#08 Biochemistry Hemoglobin Lecture for Kevin Ahern’s BB 450/550

#08 Biochemistry Hemoglobin Lecture for Kevin Ahern’s BB 450/550


Captioning provided by
Disability Access Services at Oregon State University. Ahern: We are moving
rapidly through stuff and I finally sat down and
looked at when the exam is and the exam is this Friday, so I guess… [classroom chatter] Ahern: It’s not? The exam is a week from Monday. I’m guessing you guys wouldn’t
be ready for it this Friday? No? There will be fewer things on it. Think about that. A smaller nice, little tidy exam. Student: And the
next will destroy us. Ahern: And the
next will kill you. [laughs] Or you will kill
me, I don’t know. The second exam
is not cumulative. The final is cumulative. I just have a couple
fairly minor things I want to say about
characterizing proteins and I do that in
recognition of the fact this is not a biophysics class. The things that are there are
really more biophysical in nature than they are
biochemical in nature. I want you to be
aware of what we can do with certain techniques. One of the techniques, last time I talked about
MALDI-TOF and MALDI-TOF is a fantastic method
as I say that I realize I didn’t post that figure for it. I will get that figure
posted showing you the set up for a MALDI-TOF instrument and you can hopefully see it
better than my words can describe. I will get that posted for you. What I want to talk about today are a couple other
very powerful techniques that come to us from biophysics. I’m not going to go in depth but you should know
the basics of them and you should be able to
understand why they’re useful for us. The two techniques
I want to talk about are X-Ray crystallography and
nuclear magnetic resonance. You’ve probably had some exposure at least with nuclear
magnetic resonance, I’m guessing in your
organic chemistry class. Has anyone had exposure to
X-Ray crystallography before? Little bit, okay. I’ll start with
X-Ray crystallography. X-Ray crystallography, both these
techniques, by the way, are extraordinarily useful
for helping us to understand relative positions
of nuclei in space. I always tell the story when
I give a tour of our facilities in ALS in Biochemstry
and biophysics to students that using the tools
of X-Ray crystallography and nuclear magnetic resonance, biophysicists can
determine the position in three dimensional
space of every atom that might exist in an enzyme
that has 10,000 or 50,000 atoms. That’s a really remarkable thing because knowledge about structure leads to understanding
about function. We’ve heard structure
function before. When we think about
how drugs get designed, the design of drugs is
happening increasingly as a result of the molecular
knowledge of the structure of the proteins that
they’re targeting. If I know the position
or I know the structure of the active site of the enzyme, the place where the
reaction is catalyzed, I know the dimensions
of the molecule I need to design to
plug up that enzyme. That knowledge of structure
is really valuable for us to have for whatever purpose. And there are purposes aside
from designing drugs as well. X-Ray crystallography
arises from the fact that X-rays get diffracted which means they get bent when
they encounter electron clouds. That diffraction
process is depicted here. To do X-Ray crystallography, one has to have
first of all a crystal and a crystal is a as
you guys see crystals you don’t think at a molecular
level what a crystal is, but a crystal is a perfectly
packed homogenous molecule that has a regular repeat to it. That is all the molecules in
there are the same composition and they’re organized
in a regular fashion. That regular repeat is what
gives rise to the crystal itself. The process of making a crystal
for a lot of X-ray crystal analyses is actually the thing
that takes the longest. A lot of different, there’s no
one formula for making a crystal. Different proteins crystallize
in different to way. Suffice to say that
making the crystal, which is already
shown right here, can be a very and
time consuming step and a frustrating
step in the process. Once one has a crystal, one can take they crystal and put in the path
of an X-ray beam. That X-ray beam will have
its rays diffracted again according to the electron
clouds that it encounters. The importance of the
regularity of the molecules in the crystal are very important because those really add up and give us the diffraction
patterns that we see. Now interpreting the
diffraction patterns obviously isn’t a thing
we’re going to do here, but sufficed to say
that diffraction gives, this is what a
diffraction pattern of a given crystal might
look like and we say wow, there are some spots. These are the spots
correspond places where X-rays got diffracted to. So a biophysicist
can take information from a diffraction
pattern and work backwards, ultimately to determine where
the individual electron clouds where it caused the
diffraction pattern to exist. So X-Ray crystallography
is extraordinarily powerful because it does give us three
dimensional information about the position and orientation
of electron clouds and a working map might look
something like what you see right here. Here are patterns of electron
clouds and then within there we decide what are the individual
atoms that correspond to there, you see different carbons
and hydrogues and oxygens and so fourth
scattered through there. And interpreting these patterns
can again be a very time consuming process it’s very
computationally intensive. The result is at the end of this that one has that structural
