CLASS: FUNdamentals Scribe: Ebony Lewis
8-15-2011 10:00-11:00 Proof:Joe Vaughn
Ryan Hemoglobin Structure and Function Page 1 of 8
Proofer Note: This lecture and the second lecture by Ryan seem a little long. But all the material in the transcript is relevant and could not be cut out. Every word out of Ryan’s mouth was lecture material (no stories) and he pretty much talked non-stop. Also beware that he often talked a lot for many slides, and a lot of the information he expanded on is not in the slides, but it is all included in these two transcripts.
a. Hemoglobin Structure and Function (S1)
a. Hemoglobin is an excellent example of quaternary structure and also of allosteric regulation.
b. Red Blood Cells Contain Hemoglobin (S3)
a. We have about 5 million RBC’s (red blood cell) per microliter of blood in our body, and if you add all microliters of blood we have, you will see the total of red cells in our body is about 2.5 x 10^13.
b. Human body has total of 1 x10^14 cells. So red blood cells represent about 25% of all cells.
c. Inside each RBC, 95% of the cytosolic proteins is hemoglobin, a major protein of the body.
d. 85% of the total body heme is found in the RBC.
a. Heme is a component of each globin chain in hemoglobin. In the middle of each heme is an iron atom, thus 70% of the total body iron is in your RBC’s.
c. Erythroid Development (S4)
a. RBC’s come from the Hematopoietic stem cells (HSC).
a. And after several divisions, producing various multiple progenitors you get the first RBC lineage cell, called the BFU-E erythro (erythrocytes), which is in the bone marrow.
b. And these then divide a few times to produce CFU-E erythro (colony forming).
b. Unsure how to isolate these. We just know that if we take bone marrow and put it in the dish with the right cytokines and growth factors, we will see a little colony of RBC’s. These results tell us that two weeks earlier when we originally put the bone marrow in there, there was a BFU-E there, because it gave us a colony.
c. This CFU-E divides several more times and then produces erythroblasts.
a. We see a series of identifiable erythroblasts in the bone marrow, starting with the pro-erythroblasts, then basophilic, polychromatic, orthochromatic. These are all in the bone marrow and all nucleated.
b. Final stage of erythroid maturation = removal of this nucleus (denucleation of RBC)
i. At this stage the RBC is called a reticulocyte
ii. It hangs out in the bone marrow, maturing for couple of days.
iii. Then exits marrow, enters circulation, living for 128 days
d. The hemoglobin content inside each RBC increases with erythroid differentiation maturation.
a. Very little in these early progenitors, but increases during maturation because of translating more and more of globin message into hemoglobin.
d. Red Blood Cells Contain Hemoglobin (S5)
a. Hemoglobin is a tetramer composed of two alpha globin chains and two beta globin chains.
b. Each alpha chain will form a dimer with a beta globin
a. These two alpha beta dimers come together to form this tetrameric hemoglobin.
c. Sickle cell anemia and Cooley’s anemia are caused by a mutation in the beta globin.
a. Sickle cell caused by a single DNA base nucleotide change, causing a single amino acid change, ultimately changing the beta globin chain of hemoglobin
b. Cooley’s anemia is a severe form of beta thalassemia.
i. A thalassemia is a globin chain imbalance. Normally you have equal amounts of alpha and beta chains inside a RBC.
d. Alpha thalassemia – don’t have enough alpha chains.
e. Beta thalassemia - not enough beta chains. Cooley’s anemia is having no beta chains. You can’t survive without any hemoglobin; very serious disease.
V. The Structure of Myoglobin is similar to that of the hemoglobin monomer (S6)
a. Myoglobin, found in the muscle cells, is another globin that binds oxygen.
i. It’s a monomer, it doesn’t pair with another globin to form a dimer or tetramer like hemoglobin.
b. We see alpha-beta chains of myoglobin (Mb), just like hemoglobin (Hb). Figure shows heme molecule in red. This a porphyrin ring with an iron in the middle of it, which is where oxygen binds.
VI. Hemoglobin and Myoglobin (S7)
a. Both Hb and Mb bind oxygen. Hb is an oxygen transport molecule that binds oxygen in the lungs and delivers it to the tissues.
b. Myoglobin is an oxygen storage protein in the muscle that provides muscle cells with oxygen when we are actively using them.
a. Hemoglobin is a classic example of allosteric regulation.
