Unit 5 - Myoglobin and Hemoglobin Review Notes PDF

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WGU biochemistry notes unit 5 ...

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

I thought you might wish to take a look at these notes for Myoglobin and Hemoglobin. Here, I offer my perspective on these topics. There is nothing here that is not in the course material but sometimes, getting it from a different person helps.

Here are links to the recorded cohort for this unit and the relates slides. Myoglobin and Hemoglobin Recorded Cohort - Myoglobin and Hemoglobin Slides

Myoglobin and Hemoglobin STRUCTURE A crucial difference between myoglobin and hemoglobin is in their structures. Myoglobin has a single polypeptide chain, a single heme group with a single iron atom at its center and can bind just one molecule of oxygen (O2).

Myoglobin

(don’t worry about how it is drawn here, my point is ONE polypeptide chain, shown in pink). Myoglobin is an example of a protein that has tertiary structure (and secondary and primary, of course!).

Hemoglobin has four (4) separate polypeptide chains (2 alpha chains and 2 beta chains) that we call “subunits.” Each polypeptide chain has a single heme group with a single iron atom, and can bind one molecule of oxygen (O2).

Since there are four subunits, hemoglobin can bind a total of four oxygen molecules (4 x 1 = 4). © 2016-2020 RBRawson Ver. 04112020

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

Hemoglobin Again, I only want to emphasize the fact that hemoglobin has four (4) subunits.

AFFINITY One point to be clear on is the concept of “affinity”. “Affinity” is simply a way of describing how tightly (or loosely) one molecule binds to another, in this case, oxygen and hemoglobin. We often talk about hemoglobin “binding oxygen tightly” or “releasing oxygen easily.” Well, if it binds more tightly, it releases less easily and if it binds less tightly, it releases more easily. These are just two ways of describing the same thing, depending on which way you look at it, from the binding or the releasing point of view. To have a measure that doesn’t depend on our point of view, we can talk about “affinity”. Higher affinity means tighter binding and less releasing of oxygen; lower affinity means looser binding and easier releasing. Recall that pH has a strong influence on hemoglobin’s affinity for oxygen. © 2016-2020 RBRawson Ver. 04112020

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

Remember: lower pH, lower affinity; higher pH, higher affinity.

Changes to the Heme Group and to Hemoglobin If we were to look at a top-down view of the heme group in hemoglobin or myoglobin with oxygen bound to the iron atom, it might look something like this:

The two red spheres represent a molecule of oxygen (O2) and the black sphere beneath it represents the iron atom. The scarlet color refers to the color of the heme group when oxygen is bound. Then if we were to look at it from a more side-ways perspective (without oxygen, this time, so it’s maroon), it might look like this:

Now, let’s look at it straight from the side. Here are some side-view diagrams of the heme, with (red) and without (maroon) molecular oxygen (O2; two red spheres) bound to the iron atom (black sphere; Fe++).

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

Here, the heme group is flat, or “planar” because the oxygen is bound to the iron atom. Without oxygen, the heme group becomes bent (dome-shaped, nonplanar), like so:

Cooperativity A key concept here is that of cooperativity. This is the phenomenon whereby the binding of the first oxygen molecule to hemoglobin makes it easier for the second oxygen to bind. These two, being bound, make the third one even easier to bind and the fourth oxygen to bind binds about 300 times more easily than this first one did. That is, each oxygen that binds to hemoglobin makes the next one bind more easily . That is cooperativity and this behavior helps hemoglobin do its job as a pick-up and delivery system for oxygen better than it otherwise could.

R-to-T TRANSITION In order to explain this, let’s make sure we have the necessary background information too. Hemoglobin has 4 subunits and each subunit can change its shape a little bit. This is because (as shown above) the heme group changes its shape when oxygen binds to the iron atom. The heme group is shown below, again, from a side-on view. In this view, we see that, in addition to being bound to the heme group, the iron atom is also bound to the side chain of an amino acid (histidine, His).

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

When oxygen is not binding the iron atom, the iron atom is a little too big to fit neatly into the hole for it in the center of the heme group. As a result, the iron pops up out of the heme group and the whole thing gets bent into a domed shape (bent, non-planar). Consider a sideways view of what that would look like (shown above).

