9 Polyprotic Acid-Base Equilibria

PROTEINS ARE POLYPROTIC ACIDS AND BASES

60
20
50 COO–
COO–

CH2 CH2
30
70 40 Heme CH2 CH2
H
group C
C C
110 H3C C C CH3
C C
10
C N N C
HC Fe CH
90 153
130 C N N C
H2C
C C CH3
80 140 C C C
150 C
H C C
1
H
CH3 C
H CH2
(c) Space-filling model
(a) Myoglobin backbone (b) Heme structure of myoglobin

(a) Amino acid backbone of the protein
myoglobin, which stores oxygen in muscle
tissue. Substituents (R groups from Table 9-1)
are omitted for clarity. The flat heme group at
the right side of the protein contains an iron
atom that can bind O2, CO, and other small
molecules. [From M. F. Perutz, “The Hemoglobin
Molecule.” Copyright © 1964 by Scientific
American, Inc.] (b) Structure of heme. (c) Space-
filling model of myoglobin, with charged acidic
and basic amino acids in dark color and
hydrophobic (nonpolar, water-repelling) amino
acids in light color. White amino acids are
Proteins perform biological functions such as structural support, catalysis of chemical reac-
tions, immune response to foreign substances, transport of molecules across membranes, and
control of genetic expression. The three-dimensional structure and function of a protein is
determined by the sequence of amino acids from which the protein is made. The diagram below
shows how amino acids are connected to make a polypeptide. Of the 20 common amino acids,
three have basic substituents and four have acidic substituents. Myoglobin, shown above,
folds into several helical (spiral) regions that control access of oxygen and other small mole-
cules to the heme group, whose function is to store O2 in muscle cells. Of the 153 amino acids
in sperm-whale myoglobin, 35 have basic side groups and 23 are acidic.
H H H
  
ç

CO2
ç

hydrophilic (polar, water-loving), but not H3N C CO2  H3N C  H3N çC CO2 mino acids
charged. The surface of this water-soluble
ç
ç

protein is dominated by charged and R1 R2 R3

hydrophilic groups. [From J. M. Berg, J. L. A side group
(also called a 2H2O
Tymoczko, and L. Stryer, Biochemistry, 5th ed.
substituent)
(New York: Freeman, 2002).]
H O H O H
 ¡ ¡ A polypeptide
ç

H3N C C N C C N C CO2 (A long polypeptide

is called a protein.)
ç
ç

R1 H R2 H R3
N-terminal C-terminal
residue Peptide residue
bond

P olyprotic acids and bases are those that can donate or accept more than one proton. After
we have studied diprotic systems (with two acidic or basic sites), the extension to three
or more acidic sites is straightforward. Then we step back and take a qualitative look at the
big picture and think about which species are dominant at any given pH.

Proteins are Polyprotic Acids and Bases 185

 9-1 Diprotic Acids and Bases
Amino acids from which proteins are built have an acidic carboxylic acid group, a basic
amino group, and a variable substituent designated R. The carboxyl group is a stronger acid
than the ammonium group, so the nonionized form rearranges spontaneously to the zwitterion,
which has both positive and negative sites:

A zwitterion is a molecule with both positive Amino group H2N H3N Ammonium group
and negative charges.
CH R CH R
O
Carboxylic acid HO C C Carboxyl group

O O
Zwitterion

pKa values of amino acids in living cells are
somewhat different from those in Table 9-1
because physiologic temperature is not 25ⴗC
and the ionic strength is not 0.
At low pH, both the ammonium group and the carboxyl group are protonated. At high pH,
neither is protonated. Acid dissociation constants of amino acids are listed in Table 9-1, where
each compound is drawn in its fully protonated form.
Zwitterions are stabilized in solution by interactions of ONH3 and OCO2 with water.
The zwitterion is also the stable form of the amino acid in the solid state, where hydrogen
bonding from ONH3 to OCO2 of neighboring molecules occurs. In the gas phase, there are
no neighbors to stabilize the charges, so the nonionized structure in Figure 9-1, with intra-
molecular hydrogen bonding from ONH2 to a carboxyl oxygen, predominates.
Our discussion will focus on the amino acid leucine, designated HL.