VII. Globin Subunit Conformational Structures (S8)
a. Diagram of the three globins: Myoglobin (a monomer), alpha-Globin of hemoglobin, and beta-globin of hemoglobin.
b. Number amino acids vary from chain to chain, but they all have basically the same structure. They all bind the heme group.
c. All of these proteins are alpha helical in structure. These helixes are named, they are given letter designations, (starting from the amino end) A, B, C, through H.
d. Heme group is nestled between the E and F helixes. When referring to different amino acids, refer to them with the helix letter designation followed by the amino acid number.
VIII. Myoglobin Structure (S9)
a. Myoglobin is a storage protein for oxygen in the muscle.
i. The polypeptide, again these two helixes, E and F helix, cradle this heme group.
ii. The iron in this heme group is a ferrous ion, the 2+ form, the form that binds oxygen.
iii. If you oxidate this to the 3+ form, the ferric iron, it can’t bind to oxygen anymore. That’s referred to as metmyoglobin. Metmyoglobin will have to be reduced back to the +2 state in order to bind oxygen again.
IX. Mb and Hb use for porphyrins to bind Fe 2+ (S10)
· On the right is the structure of heme. It is made from protoporphyrin 9, which coordinates with iron in the middle of the pyrrole rings. It’s a planar molecule, and the heme is nestled between these nitrogens (coordinates with 4 nitrogen ligands).
· And it has two more ligands out of the plane of the board and behind it. One of these coordinate with the histadine, (of the myoglobin or hemoglobin chain), and the other with the oxygen.
· So this protoporphyrin 9 is composed of 4 pyrrole rings which are linked with these methylene bridges.
i. They have multiple proprionic acid, methyl, and vinyl side chains. And again there’s the ferrous iron that coordinates between the nitrogens.
X. Intracellular Free Heme Control (S11)
a. How do you control the heme level inside the cell?
i. RBC’s have the majority of the heme in the body, but there are many other proteins that also utilize heme.
ii. RBC is specialized to synthesize lots of heme and to import lots of iron to make that heme.
b. So there’s a series of enzymatic steps to make heme.
1. First step and last three steps are found in the mitochondria. The other 4 steps are found in the cytoplasm.
2. Heme starts with the first enzyme ALAS. Glycine and Succinyl CoA are condensed to form Alamino lamntic acid (word is unclear?). This goes to the cytoplasm to be worked on by 4 other enzymes, and then it goes back into the mitochondria for the final three steps.
3. Last step uses ferrochelatase to take the iron and puts in in the proroptophyrin 9 to make Heme. The iron has to come from the blood stream and the red cell has up regulated or as an erythro-specific transparent receptor (??) which will bind up this iron, import it into the cell, move it into the mitochondria so it can bind protoporphyrin 9.
a. Once the heme is made, it transfers into the cytoplasm and it binds to heme proteins (once again in red blood cells most of this will be hemoglobins).
4. So for hemoglobin, you get high levels of expression of the alpha and beta globin chains. The DNA message comes out in the cytoplasm and is translated into proteins. Those alpha beta chains come together to form a tetrameric hemoglobin, and most of the heme is found by the hemoglobin.
· Excess free heme can be degraded by the enzyme heme-oxygenase,
i. This degradation will liberate carbon monoxide, iron, and billiverdin.
i. Billiverdin is reduced to Billirubin. The iron is re-utilized by an erythropoiesis in the bone marrow.
ii. Much of this occurs in the macrophage, which ends up scavenging most of the free hemoglobin. The iron is re-utilized and the rest of the heme is degraded to billiverdin.
f. Heme can also be exported out of the cell by FLVCR (full name isn’t important), an exporter in the membrane.
a. excess heme is not good for the cell. A lot of free heme can regulate the expression of different proteins in the cell
b. but if you have too much free heme (due to malfunctioning exporter), it can cause the early destruction of the erthyroblasts in bone marrow.
c. Important to maintain heme at just the right level.
