Myoglobin: Oxygen Storage and Hemoglobin Transport
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First protein structure ever solved?
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myoglobin
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Function of myoglobin?
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Oxygen storage in the muscle
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Function of hemoglobin?
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It's a RBC protein (nearly 1/7th of the protein in the RBC) that functions to transports oxygen from lungs to tissue
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Which types of secondary structure are present in this? How many domains? Which amino acids do you expect in the interior? Which amino acids to expect on the surface?
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Alpha helix and random coil 1 domain Hydrophobic aa on the inside, such as: Leu, Ile, Met, Ala, Trp, Phe, maybe Tyr Hydrophilic on the surface: any with polar or charged side chains
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Similarities between primary sequences, secondary and tertiary structures between hemoglobin and myoglobin?
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Primary sequence: 18% identical and 38% similar Secondary and tertiary are very similar
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Where do differences between myoglobin and hemoglobin structures appear?
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in the quaternary structure
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Quaternary structure of hemoglobin and myoglobin?
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Myoglobin is a monomer Hemoglobin is a tetramer (two alpha subunits and two beta subunits)
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What are the different isoform chains possibilities in hemoglobin?
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In human adults, it's usually beta In fetuses, its typically gamma The minor form in human adults is delta
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Where do you expect most residues involved in hemoglobin tetramerization to fall in the alignment between it and myoglobin? Why?
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In positions that are NOT similar to myoglobin because tetramerization is special to hemoglobin, therefore it will involved amino acids that are different from myoglobin
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What same thing do Hb and Mb bind to?
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The same heme (prosthetic group), and the same ligand (O2)
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What is a prosthetic group?
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it stays tightly bound and associated with the protein for its entire life
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Is binding to oxygen permanent?
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No, it can associate and dissociate reversibly
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What is in the center of all hemes?
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Iron
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Why do you need proteins to be bound to free hemes?
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Because the free heme binds CO 25,000 times more tightly than oxygen, and the protein hinders the heme binding to CO, so now you have a chance to make an oxygen carrying protein. And because O2 oxidizes the iron in the free heme, and oxidized iron DOES NOT bind O2. Once again, the protein around the free heme inhibits that reaction.
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Answer the questions
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Means that there is cooperativity We know there is more than one binding site due to the sigmoidal curve The Mb curve should be hyperbolic
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Answer the questions
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This means that MYOGLOBIN binds oxygen more tightly because this is a dissociation curve You can see at concentration of 20, hemoglobin has less than 20% saturation and myoglobin has more than 80%
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What form of the tetramer in Hb does O2 first bind?
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the de-oxy form of the tetramer (lower affinity), or the T state
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When does oxygen stop binding to the low affinity form of tetramer and move over to high affinity? How does it affect the curve?
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The first O2 binds to low affinity, and then half of the second O2 binds, and by the time the third O2 is binding, it has switched to the high affinity form, so the fourth O2 binds to the high affinity form, giving rise to the sigmoidal curve
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Can you have cooperativity and allostery in the same protein? Example?
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Yes. Hemoglobin!
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What's one of the allosteric effectors in Hb? How is it made and how does it bind?
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2,3-BPG, which is synthesized from glucose in erythrocytes. High concentration of BPG is in the cell, so many hemoglobin in cell are bound to BPG, though only one BPG binds to a tetramer.
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What form of Hb does BPG bind to preferentially?
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The deoxy, or lower affinity form It does NOT bind to high affinity
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How does BPG affect the curve for Hb?
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Without it, the curve looks like Mb
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What is hypoxia?
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deficiency in oxygen delivered to tissues
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What options do deoxyHb have for binding?
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It can either bind to oxygen and go to oxy form, or stay stable as a deoxy form and bind to BPG
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What facilitates the conversion of Hb from oxy form to deoxy form?
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BPG, because it pulls the reaction to the more stable state, which is deoxy with BPG bound
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Why would Hb curve look like Mb without BPG?
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because there would not be enough deoxy around to have significant cooperativity
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Functions of BPG?
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Alters oxygen binding, and stabilizes deoxy form of Hb Gets upregulated if you're hypoxic, which is a quick way for your body to know that its hypoxic Is an issue with storing blood, because BPG is degraded over time, and it's a source of short shelf life of stored blood
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Does BPG bind better to fetal or adult Hb?
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Binds to adult Hb better
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One main difference between fetal and adult beta chains?
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Adult histidine at position two is changed to serine in fetal Hb, which is in the BPG binding site
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How does fetal Hb binding for oxygen compare to adult Hb? Why?
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Fetal binding will be higher so the fetus can effectively compete for oxygen with the mother
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BPG binding is related to O2 binding how?
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Weaker BPG binding results in strong O2 binding
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Two other allosteric effectors for Hb? What are their effects?