By contrast, when oxygen binds to the iron atom, it actually gets a little smaller, pops back into the hole in the center of the heme group, and the whole thing flattens out (planar). When the heme group moves from flat to bent, the iron atom tugs on that His side chain. This tug sort of ripples along the entire polypeptide chain of the subunit, causing each amino acid to shift its position a little bit. [NOTE: we are exaggerating the motion of the heme group to make it more obvious. In reality, the shift is less dramatic but no less important than shown here.]

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

You can think of a line of dominoes - when you push one over, eventually, every domino in the line must change its position (fall over) to accommodate that first domino's small change. The change ‘ripples along the line. In a similar way, the change in the position of that one histidine makes every other amino acid in the polypeptide chain adjust its position too, not much, but some. The result is a small change in shape for the subunit. But remember. There are four subunits and they touch one another. As oxygen binds each one, it shifts too. It’s neighbors must also adjust their positions al little to accommodate the shift of the first polypeptide chain.

The end result is that four times a little bit ends up being a pretty significant change in position. The subunits move from being close together to being a little farther apart (see below)

This is the relaxed (R) to tense (T) transition. (see about CO2 below).

You may recall that in the TENSE state, the subunits of hemoglobin are actually somewhat farther apart than in the relaxed state. This may seem counter-intuitive. Normally, we think of tense as being all uptight and relaxed as being loose.

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

Well, the terms “Tense” and “Relaxed” for hemoglobin only refer to its “willingness” to bind oxygen. They were coined decades before we ever had the detailed molecular protein structure data that show us how the subunits actually move. SO, we just must remember – the subunits are closer together in the RELAXED state and further apart in the TENSE state.

(Here we see the T and R forms of hemoglobin - below them is myoglobin for comparison)

2,3-BPG In our red blood cells, we have a compound called 2,3-BPG (2,3-bisphosphoglycerate). Because the subunits are further apart, there is more room at the center of the hemoglobin molecule in the Tense state and 2,3-BPG can slip into that hole. Then, 2, 3-BPG can bind there, interacting with specific amino acid side chains from the subunits, in between the four and help to hold the T state in place. In the diagram above, I have drawn circles around the centers of the two forms of hemoglobin to emphasize the change in size of that central cavity. Again, when 2, 3-BPG is stuck down in the hole, it is harder for hemoglobin to switch from T to R. 2, 3-BPG stabilizes the T state. Once there, it can interact with the side chains of some of the amino acids of the hemoglobin subunits and these interactions help to stabilize the Tense state. This is very much the same way that interactions between amino acid side chains help to stabilize tertiary structure in the first place. Only this time, it is not side chain:side chain interactions but side chain:2,3-BPG:side-chain interactions that are stabilizing things.

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R. B Rawson

Myoglobin and Hemoglobin Review Notes

You can imagine 2,3-BPG acting as a “bridge” that permits interaction between two amino acid side chains that are too far apart to interact directly. When each interacts with 2,3-BPG, the Tense form is stabilized. These interactions help to stabilize the Tense state and, thus, hemoglobin spends more time in the lower affinity Tense form than it would without 2,3-BPG. In mammals, fetal hemoglobin is composed of two alpha and two gamma subunits. The gamma subunits have different side chains in the key places as compared to the adult beta subunits and, therefore, fetal hemoglobin does not interact with 2,3-BPG. As a result, it spends less time in the low affinity Tense state (and more in the higher affinity Relaxed state.) Thus, fetal hemoglobin has a higher average affinity for O2 than does adult hemoglobin . This is important. If the fetus’s hemoglobin had the same affinity as the mothers, we would be just as likely to transfer oxygen from the fetus to the mother’s blood as from the mother to the fetus. Clearly, that is not going to work because the fetus has no access to oxygen except through its mother. Therefore, we can understand why the fetal hemoglobin must have higher affinity for O2 than does adult hemoglobin. At birth, the neonate needs to replace fetal hemoglobin with adult hemoglobin, since it is now breathing on its own. This means destroying the fetal hemoglobin and its heme groups and building adult hemoglobin. One of the breakdown products of heme is bilirubin. The transition from fetal to adult hemoglobin underlies the bilirubin crises in some neonates.

