haemoglobinopathies?
inherited disorders affecting haemoglobin synthesis, structure and function
How many mutant haemoglobins are there?
400 mutant haemoglobins, 95% due to single amino acid change. most common monogenic diseases worldwide
What can mutations affect?
- Amount of Hb synthesised
- Structure (subunit interfaces)
- Stability of Hb- leading to haemolytic anaemia
- Affinity for O2
- Affinity for regulators
What is a position mutation?
- Mutations in critical residues will affect function (e.g. in protein structure or at exon splice site or promoter region)
What are conservative & non-conservative substitution mutations?
Conservative= maintains properties (e.g. change of one non-polar residue for another)
Non conservative= changes properties ( e.g. Leu to Lys may have a major effect on protein structure and function)
