Biochemistry Acid/Base Behavior of Amino Acids

Hello and welcome back to Educator.com, and welcome back to Biochemistry.0000

Today, we're going to continue our discussion of amino acids, and we're going to talk about the acid-base behavior of amino acids.0004

This is profoundly important.0012

If you understand this, then you'll pretty much understand all of protein behavior; so let's just jump in, and see if we can completely wrap our minds around this thing.0015

OK.0028

Amino acids have two groups that are ionizable.0030

Let me go ahead and do blue here.0035

Amino acids have two groups that are ionizable.0039

OK.0055

What we mean by that is the following: that is two groups that can release and/or accept hydrogen ion depending on what the pH is.0057

In other words, an amino acid is a diprotic weak acid.0088

It has one H that it can give up, and it has another H that it can give up under different pH conditions.0093

Maybe it has already given them up, and maybe this time it will act as a weak base, and will actually take the protons- that is all this means.0100

When we say it has two groups that are ionizable, that means it has two groups that can release or accept the proton depending on what the pH happens to be.0106

In other words, it's a diprotic weak acid, weak base.0115

OK, so let's take a look at alanine.0118

Let's look at an example.0120

Let's look at alanine.0122

OK.0128

And again, the structure of alanine is: we have that, we have that, we have our amino group, and we have CH₃.0129

However, I'm going to write it, fully protonated form, COOH.0141

In fact, I'm going to actually draw out the carboxyl groups so we know exactly what we're looking at - COOOH - that's it.0147

This is our first ionizable group - that's one hydrogen that can go away - and here we have our second ionizable group, so every amino acid has at least two ionizable groups.0156

One of this Hs can go away- that's all this means.0166

OK.0171

Let's follow the titration of an acidic solution of alanine with a strong base.0172

This is going to be your standard weak acid strong base titration of a weak acid with a strong base, and see what happens as we raise the pH because that's what we're doing.0200

When we're adding base to a solution, we're raising the pH; and what we're going to be doing is, we're going to be pulling off these hydrogens one at a time as the pH rises.0225

OK.0236

Again, alanine as all amino acids - I'll just write AA - are diprotic weak acids just like carbonic acid H₂CO₃.0239

H₂CO₃ releases one hydrogen to become bicarbonate, releases another one to become carbonate.0261

Amino acids, one of the Hs is released from the carboxylic acid group, another H is released from the NH₃⁺ group- that's all that's going on here.0266

Let me see how it is that I actually wrote that weak diprotic acids, so I'm going to write it like this, then I'm going to go back to blue.0279

I'm going to use the three-letter designation, and I'm going to write the carboxyl group, and I’m going to write the NH₃⁺ group.0289

This includes the particular R-group, so this is what we're looking at right here.0297

That's one ionizable group; that's another ionizable group.0303

Let's go ahead and see.0307

Let me go to the next page.0310

Let me redraw this so we have it on the same page: COOH, and we have NH₃⁺- there.0311

OK.0323

Now, the carboxyl group, this one right here, the COOH group, is the stronger acid of the two groups.0325

It's the stronger acidic group of the two, and what that means is, it loses its hydrogen ion first; so it loses its H⁺ first- that's it - and to become COO^-, right?0342

When it loses its hydrogen ion the negative charge stays back, so it becomes COO^-.0376

OK.0383

The NH₃⁺ group loses its H⁺ second to become NH₂, so when it loses its hydrogen ion, it's taking its plus charge with it, and it leaves this one uncharged.0385

OK.0407

Now, let's go ahead and follow the titration here, so let me draw this out; and I want to make sure that I leave room at the bottom to draw a structure.0408

This axis is going to be the pH, and this axis is going to be just milliliters of OH^-.0421

This is milliliters of OH^-, like I'm an adding a sodium hydroxide solution or something; it’s just a volume as I add.0432

Here is what its look like.0439

I'm going to go ahead and go, something like that.0441

Here is what's happening.0451

OK.0453

I'm going to mark off a couple of points.0454

I'm going to mark an X there.0457

I'm going to mark an X there, and I'm going to mark an X there.0458

Now, I'm going to go to red- there is that, there is that, there is that, this is pK_a1.0461