The substituent R in leucine is an isobutyl  pKa1  2.328  pKa2  9.744

group: (CH3)2CHCH2O H3NCHCO2H #£££££4 H3NCHCO 
2 #£££££4 H2NCHCO2
Leucine
H2L HL L
The equilibrium constants refer to the following reactions:

We customarily omit the subscript “a” in Ka1 Diprotic acid: H2L Δ HL  H Ka1 ⬅ K1 (9-1)
and Ka2. We will always write the subscript “b”  
HL Δ L H Ka2 ⬅ K2 (9-2)
in Kb1 and Kb2.
 
Diprotic base: L  H2O Δ HL  OH Kb1 (9-3)
 
HL  H2O Δ H2L  OH Kb2 (9-4)

Recall that the relations between the acid and base equilibrium constants are

Ka1 ⴢ Kb2  Kw (9-5)

Relations between
270 pm Ka and Kb:
Ka2 ⴢ Kb1  Kw (9-6)

288 pm
FIGURE 9-1 Gas-phase structure of alanine,
determined by microwave spectroscopy. [From
S. Blanco, A. Lesarri, J. C. López, and J. L. Alonso, “The
Gas-Phase Structure of Alanine,” J. Am. Chem. Soc.
2004, 126, 11675.]
We now set out to calculate the pH and composition of individual solutions of 0.050 0 M
H2L, 0.050 0 M HL, and 0.050 0 M L. Our methods are general. They do not depend on
the charge type of the acids and bases. That is, we would use the same procedure to find the
pH of the diprotic H2A, where A is anything, or H2L, where HL is leucine.
The Acidic Form, H2L
Leucine hydrochloride contains the protonated species, H2L, which can dissociate twice
(Reactions 9-1 and 9-2). Because K1  4.70  103, H2L is a weak acid. HL is an even
weaker acid, because K2  1.80  1010. It appears that the H2L will dissociate only partly,
and the resulting HL will hardly dissociate at all. For this reason, we make the (superb)
approximation that a solution of H2L behaves as a monoprotic acid, with Ka  K1.
With this approximation, finding the pH of 0.050 0 M H2L is easy.

 Ka  Ka1  K1 

H2Lⴙ can be treated as monoprotic, with H3NCHCO2H #£££££4 H3NCHCO2  H
Ka = Ka1. H2L HL H
0.050 0  x x x

186 CHAPTER 9 Polyprotic Acid-Base Equilibria

 Ka  K1  4.70  103
2
x
 Ka 1 x  1.32  102 M Solve for x with the quadratic equation.
Fx
[HL]  x  1.32  102 M
[H ]  x  1.32  102 M 1 pH  1.88
[H2L ]  F  x  3.68  102 M

TABLE 9-1 Acid dissociation constants of amino acids

Carboxylic acidb Ammoniumb Substituentb Formula

a a
Amino acid Substituent pKa pKa pKa mass
Alanine (A) ¬CH3 2.344 9.868 89.09

NH2
•
Arginine (R) ß CH
C 2CH2CH2NHC ç 1.823 8.991 (12.1c) 174.20
NH2
O
¡
Asparagine (N) ß CH2CNH2 2.16c 8.73c 132.12
Aspartic acid (D) ¬CH2CO2H 1.990 10.002 3.900 133.10
Cysteine (C) ¬CH2SH (1.7) 10.74 8.36 121.16
Glutamic acid (E) ¬CH2CH2CO2H 2.16 9.96 4.30 147.13
O
¡
Glutamine (Q) ß CH2CH2CNH2 2.19c 9.00c 146.15
Glycine (G) ¬H 2.350 9.778 75.07

NH
Histidine (H) ß CH2 (1.6) 9.28 5.97 155.16
N
H
Isoleucine (I) ¬CH(CH3 )(CH2CH3 ) 2.318 9.758 131.17
Leucine (L) ¬CH2CH(CH3 )2 2.328 9.744 131.17
Lysine (K) ¬CH2CH2CH2CH2NH 3 (1.77) 9.07 10.82 146.19
Methionine (M) ¬CH2CH2SCH3 2.18c 9.08c 149.21