information that’s very useful. Again, that’s just a very cursory description of X-Ray
crystallography. X-Ray crystallography
has its advantages and it has its disadvantages. The advantage of X-Ray
crystallography is if you get crystals, then you can determine
these positions very nicely. Sometimes you can’t always get
crystals and that’s one limitation. And the other is crystals may or
may not correspond to the natural, whatever that means,
shape of the solution. So we think about the
enzymes we have in our cells, most of them are dissolved
the in water of the cytoplasm. So when I’m making a crystal, I’m basically taking
it out of the solution so one thought is
well is this reflecting the actual structure it has
when it’s in the solution? So to partly address
some of those concerns, this additional technology
of nuclear magnetic resonance is very useful because nuclear
magnetic resonance analysis allows one to determine molecular
structures in aqueous solution. They work in different ways
and nuclear magnetic resonance relies on the fact that
certain nuclei have spins that are characteristic
of them and those spins can be altered in the presence
of an electromagnetic field. So understanding the energies
it takes to alter those spins of given nuclei
for example protons. Protons are very
commonly used in analysis to understand the changes
in those spin gives us some knowledge
about the structure. I’ll just show you a
very brief example here. Here’s a nucleus that
has a characteristic spin. There are two possible
spins that can exist. One has a slightly higher
energy than the other and the difference
between that energy is what is excited using the
electromagnetic radiation. It turns out the different
nuclei have different spins corresponding to the
electronic environment in which they find themselves. This depicts the nuclear
magnetic resonance signal of a very simple molecule. This is ethanol. We can see that ethanol has three
different kinds of protons in it. It has methyl protons that
are farthest off in the end. It has methylene protons
in the middle here and it has hydroxyl on the end. These give rise to
characteristic signals. These signals have
known positions, we know where
hydroxyl protons arise, we know where methyl
protons arise, etc. And so we can examine the
spectrum that comes from this. The spectrum is called the chemical
shift which I won’t go in to. This molecule has some hydroxyl, it has methylene, it
has methyl protons. As you can imagine for a
molecule like a protein, it’s not not nearly as this. We might get very
complicated spectra and in fact we do get
complicated spectra. So this is a little bit more
challenging to interpret. We’re not going to do
that obviously here. But sufficed to say that analysis
of nuclear magnetic spectra does allow,
ultimately a scientist, to allow which signal corresponds to which groups
inside the protein. Now understanding the different
kinds in this case of protons that exist in a protein
is useful information but one of the things
we’re interested in as biochemists is
how do proteins fold. Because remember that folding
really gives the protein its characteristic shapes so we
would like to get more information because the knowledge of
different protons we have in the protein really isn’t
sufficient enough to tell us structure. One of the techniques done
with that is an enhancement as it were of nuclear
magnetic analysis. It’s called the nuclear
overhouser effect and it arises from the fact that, this clip doesn’t work, it arises from the
fact that nuclei, when they interact
with each other, also have effects. In this case, here are
two protons that, as a result of folding, have been brought
into close proximity. And if they’re brought
into close enough proximity they actually do affect
the signal of the other one. This requires a very
sophisticated analysis called 2D and that’s obviously
a lot more complicated than 2D gel electrophoresis
I’m not going to go into that. But sufficed to say that
with this type of an analysis, one generates some even
more interesting spectra but this information
that we see here now tells us not only what
kinds of protons that we have but how close those
protons are to each other. That’s very, very useful when
one goes to trying to determine the overall structure
of a protein molecule. So that can be very, very useful. Commonly these two techniques
are used in combination with each other to help
elucidate molecular structure. There is a biophysics
course in 5 minutes. How’s that? Questions or comments about that? Let’s get away from
biophysics and talk about, this is the lecture
I’m going to give today is most of the most
popular lectures I give throughout
the entire term. It’s the lecture on hemoglobin. And hemoglobin is, I
hope to convince you by the end of the
lecture on Friday, one of the most magical
molecules in our body. It is absolutely
incredible the abilities that are built in to the
structure of this protein. I start talking
not about hemoglobin but about a related
protein called myoglobin and I introduced
myoglobin to you before as a protein related
to hemoglobin. It’s found in our
muscle cells primarily and there it serves the
function of storing oxygen. It’s a very good
way to store oxygen. Hemoglobin is very
good at delivering, that is picking up and
dropping off oxygen. The difference you
recall structurally I hope between the two proteins
and that myoglobin has a single protein subunit. And hemoglobin has
four protein subunits. So hemoglobin has quaternary
structure, myoglobin does not. And this quaternary structure