Here is what happens: we're going to be starting at a low pH, an acidic solution of alanine.0472

This H is attached, and this H is attached, so what we have is this particular molecule.0479

As I add hydroxide, add hydroxide, add hydroxide - well, you remember a weak acid, all of a sudden, what's going to start happening is that base is going to start pulling off this hydrogen ion from the carboxylic acid part of the amino acid, and it’s going to leave carboxylate, well, remember what we said the pK_a was? - the pK_a of a given weak acid of a given group, it is the pH at which the acid form and the base form are in equal concentration.0486

So, I’m just going to write a couple of things down, but I'm going to write the reaction underneath, and it will all come together.0522

The pK_a1 for alanine is 2.34.0528

Well, so that's the pK_a of that group.0534

This one up here, I'll call it pK_a2, that is the pK_a of the NH₃⁺ group; and it happens to be 9.69 for alanine.0546

Now, I’m going to write something here called PI = 6.01, and here's the reaction that's taking place.0561

I’m going to write: ALA, COOH, NH₃⁺.0571

OK.0586

This is going to be ALA, COO^-, NH₃⁺, and then we have ALA, COO^-, and we have NH₂.0588

This is a +1 charge; this is a 0 charge, and this is a -1 charge.0608

Here's what's happening: as we proceed with the titration, we start with this form, this form right here, OK; the OH is protonated, and the NH₃ is protonated, we add base, we add base, we add base, were going to pull off some of this hydrogen.0621

Well, at a pH of 2.34, there is an equal amount of this form, and this form, the form with the H pulled off.0634

This is the pK₁- that's this one.0643

Now, that's this right here.0650

This the first buffering region.0653

Remember, where it's flat, that's the buffering region.0657

That is when you have the base form and the acidic form.0658

In other words, the deprotonated form and the protonated form in a concentration that allows you to actually buffer.0662

It resists changes in pH, that's why it looks like this.0671

We are actually adding a hydroxide, but the hydroxide is being eaten up by this H.0675

This H is neutralizing the OH that is added, that is why the pH isn't changing, but at a certain point, this H, all of a sudden, there is no more H for the hydroxide that we add to eat up; so it actually jumps up, the pH jumps up.0680

Now, at this point, it's all in this form: negative charge on the carboxylate, positive charge here, there is a zero total charge.0695

So here, this molecule is positively charged, this is positive 1.0706

At this point it is that, and I'll talk about what the PI means in a minute.0710

I stands for isoelectric point.0714

Isoelectric means there is a zero charge- equal electricity.0715

OK, now, but notice this hydrogen is still attached.0721

I'm going to keep adding hydroxide, now, what’s going to happen is now, the hydroxy is going to pull off this hydrogen; so now, it's going to be converted to this, or this hydrogen is gone, now, this hydrogen is gone.0725

Well, during that, we have our second buffer region.0738

Now, it's the amino group that's acting as a buffer, and again, it's buffers well between about 8.69 to 10.69, here, buffers well from 1.34 to 3.34.0742

That’s the buffering region- one unit above and below the pK_a;one unit above or below the pK_a.0756

The reaction that's taking place is this: I'd begin with a fully protonated form, I add hydroxide, I pull off the first hydrogen, I pull it all the way off, now, I am here, now, I start pulling off the second hydrogen from the amino group, and I get to a point once I've pulled off everything, now I'm over here, now, my molecule, my amino acid has a negative 1 charge- that's all that's going on when we titrate an amino acid.0764

One hydrogen to give up, second hydrogen to give up; two buffering regions, two ionizable groups.0794

OK.0803

Now, when an amino acid exists as follows: when it exists like this C, COO^-, NH₃⁺, H, and R, I'd switched the NH₃ and the R, in this particular case, this is not a Fischer projection, I wanted to see, I wanted it to be - well, you know what, actually I don’t need to do that, why don't I just stick with what we've done.0805

We have our R-group down here, and we have our NH₃⁺ like that.0857

When it has a zero charge, when it exists in this form, it's called the zwitterion- that's it that's the name for it.0863

When the carboxyl group has been ionized but the amino group has not been ionized, negative charge, positive charge, the total molecule has a zero charge- it’s a zwitterion.0872