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Myoglobin and Hemoglobin Review Notes

Putting Key Pieces Together I tell myself a silly little story to help me remember this. I came up with it when I first took biochem. Imagine a marathon runner. Her leg muscles will be operating aerobically, consuming great amounts of oxygen (O2). Her lungs will be inhaling and exhaling to get that O2 and get rid of CO2.

Think of it this way: ●

In this example the legs are down lower in the body.

●

Because of all the metabolic activity, the pH of the blood there will be lower (more acidic – higher concentration of H +).

●

Hemoglobin’s affinity for oxygen will be lower.

●

And I like to imagine that hemoglobin is feeling ‘low’, so it is tense – “go away, oxygen! Don’t bother me!”

●

The lungs (in this example) are higher in the body.

●

The pH in the lungs is higher (more basic or alkaline – lower concentration of H+)

●

And hemoglobin’s affinity for oxygen is higher.

●

And I like to think that hemoglobin is feeling ‘high’, so it’s relaxed – “C’mon, oxygen. Let’s get together!”

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Myoglobin and Hemoglobin Review Notes

The pH Scale

Think of an old-fashioned double-pan balance scale. If we started out with equal amounts of H+ on each side, the pans would be level. Think of that as neutral pH (7.0). Then, as we remove H+ from one side and add it to the other, the side with more and more H+ gets lower and lower. The side with LESS H+ gets HIGHER. The point is this:

More H+ = lower pH; less H+ = higher pH. Remember: lower pH, lower affinity; higher pH, higher affinity. Here is a mnemonic device – “Tissues = tense.” Then we have – “Respiratory system = relaxed.”

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Myoglobin and Hemoglobin Review Notes

THE BOHR EFFECT When we discuss the Bohr effect, we often encounter graphs like the ones shown here. Some students are put off by graphs, but we can make these easier to understand, even though they look a bit complicated.

The first thing to know is that, whatever numbers or units are shown along the bottom (X-, or horizontal-) axis of the graph, what it means is always the same, We are starting with absolutely NO oxygen present in the experiment on the LEFT of the graph. As we move to the RIGHT, we are adding more and more oxygen to the experiment, up to some high level (usually about 20%, the amount of oxygen in the air that we breathe).

Along the left-hand side of the graph (the Y-, or vertical-) axis, we are measuring just how many of all the sites on hemoglobin available for binding oxygen are actually filled with oxygen. (Remember, hemoglobin has four sites for binding oxygen; if we had 10 hemoglobin molecules in a test tube, we’d have 40 oxygenbinding sites, for example. If 20 of those sites were actually binding oxygen and the other 20 were not, we’d be at 50%, halfway up the vertical axis.) Our curves for hemoglobin (the green, blue, and yellow lines on this graph; the stretched-out, S-shaped [‘sigmoidal’] curve) always starts at 0. This makes sense, when there is no oxygen around to bind, there is no oxygen bound to hemoglobin. Then, as we add more oxygen, hemoglobin can bind some of it. For any given amount of oxygen, hemoglobin will bind more of it when it has higher affinity and less of it when it has lower affinity.

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Myoglobin and Hemoglobin Review Notes

What this means is that as the curve is shifted further and further to the right, that indicates that hemoglobin has lower and lower affinity for O2. By contrast, the further the curve is to the LEFT, the HIGHER the affinity of hemoglobin for oxygen.

I wanted to touch on one aspect of this in a bit more detail. Imagine you get a question that asks something such as, “What is the percent saturation of hemoglobin at pH 7.4 when the oxygen concentration is 40 torr?” To answer these kinds of questions, we need to look at the graph of the Bohr effect. In order to find out the fraction of hemoglobin’s oxygen-binding sites that are actually binding to oxygen, (% saturation of hemoglobin with oxygen) at pH 7.4 when the oxygen concentration is 40 torr, we actually DO NOT need to “calculate” anything. We can use the graph itself to answer this question. Let’s remove the other curves for now so that this may be seen a little more clearly. We can add them back later, once we understand the process.