that myoglobin has is, that hemoglobin has
is what gives rise to all of the properties
that the molecule has. Well, you’ve seen
myoglobin before, it’s mostly alpha
helical structures, it looks something like this. There’s the amino terminus and
there’s the carboxyl terminus. And here you see
alpha helix bend, alpha helix bend,
alpha helix bend, a lot of alpha helices here. We see the amino acids,
we see 146 amino acids. Myoglobin was I believe
the first protein whose structure of this
nature was actually determined and so that has some
biochemical significance. Not of any concern
to us at the moment. But the other concern for us is it has an oxygen binding
group in it called a heme. So the heme, and yes,
myoglobin has a heme just as hemoglobin has a heme. The heme is located right here. Yes, sir? Student: So wait, is this
myoglobin or this hemoglobin? Ahern: Actually, I’m sorry,
this is the beta chain. I have a link to it
that says myoglobin. They’re very, very similar so this
is the beta chain of hemoglobin. We can think of this as myoglobin because as I said the structure
is very similar between the two. Thanks for noticing that. Anyway, both myoglobin
and hemoglobin have a heme. Hemoglobin you
recall has four chains to call beta and to call alpha. This is one of the
betas right here. Now the heme turns out to be really
important for several reasons. The number one being of
course that it’s the place where the oxygen is bound
by this protein subunit. There’s the heme. Heme is a flat ring. It is something we refer to in
these proteins as a prosthetic group. Sounds like a very mouthful name. Prosthetic group
is simply a molecule bound to a protein that helps
the protein do what it does. It’s a non-amino acid. So it’s a non-amino
acid bound to a protein that helps the
protein to function. The heme group of
hemoglobin and myoglobin, the two are essentially
identical and they’re very, very similar in
structure to chlorophyll. The electron gathering
component of chlorophyll that we find in plants. The difference in plants that
instead of having iron in middle, we have a magnesium
in the middle. Student: You said this is planar? It has like 20 carbons
and stuff in the middle. How is it possible? Ahern: How is it possible? Well, if we’re talking
about an exact plane, there’s nothing
that’s exactly flat. Generally, it’s a flat structure. You can see when I
talk to you about this that the places actually pucker. So it’s planar, but I wouldn’t
say it’s a perfect plane, no. Alright, that puckering that we
will see is very, very important. It’s actually seen right
here in this figure. What I’m getting ready
to tell you about here occurs in both
myoglobin and hemoglobin but the impact is felt in hemoglobin
because of its four subunits. What you see happening on the
screen happens in both proteins. Let me describe to you
what’s going on here. If you we look at the
deoxyhemoglobin on the left, that’s the way it normally sits. Shannon says, “well
that’s not exactly planar.” And I say well okay, look. It is slightly puckered. We can imagine it being a
little concaved downwards like my hand is. When the oxygen binds and we
see oxygen bound over here, there is a very,
very tiny change. So instead of being
slightly puckered, it flattens a bit. Why does that happen? It happens because the
oxygen pulls up the iron atom. The iron atom
physically gets lifted. This change is minuscule. We’re talking
fractions of angstroms. Very, very tiny change. Yes, sir? Ahern: His question is, “Is this just the heme group?” It turns out this movement
affects a lot of things. It’s a very good question. For the moment, we’re thinking
only about the iron atom. The heme group
itself is not moving. It’s the iron atom that’s moving. So the iron atom moves up a very
tiny fraction of an angstrom. And if you look at the structure, you’ll notice that the iron atom
is not floating freely there. It is in fact attached to
an amino acid beneath it. This amino acid that it’s
attached to is a histidine. Now, if I pull up on iron and
iron’s attached to histidine, you can do the math and
figure that the histidine is probably moving a fraction
of an angstrom as well and you’d be exactly right. And you’d say
histidine is attached to another amino
acid in the protein, is it moving also? Yep, so the foot bone, the toe bone’s connected
to the foot bone, and the foot bone, this isn’t going to
be a song by the way. The foot bone’s connected
to the ankle bone, and the ankle bone is
connected to the shin bone and by pulling on the toe, I’m ultimately going
to affect the hip. Even if it’s by a
very tiny amount. And this very tiny amount, I can’t emphasize enough the
importance of this very tiny change because I’m going to hopefully
convince you by the end that the result of this
very tiny amount of movement allows us to be animals. Without this movement, animal
life is essentially not possible. This is a scary thought. Why is it that this makes
animal life possible? We’ll talk about that. Hemoglobin of course doesn’t
exist as a single subunit, it exists as 4 subunits. All 4 subunits have a, each subunit of the 4 has
a heme group of its own. So when this guy binds an oxygen, and by the way, because
it’s a schematic, they’re not showing
the connection, but in each case it’s
connected to a histidine. When this guy binds to an oxygen, let’s see I’ve got this
hemoglobin that’s got no oxygen whatsoever. This guy binds an oxygen, it’s going to cause the iron
atom to move a slight distance, it’s going to cause that histidine
to move a slight distance. It’s going to cause that entire