14:45OK.0884

In this form, the COO^- group, it can, if it has to, it can accept a proton, it can accept a hydrogen ion, so the amino acid can behave as a base.0885

The amino acid, as a whole, can behave as a base because there is a group that can accept the hydrogen ion- that carboxylate group.0920

Now, the NH₃⁺ group can give up a hydrogen ion, so the amino acid can also behave as an acid if it has to.0933

In other words, it can be both an acid or a base depending on the pH, depending on the conditions at the time, the condition surrounding the amino acid.0967

OK.0976

It is amphoteric.0979

An amphoteric substance is something that can behave as both an acid and a base depending on the environment.0981

Amphoteric is the adjective or ampholyte is the noun, so an amino acid is an ampholyte- it is an amphoteric substance.0987

OK.1003

Now, let me go to red.1004

The pH at which an amino acid is a zwitterion is called is called the PI; it’s called the isoelectric point.1008

When the first hydrogen from the carboxyl group has been completely pulled away, but none of the hydrogens from the amino group had been pulled away, the total charge on the amino acid is 0, -1, +1, they cancel the zero- it is a zwitterion.1037

The pH at which that happens, that's called the PI- the isoelectric point.1055

In the case of amino acids that have two ionizable groups, the PI is just the arithmetic mean between the two pK_as- the pK_a for the carboxyl group, the pK_a1, and the pK_a2, which is the pK_a for the amino group.1061

You just add them together, divide by two, and you'll get your PI.1078

OK.1083

Now, notice how PI for alanine is 6.01.1087

Now, PIs for most amino acids or most amino acids will be in this range.1106

Now, you understand why we wrote amino acids the way that we did with the COO^-, but the NH₃⁺.1124

This is why at pH equal to about 7, we wrote our amino acids as C, H, COO^-, NH₃⁺ and R because at normal physiological pH, somewhere in the neighborhood of about 7 to 7.4 amino acids, they exist as zwitterions.1134

So, free amino acids exist in this form under normal physiological conditions.1183

OK.1188

Now, I hope that made sense.1189

You have this amino acid, it has a carboxylic acid group, it has an amino group that's protonated under conditions of low pH.1193

Both of the groups are protonated under conditions of really, really high pH.1203

Both of them are deprotonated somewhere in the middle, which is normal physiological pH.1207

The COOH group is deprotonated, but the NH₃⁺ group is still protonated that carries positive charge to the COO^- carries the negative charge.1212

Your total amino acid is zero charge zwitterion it can act as acid or base.1222

it can go both ways depending on what needs to happen in that particular environment.1226

That's what makes amino acids so incredibly powerful.1232

OK.1236

Now, some amino acids have three ionizable groups, and I'll go ahead and list them.1237

They are tyrosine, cysteine, lysine, histidine, arginine, aspartate and glutamate.1254

These amino acids have three ionizable groups because their R-group also contains something that can release or accept a proton.1281

Now, for these amino acids, you have 3 pK_as.1290

We call them pK₁ for the carboxylic acid group, pK₂ for the amino group, and pK_R, we can call it pK₃, pK_R.1294

They say pK_R because it happens to be the group that's attached to the R-group.1305

It can be either carboxylate or it can be an amino.1310

OK.1314

Now, let's see what we've got.1316

Now, as always, the COOH that's attached to the alpha carbon, attached to alpha C, always ionizes first; so that doesn't change.1318

The pK₁ always refers to the, in other words, you have an amino acid.1347

OK.1361

That particular H is the one that always ionizes first.1363

Now, well, for NH₃⁺ and the particular R-group, it's a toss-up.1366

Sometimes the NH₃ will ionize second, and then sometimes the R-group will ionize second; and then the NH₃ as the pH is rising, so sometimes this will have a lower pK_a than that one, ionizes first, sometimes this R-group will have a lower pK_a than this group.1378

It means it ionizes first; it loses a proton first- it's just depends.1397

OK.1404

Sometimes, one or the other will ionize first, will lose its proton first.1410

A titration curve for a triprotic amino acid on that list is going to end up having three plateaus.1424

It's going to look something like this; in general, it's going to look something like this, something like that: pK_a1, pK₂ or pK_R, depending on which one is first, and pK₃ and somewhere in here you're going to have your PI.1434