Phenylalanine (F) ß CH2 ß 2.20 9.31 165.19


H2N Structure
Proline (P) ∂ of entire 1.952 10.640 115.13
HO2C amino acid

Serine (S) ¬CH2OH 2.187 9.209 105.09

Threonine (T) ¬CH(CH3 )(OH) 2.088 9.100 119.12
ß CH2
∂

å
Tryptophan (W) 2.37c 9.33c 204.23
N
H
Tyrosine (Y) ß CH2ß ß OH 2.41c 8.67c 11.01c 181.19
Valine (V) ¬CH(CH3 )2 2.286 9.719 117.15

a. The acidic protons are shown in bold type. Each amino acid is written in its fully protonated form. Standard abbreviations are shown in parentheses.
b. pKa values refer to 25 C and zero ionic strength unless marked by c. Values considered to be uncertain are enclosed in parentheses. Appendix G gives pKa for ␮  0.1 M.
c. For these entries, the ionic strength is 0.1 M, and the constant refers to a product of concentrations instead of activities.
SOURCE: A. E. Martell and R. J. Motekaitis, NIST Database 46 (Gaithersburg, MD: National Institute of Standards and Technology, 2001).

9-1 Diprotic Acids and Bases 187


Hydrolysis is the reaction of anything with
water. Specifically, the reaction Lⴚ ⴙ H2O Δ
HL ⴙ OHⴚ is called hydrolysis.
Lⴚ can be treated as monobasic, with Kb = Kb1.
188
What is the concentration of L in the solution? We have already assumed that it is very
small, but it cannot be 0. We can calculate [L] from the K2 equation, with the concentrations
of HL and H that we just computed.
[H ][L ] K2[HL]
K2  1 [L ]  (9-7)
[HL] [H ]
(1.80  1010 )(1.32  102 )
[L ]   1.80  1010 M ( K2 )
(1.32  102 )
The approximation [H] ⬇ [HL] reduces Equation 9-7 to [L]  K2.
Our approximation is confirmed by this last result. The concentration of L is about eight
orders of magnitude smaller than that of HL. The dissociation of HL is indeed negligible
relative to the dissociation of H2L. For most diprotic acids, K1 is sufficiently larger than K2
for this approximation to be valid. Even if K2 were just 10 times less than K1, [H] calculated
by ignoring the second ionization would be in error by only 4%. The error in pH would be
only 0.01 pH unit. In summary, a solution of a diprotic acid behaves like a solution of a
monoprotic acid, with Ka  K1.
Dissolved carbon dioxide is one of the most important diprotic acids in Earth’s ecosystem.
Box 9-1 describes imminent danger to the entire ocean food chain as a result of increasing
atmospheric CO2 dissolving in the oceans. Reaction A in Box 9-1 lowers the concentration of
CO23 in the oceans. As a result, CaCO3 shells and skeletons of creatures at the bottom of the
food chain will dissolve by Reaction B. This effect is far more certain than the effects of
atmospheric CO2 on Earth’s climate.
The Basic Form, L
The species L, found in a salt such as sodium leucinate, can be prepared by treating leucine
(HL) with an equimolar quantity of NaOH. Dissolving sodium leucinate in water gives a
solution of L, the fully basic species. Kb values for this dibasic anion are
L  H2O Δ HL  OH Kb1  Kw/Ka2  5.55  105
HL  H2O Δ H2L  OH Kb2  Kw/Ka1  2.13  1012
Kb1 tells us that L will not hydrolyze (react with water) very much to give HL. Furthermore,
Kb2 tells us that the resulting HL is such a weak base that hardly any further reaction to make
H2L will occur.
We therefore treat L as a monobasic species, with Kb  Kb1. The results of this (fantas-
tic) approximation are outlined as follows:
Kb  Kb1 
 
H2NCHCO
2  H2O #£££££4 H3NCHCO2  OH
L HL OH
0.050 0  x x x
Kw
Kb  Kb1   5.55  105
Ka2
x2
 5.55  105 1 x  1.64  103 M
Fx
[HL]  x  1.64  103 M
[H]  Kw /[OH]  Kw/x  6.11  1012 M 1 pH  11.21
[L]  F – x  4.84  102 M
The concentration of H2L can be found from the Kb2 (or Ka1) equilibrium.
[H2L  ][OH  ] [H2L]x
Kb2    [H2L]
[HL] x
We find that [H2L]  Kb2  2.13  1012 M, and the approximation that [H2L] is insignif-
icant relative to [HL] is well justified. In summary, if there is any reasonable separation
between Ka1 and Ka2 (and, therefore, between Kb1 and Kb2), the fully basic form of a diprotic
acid can be treated as monobasic, with Kb  Kb1.
CHAPTER 9 Polyprotic Acid-Base Equilibria