chain to very slightly shift. That very slight shift changes
the overall shape of this subunit. And guess what? That shift affects how it interacts
with its adjecent subunits. And the adjacent subunit now becomes more favorable
for binding oxygen. So this one, when we have
hemoglobin that’s empty of oxygen, it’s not real keen
on binding oxygen, but once one of
them binds oxygen, these changes get communicated
between the subunits and each additional oxygen is
increasingly favored for binding. This phenomena I’ve just
described to you is called cooperativity. The bonding of one
molecule to a protein affecting the binding of others. In this case, it’s positive cooperativity. It’s favoring more binding. Now this is really important. We have to, we are animals, we are moving creatures, we have to have an
adequate oxygen supply. Plants don’t have this issue. Plants don’t have to get
up and run around and jump and go chase things or
run away from things. Their oxygen needs
are more constant. Ours are rapidly changing. We need oxygen, we need it now. When our hemoglobin
gets to our lungs, it doesn’t have an awful
lot of time to be there. We want it to load up on
oxygen as much as it can and take that oxygen out to
the tissues where its needed. Cooperativity as we will see, plays a very big role in
the loading up of hemoglobin. So we’re loading up hemoglobin. If we can’t load up hemoglobin, we don’t have enough oxygen, we can’t go run,
we can’t go escape, we can’t do things
that animals do. Very, very important. The other thing I want you
to look at in this structure, it’s actually easier
to see right here is that when we look at hemoglobin
from above as we are in this case, we see that hemoglobin is
shaped sort of like a doughnut and there’s a little
hole in the middle. That little hole turns out
to be extremely important. Extremely important. So I’m going to talk back
and talk about that hole but before I do that I want
to tell you a little bit about the needs of
oxygen in the cell and how hemoglobin
helps to supply those. Questions on this
before I move forward? Everybody understands
what cooperativity is? Yes, sir? Student: Does the inverse occur
when it unbinds the oxygen? Ahern: Good question. Does the inverse occur
when it unbinds oxygen? The answer is to some
extent, yes it does. Loss of one will favor the
loss of additional ones. So it would be a
negative cooperative. So you start to see where
this is heading, right? If we look at the oxygen binding
of myoglobin, this is a plot. Again, whenever
I show you a plot, I always want you to
know what the axes tell us because without the axes, the plot has no meaning. This is the fractional
saturation meaning what fraction of all
the myoglobin molecules in the solution have
an oxygen bound to them? Myoglobin can only bind
one oxygen per protein because there’s only one subunit
and each heme only binds one oxygen. Either it’s bound
or it’s not bound. What percentage of those
guys are bound with oxygen? What we see that it
takes very little oxygen. This is very low, the pressure
of oxygen on the X-axis. Very little oxygen for
us to get 50% saturated. What does that mean? It means that myoglobin
when it has the chance is grabbing a hold of oxygen. It’s very good at storing oxygen. It grabs it, it holds
onto it very well. Well that’s nice, but it’s not ideal
for delivering oxygen because if myoglobin
didn’t give up its oxygen til the oxygen
concentration got very low, it could travel all the
way through the body, get all the way to the lungs and
it hasn’t given up its oxygen. “No, it’s mine. I’m the big kid I
get the quarter.” Right? “I’m not going to
give this up for you.” Myoglobin only
gives up its oxygen when the oxygen
concentration gets very low. How many people have
UPS on their computer? Anybody know what a UPS is? It’s an uninterruptible
power supply. It’s there to give you
power when the power goes off so you have a chance to
shut down and save your work. Myoglobin is the
UPS for your muscles. When you’re working very hard, it’s very easy for
you to use oxygen faster than your
blood can deliver it. Well oxygen is important. It’s not essential, but it’s important so that
more oxygen your muscles have, the better off they are because
muscles need it for contracting, we gotta run away from something, we gotta beat something
up, we gotta do whatever, hopefully we’re not
doing too much of that. If hemoglobin can’t supply
all the oxygen that’s needed, we want something there to back
it up and this is backing it up. When the oxygen concentration
starts getting very low, myoglobin says, “Oh,
here’s some oxygen.” That’s the only time
myoglobin gives up oxygen. When the concentration
gets very, very low. But it helps us
when we need that. Let’s compare that
with hemoglobin. Ahern: That’s the
oxygen concentration. Oh, here? How much does it take to
get half of it saturated, half of it bound to oxygen. Is P 1/2 just
refers to 50% of it. So very, very low number there. If we compare this
with hemoglobin, hemoglobin has a
different looking curve. So the curve that
corresponds to myoglobin is what we call hyperbolic. It’s a hyperbolic curve. It’s a hyperbolic function
that will fit that curve. The curve that hemoglobin
gives is called sigmoidal because it has sort of a S shape. It’s sigmoidal. Look at this. At low oxygen concentrations, there’s not very
much oxygen bound. When hemoglobin travels
through our body, it goes from places of
high oxygen concentration, our lungs, high
oxygen concentrations, essentially 100% of it