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H+: weakens O2 binding CO2: lowers O2 affinity by binding at N terminal amines, though it is reversible
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How do all allosteric effectors affect oxygen binding for Hb?
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Make it weaker
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Answer the questions
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The O2 affinity is lower The midpoint of the new curve is higher pO2 You rationalize it because high H+ and CO2 build up in tissue is using lots of glucose to make energy. The final step in ATP production requires O2. Therefore, the buildup of H+ and CO2 sends the message that "this tissue has used a lot of O2 and could use more"
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How do Hb and Mb moonlight?
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As an enzyme that changes NO to NO3+ (nitrous oxide to nitrate)
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How does Hb and Mb converting NO to NO3+ affect the body?
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It turns the blood chocolate brown
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What are the three physiological allosteric regulators that affect Hb? What are their binding sites and effects?
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BPG: binds to deoxy Hb histidine and lowers O2 affinity and enhances cooperativity H+: weakens O2 binding; binding site unknown (too small to crystallize) CO2: lowers O2 affinity; binds covalently and reversibly at the N-terminal amines
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Define the Bohr Effect
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An effect by which an increase of carbon dioxide in the blood and a decrease in pH results in a reduction of the affinity of hemoglobin for oxygen.
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Easiest way to change oxygen concentration available?
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Go up and down in altitude
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At sea level, what is the O2 saturation?
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about 94%
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O2 saturation at Pikes Peak?
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70%
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O2 saturation of a commercial airplane?
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about 35%, which is why there has to be pressurized cabins and oxygen masks in planes
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What happens to Hb curve in the presence of drugs that oxidize Fe2+ to Fe3+, such as benzocaine?
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If all four subunits undergo this conformation to Fe3+ at the same time, then that makes the top part of the curve drops, because all of the oxygen will never be bound. The apparent Kd is at the same concentration though, so only the last part is changed
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What is methemoglobinemia?
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When Fe2+ is converted to Fe3+, which cannot bind O2, and iron can't bind the oxygen, which makes the blood dark brown
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Where is benzocaine found?
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In many OTC, especially numbing medicine for babies when they're teething
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Antidote for poison caused by Fe2+-->Fe3+? (iron poisoning)
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methylene blue
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Normal O2 levels in a room?
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21%
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When can you start feeling the symptoms of too much CO?
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at 0.0035%
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What happens at 1.3% CO?
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You will die in three minutes
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Why is CO so dangerous?
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It poisons more than just hemoglobin The heme binds to CO very tightly, even with the proteins reducing the binding and binds ANY protein with a heme It is substoichiometric, which means it only has to bind at one site in order to poison all four subunits of hemoglobin
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Effect of CO binding to heme protein on hemoglobin?
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Precludes O2 binding at one site Enhances O2 binding at other sites so O2 can't be released where it's needed
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How does CO binding to heme protein affect the curve?
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Will lower how much O2 can be bound, so it drops the top AND shift curve to left, which means the hemoglobin can't release the O2 when it reaches the muscle, increasing CO poisoning toxicity
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What is thalessemia? Explain what happens during the disease
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It is the loss of one subunit. When the quaternary structure of Hb is messed up, such as making unequal amounts of alpha and beta subunits, meaning there are leftover subunits, which can aggregate and precipitate. There is also the issue of not having enough normal Hb, so you have to get rid of the leftovers, and you're anemic from not enough Hb
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What are hemoglobinopathies?
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Point mutations
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Example of hemoglobinopathy?
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Sickle cell anemia
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How are hemoglobinopathies named?
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generally by the location where they were found
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What is the mutation of Hb Kansas?
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in the beta subunit, an asparagine is changed to a threonine at the 102nd position
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Clinical symptom of Hb Kansas?
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Anemia
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Functional study of Hb Kansas?
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Bohr effect is normal, but O2 affinity and cooperativity are decreased
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Stability of Hb Kansas?
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Protein became unstable, and became more likely to dissociate from tetramer to dimer and more likely to unfold, which led to it's degradation
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Occurence of Hb Kansas?
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Only 10 cases found, mainlywith Japanese families
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How does the curve change with Hb Kansas?
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The curve becomes more hyperbolic (it's a linear scale on x axis) from diminished cooperativity Decreased affinity shifts midpoint to right (weaker) Loss of some Hb tetramers due to dissociation reduces the amount of Hb that is available to transport O2
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What chemical changes happen when asparagine changes to threonine?
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Not too much difference chemically
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Features of an ideal Hb substitute?
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One that can withstand high pressure Bigger, so there is no NO zone so it can't go between cells by adding PEG all around periphery Cross link tetramer so it stays together
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Define porphyrin ring?
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Any of various heterocyclic compounds, derived from pyrrole, that occur universally in protoplasm, contain a central metal atom, and provide the foundation structure for hemoglobin, chlorophyll, and certain enzymes.