Now, you can't just add them and divide by three in this case.1452

We have to experimentally determine what the isoelectric point is for these, but that's not a big deal.1455

And again, if you look in your book, you will actually see a list- all of the amino acids.1461

It will list their three letter designation, single letter designation.1468

It will give you the molar mass.1471

It will give you pK₁, pK₂, pK_R, and then it will give you the PI and maybe some other information too.1472

OK.1480

Let's go ahead and do an example here.1481

I think this is probably the best way to do it.1483

Let me go ahead and do it on the next page.1485

So, example, we're going to take a look at aspartate.1488

In the case of aspartate, our pK₁ is less than our pK_R, is less than our pK₂; so in this particular case, the R-group, the carboxylic acid ionizes first, releases its hydrogen, then the R-group will release its hydrogen, then the amino group the alpha-amino group will release its hydrogen as we titrate.1495

I'm going to draw out the reactions; I'm not going to do the titration curve.1519

I’m going to write out the reaction- that's what's important.1521

We want to get the structures correct: H, NH₃⁺, we have CH₂ ,and we have COOH.1526

OK.1540

We have that one, and it's going to be H₃N⁺.1542

And again, I’m hoping that you're actually confirming all of this because there is a whole bunch of structures going on, so I might miss an H, I might miss a C, I might miss an N.1547

I hope you are confirming this.1557

OK.1560

This is C, this is COO^-, and this is CH₂, and this is going to be COOH, so this is pK₁.1562

OK.1572

This group right here loses first.1573

Now, our second ionization is going to be pK_R, so this H is going to go next1576

What we have is NH₃⁺, alpha carbon, COO^-, H, we have CH₂, and we have COO^-.1584

Now, we have our final equilibrium which is going to be pK₂, which is going to be the amino group, and we are going to end up with a C, a COO^-, an H, a COO^-; and then will going to have an NH₂ neutral.1597

Notice, it went from plus to neutral, because it gave up an H; that's what's happening her- an H is being lost in each case.1619

In terms of the biochemical, an H⁺ is leaving - actually you know what I should do it on the upper arrow, not the lower arrow, the lower arrow is the one that is coming in - so, H⁺ is going away, a second H⁺ is going away, a second H⁺ is going away.1631

This is pretty typical biochemical nomenclature.1660

They actually show things coming in and going out of a reaction on the arrows, but again we'll talk a little bit more about that.1663

This is pK_R, now, let's do some numbers: pK₁ = 1.88, the pK_R = 3.65, the pK₂ = 9.60, and its isoelectric point happens to be at 2.77.1669

So, at a pH of 2.77, it actually exists in this form- 0 net charge.1694

That's all that is going on here which makes sense because you're looking at 1.88 and 3.65, because each of this contributes a negative; the only positive charge comes from this thing right here, so this PI is going to be lower than you would expect.1707

Notice the PI of alanine was 6.01- this one is a lot lower.1722

OK.1727

Let's do another example.1731

This time we'll do an example where the alpha-amino group actually ionizes before the R-group does1734

Let's do tyrosine, which is actually kind of interesting in the case of tyrosine but...so pK₁ is less than pK₂ is less than the pK of the R-group, so tyrosine.1739

Let's go ahead and write these equilibriums.1756

We start off with COOH, everything is protonated, we have NH₃⁺, we have CH₂, we have our phenol group or benzene, then we have OH, so everything is protonated, everything is good.1760

Now, first hydrogen to go is that top hydrogen, the alpha carboxylic acid, the carboxylic acid attached to the alpha carbon.1780

We have H₃N⁺, C, COO^-, this is H, this is CH₂, this is that, and we have OH, that's our first, this is pK₁.1789

Now, for pK₂, this time it is the amino group that ionizes next, so it becomes H₂N, neutral C, we have COO^-, we have H, we have CH₂, we have our benzene group, and then we have our hydroxy attached to the benzene, which is still protonated.1808

That one has not been released yet.1829

And now, of course, we reach our final equilibrium, which is going to be pK of the R-group.1832

Now, the R-group is going to release its hydrogen.1838

We have C, we have COO^-, we have H, we have NH₂, we have CH₂, we have our benzene group, and we have O^-.1842