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Myoglobin and Hemoglobin Review Notes

We start by drawing a line UP from the 40 torr mark on the X-axis (the horizontal, bottom axis of the graph) until we hit the pH 7.4 curve. Then, we draw a line straight over to the Y-axis (vertical, left hand axis), which tells us the % saturation of hemoglobin with oxygen (0 to 100%, shown in increments of 25 percentage points. We can see that the line hits the vertical axis below the 75% mark but close to it Let’s call our answer 73%. (It might be 74 or 71%, but that is close enough for our purposes. If the answers to choose from were 1.) 2.) 3.) 4.)

80% 72% 66% 41%

I would pick (2.) 72%. That is, if they ask you to use a graph like this, they won’t make you pick between two really close choices (like 72 versus 73%!). There will be one answer that is obviously close to the answer we found on the graph. So, we will say that when the oxygen concentration is 40 torr, at pH 7.4, hemoglobin is about 74% saturated with oxygen.

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Myoglobin and Hemoglobin Review Notes

CO2 Let’s look at carbon dioxide and our blood. As for resources for the CO2 → H2CO3- equilibrium, these figures are a bit less complicated than most about this topic. H2CO3 is also known as carbonic acid. In our bloodstream When carbonic acid is formed from CO2 and water, it immediately becomes bicarbonate ion and a proton (H+). We write it this way:

H2CO3- → HCO3-+ H+ The conversion of CO2 and water to HCO3-+

H+ is important because carbon dioxide is a gas and is not

particularly soluble in watery solutions, such as blood. Anyone who has ever bumped a can of soda pop before opening it knows that CO2 can come out of solution pretty darn fast (Whoosh! What a mess!) By contrast, bicarbonate and H + are very, very soluble in water. We can carry a whole lot more of bicarb and H+ that we possibly could of CO2.

In our bloodstream, a special enzyme called carbonic anhydrase converts a molecule of water and a molecule of into carbonic acid, H2O + CO2 --> bicarbonate and H+. We write that out:

H2CO3-. Carbonic acid immediately then becomes

H2CO3- --> HCO3-+ H+

This bicarbonate ion, HCO3- , is much, much, much more soluble in water than carbon dioxide is. When carbonic acid is dissolved in watery solutions such as our blood plasma or inside our cells, it loses one of its hydrogens (as a hydrogen ion, H+) and becomes ionized . The hydrogen ion carries a positive charge while the bicarbonate ion has a negative charge.

For reasons that are not worth explaining, we refer to the ionized form of carbonic acid as “bicarbonate” and its formula is HCO3- (That “ -ic acid” to “ -ate” ending for the acid versus the ion is standard terminology. We refer to ionized glutamic acid, for example, as glutamate, or lactic acid as lactate).

Here is a summary figure of getting CO2 from our tissues and out through our lungs, via bicarbonate in our blood cells. Note that the background color here is not the color of the blood, but the color of the pH indicator dye, litmus, which is dark pink when acidic and blue when basic.

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Myoglobin and Hemoglobin Review Notes

Converting carbon dioxide to bicarbonate means that we can transport a lot more bicarbonate than carbon dioxide and we don’t need to worry about it bubbling out of solution and clogging our capillaries. However, this comes at the price of all that carbonic acid releasing protons and lowering the pH of our blood cell. Being an acid means that carbonic acid gives up a hydrogen ion, thus increasing the concentration of H+ in solution. Increased concentration of H+ means lower pH. Thus, the more carbonic acid you have in solution, the lower the pH of that solution. More acid = lower pH.

When our blood cells reach our lungs, carbonic anhydrase runs the exact same reaction backwards . It reconverts the bicarbonate to CO2. This reduces the amount of carbonic acid in the cells and, with less acid around, the concentration of hydrogen ions also drops. As the concentration of hydrogen ions drops, the pH rises from 7.2 back to 7.4. A lower concentration of hydrogen ions means higher pH. We find a more acidic pH of 7.2 in the blood when it picks up the carbon dioxide waste product from t...