gets bound with oxygen. As it travels through the body, the oxygen concentration
starts dropping because the cells are using oxygen
and hemoglobin is the only source, oxygen concentration drops and hemoglobin starts
letting go of its oxygen. It’s a perfect
system for delivery. We see it cooperatively
binding oxygen to begin with and we see cooperatively
letting go of oxygen. Binding in a negative sense when the oxygen
concentration starts to fall. Hemoglobin, because of
cooperativity can satisfy an immense, or a diverse set of
oxygen concentrations as they occur in our bodies. This is essential for an animal. I just cannot, I keep
coming back to that, but I can’t
emphasize that enough. It binds like myoglobin at the
very highest concentrations. It will get 100% bound. But as oxygen
concentration falls, it’s down over here. And that’s pretty cool. Then by the time it’s
dumped its oxygen, it goes back to the lungs
and it gets more oxygen. So let’s think about
that a little bit. No, I’m not going to ask you
to draw this particular figure although you should be familiar
with any of these figures. We see the differences
in oxygen concentration, this figure is nice in that
it shows us the concentration of oxygen roughly
that occurs in tissues and the concentration of oxygen
that roughly occurs in lungs. We see that again, way up here
in the concentration of the lungs, myoglobin and hemoglobin
are essentially the same. And then way out over here, when this thing gets
out to the tissues, only what is 38%, no,
I’m sorry, what is this, about 30% or so of the hemoglobin
is actually still bound to oxygen. So that flexibility of
hemoglobin for oxygen is very, very valuable for us and allows
us to things like sitting here, or getting up and running
if we have to go running. And both of those work. Okay, if I exercise,
blah blah blah, if I rest, I have different needs, if I
have lungs, same sort of thing. This shows us the
quaternary changes that happen as a result of
oxygen binding to hemoglobin. Quaternary changes
mean the four subunits are actually changing
on oxygen binding. Notice that doughnut
hole that I had before. The doughnut hole
has largely closed up. It turns out that these two
different states of hemoglobin have names that we give. And we’re going to hear
more about these names later. They’re called the R
state and the T state. I need to define them for you. The one on the left
is called the T state. The T stands for tight. I like to think about it as
people I know who are uptight. People you know who are uptight, you can just sense it around them
and they can’t take anything more. They’re very rigid. Give me some oxygen, “No, I don’t want any oxygen!” Okay, tight structures. Ahern: What’s that? Student: [inaudible] the right? Or the left? Ahern: The left? Yeah, that’s tight, yeah. They’re not very flexible. They have poor binding of oxygen. So when hemoglobin has no
oxygen, it’s in the T state. It doesn’t want to
bind more oxygen. On the other hand, once it’s bound and it’s
gotten full of oxygen, its structure changes to
what we call the R state. R is the relaxed state. The relaxed state, “yeah, come
on, we’ll take this, we’ll have it, I can take a lot of stuff, man.” Right? [class laughing] I grew up in the 60s, guys, you gotta give me credit
for the language at least. So the relaxed state is a high
affinity binding for oxygen. Once we put one oxygen on there, we start flipping
it into the R state and we’ve got affinity
to find more oxygen. We flip it into the T state, it doesn’t want to bind oxygen. That’s kinda good. If you think about it. Let’s imagine that
I’m a hemoglobin that’s floating around and
I’ve just gone to let’s say the muscles where they’re
exercising pretty heavily and I dump all my oxygen. On the way back to the
heart or to the lungs, I pass through the kidney. Do I want the hemoglobin taking
the oxygen away from the kidney? That wouldn’t be a
good career move, right? So I only want it to flip when
there’s a high oxygen concentration. That’s what’s going to happen
when it gets back to the lungs. So the T state and R state really serve the body’s
needs very, very usefully. There are a couple
of ways of describing how this phenomena occurs. The way I’ve described to you
is called a sequential mains. That is the binding of the
first one affects the second one, affects the third one,
affects the fourth one. And that’s not
shown on the screen, this is a different model. If I were to show what happens, we’ve got T state
above and R state below. In the sequential model, one
of these guys turns circular, it favors the next
one turning circular, the next one turning
circular, etc. That model’s called a
sequential model of binding. Changes in the structure
of one changes the next one, which changes the next one, etc. It’s sequential. This model you see on the screen
is the opposite of the sequential, it’s called the concerted model. These are models. Models are way of
explaining things. This model says that we don’t
see one followed by the other, followed by the other,
followed by the other. Instead, what we see is
we’re either in one state or we’re in the other state and binding of things
locks them into that state. Now hemoglobin is not
a good model for this. We’ll see in next week’s lecture how an enzyme is a much
better model for this. This model and I’ll say more
about this model next week so I’m not going to
go into much here, but this model says that the
changes happen all at once and they’re independent
of the binding of anything. We see this as back and fourth. But they get locked into one vs.