This is the equilibrium that takes place.1858

This H goes first to turn into that, then this H⁺ leaves to turn into that, then this H leaves to turn into that.1861

Our numbers are: our pK₁ is equal to 2.20, and, of course, all of these numbers are available in your book or on the web- wherever.1875

I would encourage you to take a look at a table showing this stuff just to get a sense of what the numbers are for all the amino acids on a single page on a list.1887

It's a great way to get a sense of general behavior because there are just going to be some numbers that are just going to stand out.1895

They are just going to be totally different than all the others, and you're going to take a look and see what amino acid that is, and chances are, that amino acid is going to play a special role when we talk about metabolism later in the course.1901

OK.1913

pK₂ = 9.11, and pK_R = 10.07 , and PI is equal to 5.66.1915

So, at a pH of about 5.66, the majority of the amino acid exits in a neutral state- that's it.1930

OK.1939

Now, I strongly urge you to do exactly what I've done: take a couple of amino acids at random, and then just write the equilibriums for them, see what the pK_a1 is, see what’s the pK_a2 is, see what’s the pK_R is, and then arrange them, plot the hydrogens according to the order of the pK_as, and draw these out.1942

It's an absolutely fantastic way to1, familiarize yourself with the structure of the amino acids and just being able to actively draw them out, and 2, getting a sense in keeping track of which hydrogen is being ionized and where it's being ionized- very, very important.1964

OK.1984

Now, let's talk about a rule of thumb.1986

OK.1993

If you want to know whether a given group - chemical group, not amino acid group - whether a given, I should say, ionizable group is protonated, which is the acid form or deprotonated.1998

In other words, whether it’s actually has its hydrogen ion or it's lost its hydrogen ion, which is called the base form at a given pH.2037

That's often how the problems are going to present themselves.2054

We're going to say there is this particular acid and the pH of the solution is 6.7, which one of the groups is protonated, and which one is not?2057

That's how it's going to be presented, and we will do an example in a minute.2064

Here's how you do it.2068

Here's the rule of thumb: if the pH of the solution is less than the pK_a of the group - and again, we're doing this for each individual group - then the group is protonated.2070

In other words, it exits in its acid form, and, of course, the other way around if the pH happens to be bigger than the pK_a; and remember, the pK_a is a constant.2096

These things exist for a given species for a given ionizable group in that species.2111

The pK_as don't change, pHs change.2116

Then, the group is deprotonated.2128

In other words, it exists as the base form.2133

OK.2140

Now, here's the bases for this particular rule of thumb.2142

You can either learn the rule of thumb, memorize it, or you can learn this basis, which I think, is better to know the basis and to know where it comes from, because that way, you can always reason things out.2146

Well, remember the Henderson-Hasslebalch equation?2158

OK.2160

Here's the basis for the rule; let me do this in red: the pH, we said of a solution, is equal to the pK_a of the acid plus the logarithm of the concentration of the base form, the unprotonated form, over the concentration of the acid form, the protonated form.2161

Now, let me rewrite that: pH = pK_a plus the log of the base concentration over the acid concentration.2193

Well, if the pH is less than the pK_a, if this is less than that, which is a constant, that means that this number right here, the log of B over A, is a negative number, because I have to go a certain number subtracted something to get a lower number, then log of B over A is negative - in other words, it's less than zero - so if the log of something is negative, that means that the denominator is bigger than the numerator.2206

In other words, the logarithm of a fraction is negative, the logarithm of the number bigger than one is positive.2256

If the log of B over A is negative, that means B over A is a fraction.2263

If it's a fraction, that means A is greater than B.2268

That means that the denominator is bigger than the numerator, meaning - and don't worry we’ll be doing an example in just a minute - meaning there is more A than B.2272

There is more acid form than base form.2307

There is more protonated form than non-protonated form.2309

That’s all that means.2316

OK.2318

Let's go ahead and finish off with a nice example her, see what we can do.2319

Let's go back to blue.2326

Example: the cysteine solution was prepared and buffered to a pH equal to 8.2.2332

I would like you to describe the degree of protonation for each ionizable group.2361

In other words, I'd like you to tell me does this amino acids exists in what form.2382

