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.

CO

OHC

O

OCarboxyl

P

O

O

O

RO H

H

P

O

O

O

RO HPhosphate

R-SH R-S Sulfhydryl

HO O Phenolic

NH3Tertiary amine

+

N N H

+H Imidazole

H2NC

NH2

NH-

+

Guanidinium

Click to go on

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)

R-C

O

OH

H+

H+

H+

H+H+

H+

H+

H+

Strong Acid

H+

H+

H+

H+

H+

R-C

O

OH

H+

H+

H+

Weak Acid

H+

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

R-C

O

OHR-C

O

O+ H2O + H3O+

Base BaseAcid Acid

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).

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+

H2O

H2O

H2O H3O+H2O

H2O

H2O

H2O

H3O+

H3O+

H2O

R-NH2 + H3O+ R-NH3 + H2O+

H3O+

pH and IonizationIn the discussion of pK, you may have noticed the words “half-protonated, half-

ionized 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.

NaOH

pH

Fully Ionized 100%Half Ionized

50%

R-C

O

OHR-C

O

O+ OH- + H2O

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

No Ionization 0%

Ionized Non-ionized

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.

COO–

H3N-C-H+

R

pK ~2pK ~9.5

OH-

pH

2

9.5

COOH

H3N-C-H+

R

COO–

H2N-C-H

R

COO–

H3N-C-H+

R

Review the lessons in this tutorial as often as necessary to understand ionization or click to test and extend your knowledge.

Q: What structural feature is common to positive charged nitrogen groups?

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?

Test and Extend Your Knowledge of Ionization

A: All have 4 bonds to the nitrogen.

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).