the other based on what they bind. We’ll come back
to that next time. For right now, think sequential. Binding the first
changes the second, changes the third,
changes the fourth. Changing the T to R state
changes significantly the binding affinity of
hemoglobin for oxygen, which I showed you before and if we look at what hemoglobin
would look like in the T state, this is what it would look like. If it were only in the R state, this is what it would
look like and in fact, hemoglobin goes through
a transition from T to R and that’s what we’re seeing, why this curve has
a couple of shapes in it. We’re seeing a
change from T to R. That change is what we’ve
already described as cooperativity and that cooperativity is favoring
bonding in this case is oxygen. If we’re getting more oxygen or
favoring the release of oxygen if we’re getting into lower
concentrations of oxygen. This is what a
sequential looks like. Nothing bound, first
one changes this one, which causes this one to chances, which bind has caused
this one to change, which caused this one
to change its find. It doesn’t matter if it binds
to an alpha subunit or a beta subunit.
It doesn’t matter. They’re essentially the same
as far as this molecule exists. Yes sir? Student: Shouldn’t the
[inaudible] under K4 be switched where there’s a higher affinity
to drive further to the right? Ahern: All of these depend
on oxygen concentrations, so you’re exactly right. In the concentration
of the lungs, even though you’ve got a
lower going to the right, there’s enough oxygen
concentration to drive that. Student: You’re showing a
sequential increase in K1 and [inaudible] the
last one is shorter. It’s counter-intuitive. Ahern: No, because it doesn’t
want to bind that first one. I agree that this is important
for the releasing of oxygen. This, from what I’ve told
you sounds a little odd in terms of putting
that last one on there. But the reason this is the case is because that first
one doesn’t want to bind. And that’s because this
guy here is in the T state. That should answer your question. Student: Once you have
the three on there, it seems like it
should be more of a push in the equilibrium
to push [inaudible]. Ahern: Right, so his point
is that this equilibrium is favored actually in
the leftward direction and that would be
true if it weren’t in the high oxygen
concentration in the lungs. The lungs are loaded with oxygen
and that drives it to this state. We want this guy to dump off oxygen
once it gets out of the lungs. That’s why the arrow
is back to the left. Where am I at here? That’s the basics of hemoglobin but there’s so much more
that’s built into this molecule. The first one I’m going
to show you right here is a really interesting and
cool molecule called 2,3-BPG. We’ll talk more about this
molecule later in this term when I talk about glycolysis but this molecule turns out
to be a fascinating molecule. You don’t need to
know the structure but you definitely need to know at least this part
of the name: 2,3-BPG. It’s real name is
2,3-bisphosphoglycerate. I need to tell you why
this molecule is important. If my microphone works that is. Why this molecule is important. This molecule is a molecule that is released by
rapidly respiring cells. If I’m a muscle cell and I’m doing
my business I’m making 2,3-BPG, we’ll see later it’s
actually a byproduct, but that doesn’t matter
for our purposes right now. Actively respiring
cells release 2,3-BPG. So my muscle cells may
have a lot of 2,3-BPG, my nose cells may
not have so many. Okay? With me? Unless I’m sneezing, I’ve got that cold
everybody else does, I might have more 2,3-BPG. It turns out that 2,3-BPG
affects hemoglobin. If we look at hemoglobin in
the presence of 2,3-BPG and red, we see that it binds less, and they’re not exaggerating
the S so much here, they’ve sort of drawn
this to make their point. The point is that in
the presence of 2,3-BPG, hemoglobin holds
onto less oxygen. So 2,3-BPG it turns out causes
the hemoglobin to release oxygen. How does it do it? It’s very simple. 2,3-BPG has a shape that
fits exactly in that doughnut. It fits exactly in that doughnut and when it fits
into the doughnut, it favors the
conversion of hemoglobin from the R state to the T state. T state has low
affinity for oxygen, guess what hemoglobin’s going
to do when 2,3-BPG binds to it, it’s going to start
giving up more oxygen and that’s exactly what
this curve is telling us. This turns out from
a bodily perspective to be very useful
because when I’ve got actively respiring tissue
and I’ve got a lot of 2,3-BPG, what’s 2,3-BPG going to do? It’s going to bind hemoglobin
and hemoglobin’s going to say, “Okay, flip into the T state, I’m going to let
go of the oxygen.” And as concequence of
letting go of oxygen the tissues that
need the oxygen get it. That’s great. But wait, there’s more. But wait. If hemoglobin has
bound to 2,3-BPG, it’s going to be in the T state