What's the total charge on it?2388

Which group is ionized; which group in not ionized?2390

That's what is asking.2393

OK.2394

Well, let's go ahead and take a look at - first of all this is biochemistry, it's chemistry, it's organic chemistry - draw a structure.2396

OK.2404

So, cysteine, let's go ahead and draw it out as, you want to draw out the fully protonated form first and then make your decision, NH₃⁺, this is an H, this is a CH₂, and cysteine is a SH.2406

OK.2427

Again, begin by protonating all of them.2429

In other words, that's protonated, that's protonated, that's protonated- we have three ionizable groups.2431

At pH of 8.2, which one is protonated, which one is not?2436

Well, let's see what we've got.2441

We look up the pKs.2443

Well, the pK₁ is equal to 1.96; the pK_R is equal to 8.18.2446

Notice, in this case, the R-group ionizes before this group does.2456

The pK₂ is equal to 10.28.2460

Well, now, we just use our rule of thumb or reason it out.2466

pH is bigger than pK₁, right?2476

We said that pH is 8.2, and we said the pK₁ was 1.96.2478

Because the pH is bigger than pK₁, that implies that the carboxylic acid group exists as a carboxylate group.2485

It's actually been ionized; it has lost its H.2502

OK.2506

The pH which is 8.2, in this particular case, it happens to equal the pK_R- that's interesting.2507

This is 8.18, the pH is 8.2, and they are exactly the same.2519

This implies that - how shall I...I'm just going to write the group - CH₂, SH, and the CH₂, S^-, they exist in equal concentrations.2525

In this case, the pH equals the pK_a of the R-group.2550

When pH equals the pK_a of the R-group, that means the acid form, the protonated form and the base form, the unprotonated form, exist in equal concentrations.2554

So, in this case, they're both like that- it's a little bit of this, a little bit of that, half and half exist in equal concentrations.2563

Now, the pH happens to be less than the pK₂.2575

Again, the pH is 8.2, and this is 10.28.2581

Well, this implies that the alpha amino group exists as its protonated form; the pH is less than the pK_a, so it has not ripped away this hydrogen; it's still H₃N⁺, so that's it.2587

Our final answer- we have C, COO^-, H, NH₃⁺, CH₂, and SH - just want to make sure if...yes - and C, COO^-, NH₃⁺, CH₂, S^-, H, so, this is our final answer.2613

The amino acid actually exists as an equal concentration of this thing and this thing.2649

OK.2658

This group is completely ionized; this group, the amino group is not ionized.2660

The SH group- half of it is ionized, half of it is not.2666

That's what’s going on here.2672

OK.2674

I hope that made sense, and this is strictly based on the rule of thumb.2675

Let me write and OK.2678

And again, it's based on comparison of pH and pK_a; compare pH and pK_a, compare pH and pK_a of each R-group- that's all that's going on here.2683

OK.2695

Now, let's say one thing about one of the amino acids, and then we will go ahead and close out this particular lesson.2698

Histidine is special.2710

When I look, I see a pK₁ of 1.82; I see a pK_R equal to 6.00, and I see a pK₂ equal to 9.17.2717

Notice.2737

OK.2742

If you take a look at a list of all of them, this is one of the numbers that will stand out- that one.2743

It is the only amino acid whose pK_R, who's pK_a of the R-group is close to physiological pH, is close to physio pH found in intracellular and extracellular of fluids- the fluid inside the cell, the fluid outside the cell, physiological pH.2751

This is the only amino acid whose pK_R is actually close to the physio pH.2800

OK.2805

It is, therefore, it is therefore, I should say -sorry about that , let me go ahead and erase that – so, it therefore has the potential to provide good buffering capacity under physiological conditions, under physio conditions.2808

That’s it.2868

Histidine is special because its R-group has a pK_a of 6.0, which is not that far from the 7.0 or 7.2.2869

As it turns out, it has the potential to actually be a pretty good buffer in that particular range.2881

As it turns out, that's exactly what it is going to do, so keep an eye out for histidine when we start talking about enzyme reactions and when we start talking about metabolism.2889

OK. That takes care of acid base behavior for amino acids.2900

Thank you for joining us here at educator.com and biochemistry.2904

We'll see you next time, bye-bye2907