Transcript Ionization of Biological Molecules

Ionization of Biological Molecules
This tutorial extends the concepts of acid-base chemistry by showing
that [H+], i.e., the pH of a solution, affects biological functions by regulating the
ionization of biological molecules. Ionization of organic molecules feeds into
many aspects of biological phenomena. Amino acids must have a neutral
charge in order to penetrate cell membranes. Protein structure is stabilized by
close proximity of positive and negative charges on neighboring amino acid side
chains. Nucleic acids derive their acid character from ionized phosphate
groups. Fatty acids acquire their property as detergents by ionization of
carboxyl groups. The concept of ionization is vast and encompasses all the
categories of biomolecules. In this tutorial we will focus on molecules whose
positive or negative character, indeed biochemical properties, depend on pH.
What is Ionization?
Ionization refers to creating positive and negative charges on molecules.
Negative charges arise by dissociating protons from groups bearing oxygen or sulfur
atoms (click 1). Positive charges are created by acquiring protons on groups containing
nitrogen (click 1). With the exception of phosphate, all the groups shown are present in
amino acids. It will be to your advantage to know these groups for future reference.
Click to go on
C
O
OH
C
O
O
+
NH3
Carboxyl
O
+
O
NH
HN
RO
P OH
RO
OH
R-SH
HO
Tertiary amine
P OH
Phosphate
O
+ NH
2
Sulfhydryl
R-S
Phenolic
Guanidinium
C
H2N
O
Imidazole
NH-
pK and Ionization
The pK of an acid or ionizable group gauges when a proton will be picked up or
released by the group. By definition, the pK is the value for the pH when half of the total are
protonated, which of course, infers half-ionized. To illustrate, consider the ionization of a
carboxyl group (click 1). When pH = pK, half the molecules are R-COOH and the other half
are R-COO-. Acidic protons are displaced by water molecules. Because the reaction is
reversible, dissociation will depend on the [H+] in solution (click 1). Enhancing [H+]
suppresses dissociation by forcing the equilibrium to the left, which essentially cancels the
ionization of the acid. A strong acid requires a very high [H+] concentration to shift the
equilibria, whereas a weak acid (strong base) requires a relatively low proton environment
(click 1). Remember, the greater the [H+] the greater the hydrogen ion pressure, the more the
proton is on. This explains why strong acids have pK values well into the acidic range and
weak acids have very high (basic) pK values (click 1)
Acid
Base
Base
O
R-C
+ H2O
R-C
O
O
OH
Don’t mistake the meaning. Of
this diagram. It is intended to
show that a greater H+
environment is needed to
suppress the negative charge
on a strong acid, less so for a
weak acid. Spoken this way
helps you see how values for
pK are derived. Click to go on
Acid
+ H3O+
Weak Acid
Strong Acid
O
R-C
OH
H+
H+ H+
H+
H+
H+
H+
H+
H+
H+ H+
H+
H+
H+
O
H+
H+
R-C
OH
H+
Basic Groups
The ionization of a basic group is basically the same with the exception that when
a proton associates with the group, the group acquires a (+) charge. Typical of this are when
protons associate to ionize amino groups (click 1).
+
R-NH2 + HH 3OO+
3
R-NH3
+
+ H2 O
Because amine groups tend to be strong bases (weak acids), to dissociate a proton from a
charged amine, i.e., force the equilibrium above to the left, requires a very low hydrogen ion
pressure (click 1). As the proton dissociates the group loses its (+) charge. As before, H2O
and the amine group compete for the proton (click 1). Dissociation will occur only when [H+]
(H3O+)is practically nil, i.e., at very high pH.
R-NH3+
H3O+ H2O
H2O
H2O
H2O
H2O H2O H2O H3O+
H O+ H2O
3
pH and Ionization
In the discussion of pK, you may have noticed the words “half-protonated, halfionized in the description. What do we mean by “half”. The answer is of the total population
of molecules only one-half bear a charge; not that all the molecules are charged to half
power. This is important because (1) a single molecule has either a full charge or no charge
and (2) the degree of ionization is a function of numbers of charged molecules. To
appreciate how numbers change, consider the reaction of a weak acid with NaOH (click 1).
Changes are best seen as a titration curve that is generated for the reaction (click 1).
Important points to note on the curve are the midpoint and the extremes. As the curve goes
from left to right, more ionized molecules appear.
O
O
+ OH-
R-C
R-C
OH
Ionized
O
+ H2O
Non-ionized
Half Ionized
50%
pH
No Ionization
0%
NaOH
Fully Ionized
100%
At the start of the titration, no
molecules are ionized. At the
midpoint half are, and at the end
all molecules are ionized. Note
that for a given pH it is possible
to determine the ratio of ionized
to non-ionized (click 1), in
essence the ratio of salt to acid.
Click to go on
Multiple Ionizable Groups
Molecules with more than one ionizable group are common in biochemistry. A
typical amino acid at pH 7, for example, has two charged groups: a (+) amine group and a (–)
carboxyl group (click 1). Molecules with multiple groups are handled as if each group acts
independently. For example, an amino acid with an -carboxyl and -amino group has 2
plateaus in a titration curve (click 1). On a pH scale, they are 7.5 units apart. There are 3
ionized species present as 2 conjugate pairs (click 1). Note that the middle component is both
an acid and a base.
COOH
COO–
COO–
pK ~2
+
+
pK ~9.5
H3N-C-H
H3N-C-H
H2N-C-H
–
COO
R
R
R
+
H3N-C-H
R
9.5
Review the lessons in this
tutorial as often as necessary
to understand ionization or
click to test and extend your
knowledge.
pH
2
OH-
Test and Extend Your Knowledge of Ionization
Q: What structural feature is common to positive charged nitrogen groups?
A: All have 4 bonds to the nitrogen.
Q: Based on the discussion of ionization as a function of numbers, what would be the
net charge on acetate when the pH = pK? pH = 10 x pK?
A: The net charge would be negative. At 10 times pK the charge will still be negative,
but instead of half, 90.9% of the total number of molecules would bear this charge.
Q: How does knowledge of a pK value allow one to tell if a group is ionized?
A: For a monoprotic (one proton) acid, you may consider the pK as the point of neutrality.
When the value for pK equals the pH there are equal numbers of charged and uncharged
molecules. Any pH above the pK is conducive to removing protons, any below favors more
molecules with protons on.
Q: How does one evaluate the charges on a molecule when there is more than one
ionizable group?
A: More than one ionizable group means more than one pK. The net charge will depend on
what is called the “isoelectric point” or pI. The pI must be calculated by taking the sum of 2 pKs
and dividing by 2. Any pH above pI is conducive to giving a majority of the molecules a
negative character; pH below pI gives a positive character. (See you textbook for a more
detailed discussion of isoelectric point).