and when it gets back to the lungs, it’s still got 2,3-BPG, it doesn’t want to bind to
more oxygen, I’ve got trouble. Well fortunately, remember
these are not covalent bindings, fortunately 2,3-BPG
fits in that pocket. But it goes in, comes
out, goes in, comes out. Like any binding that occurs, binding and letting go
happens all the time. On the way back to
the lungs, 2,3-BPG, when it gets off of
the hemoglobin can get grabbed by cells
and be metabolized. So as hemoglobin is making its
way back to cells in most people, the cells are grabbing
it, burning it up, and hemoglobin gets
back to the lungs and it has no 2,3-BPG in it. If it had 2,3-BPG in it, you
wouldn’t bind as much oxygen. Now all of you pre-meds, everybody looks up at this point, smokers are full of 2,3-BPG. Smokers are full of 2,3-BPG. The reason that, one of the
reasons that smokers huff and puff going up stairs is that 2,3-BPG doesn’t get all
the way broken down. The hemoglobin gets
back to the lungs, uh oh. My oxygen carrying
capacity is lower, that’s why smokers huff
and puff going up stairs. They’ve got too much 2,3-BPG. The next question is
why do they have more? And that we will save and talk
about when we talk about glycolysis. Suffice to say, they have much more 2,3-BPG in
their blood than do non-smokers. Yes sir? Student: So is that a large
contributory factor to say COPD? Ahern: COPD being? Student: Chronic obstructive
pulmonary disease. Ahern: Is it a
contributor to COPD? Not to my knowledge. There are other things
that give rise to that. I’m not a medical person
so I can’t tell you that. But sufficed to say that
the primary physical observation that you could make with
respect to hemoglobin, respect to BPG in smokers
is that they huff and puff. They puff and then they
really huff and puff. If you smoke, quit doing it. Now you know at
the molecular basis why smokers are having
a hard time going up stairs. Their hemoglobin is
stuck in the T state and they can’t get it out
of that T state very well. There we go. There’s your doughnut
and there’s the binding. We’ll need to worry about the
various other stuff that’s here. Now, hemoglobin is
some pretty cool stuff. There’s a problem, though. We are not chickens. And some would say
that’s probably good. But chickens lay
eggs and the fetus that develops inside the
egg has its own resources and doesn’t have to rely
on mom except to the point where the egg is laid. Mom, however, is the source of
nutrients in mammals for food, for water, and for oxygen. Now there’s a problem. The problem is what
if mom’s hemoglobin is competing with
the baby’s hemoglobin? They both have oxygen, why should the baby’s hemoglobin
have to fight with mom for that? What turns out that fetuses
have a modified hemoglobin. They have a different
hemoglobin than adults do. They have something
called fetal hemoglobin. Adults have
alpha-2 beta-2, meaning we have
two alpha subunits, two beta subunits and that makes
up four subunits which you saw. A fetus on the other hand
has two alpha sub units and twp slightly
different gamma sub units. So you’ve got
alpha-2, gamma-2. Those two gamma subunits give
the hemoglobin of a fetus a very, I shouldn’t say very, but a slightly different
property than mom’s. Do they have cooperativity? But they have a greater affinity
for oxygen than mom’s hemoglobin. They can literally take
oxygen away from mom. Talk about a little parasite. [class laughing] A little parasite sitting
there sucking my oxygen, right? How do they do it? The gamma subunits in
addition to having a slightly different structure
cause the hemoglobin that they’re in to
not have a doughnut. That little doughnut hole
where the 2,3-BPG fits doesn’t fit 2,3-BPG anymore. The fetal hemoglobin essentially
stays in the R state all the time. Essentially stays in
the R state all the time. Now, you say that’s great, so obviously it can
take oxygen away from mom and yes, it can,
and yes, it does. But we just saw how the T state
helped to release oxygen, right? Is the fetus starved for oxygen? What do you think? No? Okay, there’s a no, there’s a no. Nobody thinks yes? No, it’s not. Why? Why is it not starved for oxygen? Student: Higher net
oxygenation level? Ahern: Higher net
oxygenation level. It does have a higher
net oxygenation level, but it also has trouble releasing
oxygen so the answer is no. The answer is simpler
than you think. Connie? Student: I mean it
just sits there, right? Ahern: Okay, and
that’s the answer. It just sits there. It doesn’t have widely
varying oxygen needs. Mom goes and climbs the stairs, she needs more oxygen than when
she’s sitting around in a chair. All the fetus does is kick. [Class laughing] So it doesn’t have widely
varying oxygen needs. It needs a relatively
constant supply of oxygen and because it does
have a high oxygen carrying capacity as you noted, there’s enough release so that
it can satisfy those needs. If it had very diverse
and very challenging needs, then you betcha there