XI. Fe 2+ is coordinated by His F8 (S12)
a. Iron interacts with 6 ligands in hemoglobin and myoglobin.
i. In 4 of these are the nitrogen atoms in the 4 pyrrole rings.
ii. The 5th one is denoted by the imizadole side chain of the amino acid His F8 (meaning that on the F alpha helix, position 8 is a histadine). This amino acid is invariant amongst all the hemoglobins known in nature.
b. When myoglobin or hemoglobin binds oxygen, the O2 molecule adds to the heme iron as the 6th and final ligand.
c. And when the O2 molecule binds to the heme, its not perpendicular to the plane, but slightly off at an angle.
i. Cause there’s another histadine on helix E that sterically keeps it from winding up. This is also important for function, b/c you need to be able to off load that oxygen. You don’t want it binded too tightly.
XII. Globin Heme Iron Liganding (S13)
a. Diagram of what this process (in previous slide) looks like.
b. This is heme, here are the 4 pyrrole rings, the heme plane. Here are the 4 nitrogens liganding to the iron. Here is the Histadine F8, and here’s the oxygen linking the 6th ligand to the iron. Again this Histadine, (His F8) is invariant throughout globin gene evolution.
XII. Oxygen Binding Alter Mb conformation (S14)
a. What happens when oxygen binds myoglobin?
i. So in deoxymyoglobin, the ferrous ion actually lies a little bit above the plane of the heme. Think of it as kind of puckered, and sitting up above the plane.
ii. When oxygen binds the iron, it pulls it back into the plane. With the oxygen bound, the iron atom is only .026 nm above the plane. Previous to that it was .055 nm, so this small change going from .055 nm to .026nm is the motion that occurs; the oxygen binds and takes that pucker out, making it a little more planar.
b. What’s the consequence of that?
i. So as this invariant His F8 side chain is pulled down, it pulls the whole F helix down a little bit. This causes a change in the shape of myoglobin.
ii. When this occurs in the multi subunit, the tetrameric hemoglobin, that small movement causes a major conformational change.
XIV. Oxygen Binding Causes Conformational Change (S15)
a. In the lighter color (on diagram) is the His F8. The heme is puckered up. Upon oxygen binding, it pulls it down, which moves the F helix, resulting in a small change. Again, myoglobin doesn’t matter so much; hemoglobin results in a BIG difference.
XV. The Conformation Change (S16)
a. He just reads off the slide.
XVI. Figure 15.20 (S17)
a. This shows an oxygen binding curve. The left side shows percent of oxygen saturation of Hb or Mb. On the x-axis is the partial pressure of oxygen.
i. With increasing oxygen pressure, have increased oxygen binding for Mb and Hb.
b. Myoglobin has this hyperbolic curve, further to the left. Every partial pressure of oxygen is to the left of the hemoglobin curve.
i. The further left you go on this curve, the tighter you’re binding oxygen. The further right you go, the lower the oxygen affinity.
c. The partial pressure of oxygen to room air is about 160 torr or 160 millimeters mercury and the arterial partial pressure of oxygen (at blood cells) is about 100 torr.
i. At the lungs, myoglobin and hemoglobin are almost fully saturated with oxygen, which means almost all the molecules are bound.
d. As you go to the venous circulation, the partial pressure of oxygen decreases down to about 30 torr.
i. Myoglobin goes from about 99% saturation to about 95%, so still most of the myoglobin is fully oxygenated.
ii. Myoglobin has a much higher oxygen affinity than hemoglobin.
e. At the Venous p02, only about 70-75% of hemoglobin is saturated.
i. So in essense its four oxygens binding to one hemoglobin molecule, (cause there’s 4 irons for the 2 alphas and 2 betas).You’ve lost on average about 1 oxygen molecule from hemoglobin going from arterial to venous.
ii. So Hemoglobin is successfully unloading an oxygen molecule, whereas myoglobin still has it held tight.
f. Now for working muscle (like exercise) where the partial pressure can get closer to 10, now you can actually off load oxygen from the myoglobin.
i. Here, myoglobin is functioning as a storage protein for oxygen, its there when you need it. So when you’re actively using your muscles, there’s a little source of oxygen there that can off load from the myoglobin.
XVII. Cooperative Binding of Oxygen Influences Hemoglobin Function. (S18)