Lecture 27

Amino Acids

In the 1820's it was discovered that heating cellulose in acid caused it to be broken down into smaller constituents. This was the "discovery" of hydrolysis. The French scientist, Henri Braconnot, tried the same treatment on gelatin. He boiled it for days in acid and managed to isolate a white crystalline substance he named glycine. This was the first amino acid to be isolated and characterized. At pH=7.0, it has the structure shown below.

It is called an amino acid for fairly obvious reasons. It has both a carboxylic acid function as well as an amine function.

All amino acids produced by living systems are called a amino acids. The carbon atom bonded to the carboxylic acid carbon is called the a carbon, Ca. If the amine residue is bonded to the Ca, then it is an a-amino acid.

All amino acids from proteins are L-enantiomers. In a Fischer projection, the most oxidized carbon is put at the top. If the amine residue is on the left, it is the L-enantiomer.

In general, the structure of amino acids is:

where R can be H, in the case of glycine, an alkyl group, or a heterocyclic ring (a cyclic group with non carbon members). All amino acids, except glycine, will be chiral compounds, because the Ca is bonded to four different substituents, The R group, the carboxyl group, the amine group and hydrogen.

Physical properties

Amino acids are crystalline solids, with very high melting points, in excess of 200o C. They are very soluble in water. All of this suggests that they exist as ionic species. At neutral pH values, they exist as zwitterions, with the carboxyl group deprotonated and the amine function protonated.

The acid/base chemistry of amino acids is summarized below:

At low pH, the amino acid is protonated at both the amine and carboxyl functions. At this pH it carries a net positive charge and can be treated as a diprotic acid, an acid with two pKa's.

At high pH, both the carboxyl and amine groups are deprotonated. At these pH values, the amino acid carries a net negative charge, and is dibasic.

At some intermediate pH, the amino acid is a zwitterions, and carries no net charge. This is called the isoelectric point of the amino acids, and is designated pHI.

At this pH value, the amino acid will be stationary in an electric field. At low pH, the amino acid carries a positive charge and will migrate to the cathode. At high pH, the negatively charged amino acid will migrate to the anode. This is the procedure used to analyze and purify amino acids and proteins. It is called electrophoresis. A solution of amino acids or proteins are placed at a given pH on a cellulose support clamped to a power source. The amino acids then migrate along the cellulose according to their pHI and the strength of the electrical field.

Titrations of amino acids

Let's titrate a solution of the amino acid, alanine, with R = CH3.

The pKa values of the two acidic groups, COOH and NH3+, are 2.34 and 9.69, respectively. These values are typical of pKa's of the carboxy and amine groups on the a carbon. We begin our titration at a low enough pH (below 2.0) to insure that the amino acid is fully protonated. We will titrate the alanine solution with the strong base, NaOH. The titration curve is shown below. Notice that it has two "waves" since we are titrating a diprotic acid.

As we add OH, we begin to deprotonate the stronger of the two acids, the carboxyl group, the group with the lower pKa. When we have deprotonated half of the COOH, pH = pKa = 2.34. This is because the Henderson-Hasselbalch equation reduces to this when [A-] = [HA]. This is the half-way point on the titration curve for this functional group, the COOH.

At the beginning of the titration, the amino acid carried a formal charge of +1, as noted at the top of the titration curve. When we halve added one equivalent of OH- , one mole of base for every mole of alanine, we have fully deprotonated the COOH. This is the first equivalence point in this titration, when all of the lower pKa acid has been neutralized. The pH at which this occurs can be calculated as approximately the average of the pKa's of the COOH and NH3+, (2.34 + 9.69)/2 = 6. At this pH, the alanine will carry no net charge. The carboxyl groups will carry a negative charge but the amine group will still be fully protonated and carry a positive charge. This is the isoelectric point of alanine.

Further addition of OH- begins to deprotonate the second acidic function, the NH3+.When half of the amine is deprotonated, we have reached the half-way point for the second acidic species. Again, pH = pKa which for the amine is 9.69. This is the second buffered region of the titration curve, with a relatively flat slope due to the presence of a weak acid (NH3+) and its salt (NH2)

An isoelectric point near pH - 6.0 is typical for amino acids that don't have acidic or basic R groups.

What about the amino acid, glutamic acid, with R = C2H4COOH?

pKa1 = 3.20 (a-COOH)

pKa2 = 4,25 (R - COOH)

pKa3 = 9.67 (a-NH3+)

It will undergo the following series of acid/base reactions:

Notice that at low pH, below pH = 2.0, glutamic acid carries a net positive charge. After the addition of one equivalent of base, the lowest pKa group will have been deprotonated, giving no net charge on a glutamic acid. The half-way point of this first wave of the titration will be at pH = pK1 = 3.2 and the pH of the first equivalence point will occur at about pH = (3.2 + 4.25)/2 = 3.7, the average of the first two pka's. This is also the isoelectric point of glutamic acid, the pHI. Addition of more OH- begins to remove the second acidic proton, from the COOH of the R-group. Shown below is the titration curve for glutamic acid with the strong base, NaOH.

At physiological pH (7.0), this amino acid carries a formal negative charge. It's isoelectric point is at pH = 3.7.

If we had looked at the titration of an amino acid with a basic R group, we would have found that the isoelectric point came later in the titration. Shown below is a summary of the acid/base chemistry of a basic amino acid, one having only one COOH group, on the Ca, but two NH3+ groups, one on Ca and the other on the R-group. This amino acid is lysine with pKa1 = 2.18 (a-COOH), pKa2 = 8.95 (a-NH3) and pKa3 = 10.53 (R-NH3). .

At low pH basic amino acids carry a net charge of +2, which then decreases as the groups are deprotonated. For the example above, the isoelectric point will occur upon addition of two equivalents of base, at an approximate pH of 9.74, (8.95 + 10.53)/2.

Every amino acid has a characteristic isoelectric point. Proteins are made of amino acids, so they also have isoelectric points, the sum of all of those of their component amino acids. As proteins carry no net forma charge at their isoelectric point, the become less soluble in aqueous solutions and tend to precipitate. With no net charge, the protein molecules are no longer electrostatically repelled. This is illustrated well in the cheese making process. Casein is a milk protein with a pHI = 4.7. Milk normally has a pH of about 6.3. Bacteria added to milk produce lactic acid, which lowers the pH. As the pH approaches 4.7, the casein molecules lose their net negative charge and precipitate, making curds.

There are 20 amino acids of biological importance. They are classified according to the nature of their R groups. One group has non-polar R groups, largely alkyl groups. These amino acids have isoelectric points near 6.0, as we saw with alanine.

A second class of amino acids has polar side chains, R groups containing dipole moments.

The third and fourth classes of amino acids are those with acidic or basic R-groups. These are all summarized on pages 186 and 187 of your textbook and pages 94-96 of the Syllabus.