would be an issue. Other questions? I’m going through
this kinda quickly. Shannon? Student: So if you’re like a mom and maybe you have a very
low blood concentration, like iron concentration, would it be smart to
take supplements for that? Ahern: If you were a
mom and you had anemia, is that what you’re saying? Student: Yeah. Ahern: Would it be smart
to take, I don’t know, iron supplements or
something like that? Yeah, for people that are anemic, that can be a consideration
with mom but again, I’m not a physician but
I’d imagine that yes, they would use that. Yes? Student: So when the fetus is
born, it becomes…[inaudible] Ahern: Yeah, yeah. Ahern: Yeah, when
the fetus is born, it has got fetal hemoglobin. So that change over happens
in the first year or two where the gamma sub
units stop being made and the beta subunits
start being made. And so the fetus transitions
to adult hemoglobin fairly early in its life. But you’re exactly right, yeah. Student: Does it change based on
how active the baby [inaudible]? Ahern: Does it change based
on how active the baby is? I honestly don’t know the
answer to that question. I don’t know. As far as I know, it’s
simply a developmental thing. Your question is whether
it responds to environment and I don’t know
the answer to that. Well we’ve gone
through a lot I thought we should finish
with a song today. What do you guys think? We haven’t done a song in awhile. I have a cold so I think
it will be worse than usual so I want you to sing
really loud today. And by the way, I have an
idea I will do with my classes. If you guys sing loud, I assure you you’ll have an
extra credit question on the exam. But if I can’t hear you… Everybody ready? Okay, everybody sing. “Biochemistry, biochemistry, “I wish that I were wiser. “I feel I’m in way over my head. “I need a new advisor. “My courses really shook me. “Such metabolic misery,
biochemistry, biochemistry. “I wish that I were wiser. “Biochemistry, biochemistry,
reactions make me shiver. “They’re in my heart
and in my lungs.” “They’re even in my liver. “I promise I will not complain. “If I could store
them in my brain. “Biochemistry, biochemistry,
I wish that I were wiser. “Biochemistry, biochemistry,
I truly am in a panic. “The mechanisms murder me. “I should’ve learned organic. “For all I have to memorize,
I outta win a Nobel prize. “Biochemistry, biochemistry,
I wish that I were wiser.” Alright, guys. See you Friday. [END]

23 thoughts on “#08 Biochemistry Hemoglobin Lecture for Kevin Ahern’s BB 450/550”

  1. Good question. The state of saturation has nothing to do with affinity of the T state. T states typically have structural features that either preclude binding of target molecules to desired sites or have structures within them that favor release of bound molecules. It is the latter case for hemoglobin.

  2. i'm a microbiology student going to give a presentation on Hemoglobin this week, though i found  your lectures very very useful but still i'm doubting myself cos i'm not biochemist student.

  3. Love the song at the end! 47:20 Wish my biochemistry professor is that cool too! Thanks for helping me 🙂

  4. hey ! thank you for the lecture, it helped me to understand so much better.
    i have a question concerning the structure of hemoglobin… if the proximal histidine would be changed in another amino acid will it damage the coopertavity of the protein ? 

  5. hello, you really helped me understand many concepts in biochemistry. i'm watching all your lectures to freshen up my knowledge before venturing to med school… I have a question sir… the cavity at the center of the Hb in T state favors binding for 2,3 BPG however when Hb is in the R state the hole shrinks… if the Hb going to tissues w/ low concentration of O2 are in an R state how will 2,3 BPG fit to it and convert the Hb in R state to T state? thank you :]

  6. I have a small question that might seem stupid. Since all the carbons in the heme group are sp2 hybridized because of the extended conjugate system shouldn't it be perfectly planar?  Does the puckering arise because of the iron alone? I did not quite understand the 0.4 A.

  7. Co operativity is the change in quartrnary structure or something else…and 2'3 BPG is the negative co oprativity inducer?..so co operativity is slower then its negative…and can myoglobin supress hypoxia and ultimately erythropoitin??

  8. I want to earnestly thank you for these lectures Professor Ahern. I admit I don't have a great proclivity for biochemistry, but the way you teach the subject is so fascinating. I wish I had greater exposure to professors such as yourself who teach with so much enthusiasm and passion – truly captivating.

  9. thank you for these amazing lectures. Never has anyone been able to make biochem as interesting as you do. I also wonder why universities pay professors who can't teach.

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