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Slide 1
 Amino acids –
charge, pI, pKa & pH
Module 3: Basics of protein building

Dr. Katrina Binger

Department of Biochemistry and Genetics Slide 2

Intended Learning Outcomes
(ILOs)
After this lesson students will be able to:
• Draw the zwitterion state of a generic amino acid.
• Compare and contrast the roles of pH, pKa and pI on
determining the charge state of an amino acid.
• Describe how changing the solution pH can alter the
charge of the amino acids Arg, Lys, Asp, Glu and His.
• Explain the significance of Histidine’s charge state on
protein function in biology.

Slide 3
Re-cap: the charged amino acids

Slide 4
Summary

• At physiological pH a generic amino acid is in a zwitterion

state: both positively and negatively charged, but overall
having a net neutral charge.
• By contrast, the amino acids Arg, Lys, Glu, Asp and His have
different charged states at physiological pH, due to the
unique chemical properties of their R-groups

Slide 6
Intended Learning Outcomes
(ILOs)
After this lesson students will be able to:
• Draw the zwitterion state of a generic amino acid.
• Compare and contrast the roles of pH, pKa and pI on
determining the charge state of an amino acid.
• Describe how changing the solution pH can alter the
charge of the amino acids Arg, Lys, Asp, Glu and His.
• Explain the significance of Histidine’s charge state on
protein function in biology.

Slide 7
Re-cap: important terms
• Acid is any chemical group/molecule that donates a proton (H+)
e.g. COOH, R-groups of Aspartate, Glutamate @ pH 7.5

• Base is any chemical group/molecule that accepts a proton (H+)

e.g. NH2, R-groups of Lysine, Arginine @ pH 7.5

• pH is the negative logarithm of [H+] (-log[H+]) in the solution High pH

i.e. pH is inversely proportional to the [H+] Low [H+]

High [H+]
Low pH Slide 8
pKa is the tendency of a molecule to
give or accept a H+

• pKa is the negative logarithm of acid dissociation constant

(Ka) (-log10Ka)
i.e. how likely the molecule gives/accepts H+

e.g. acetic acid, CH3COOH

Slide 9
Amino acids are more complicated because
they contain multiple ionisable groups

pKa1 < pKa2 < pH

pKa1 < pH < pKa2

pH < pKa1 & pKa2

Slide 10
What happens to a solution of
molecules when pH = pKa?
When pH = pKa there are equal amounts of protonated and
deprotonated molecules in the solution

e.g. acetic acid, CH3COOH has a pKa = 4.76

@ pH = 4.76
̶
= ̶

50 : 50

In this example, at pH 4.76, 50% are neutral ̶

(0) and 50% are negatively charged (-1);
meaning the average charge is -0.5

Slide 11
What happens to a solution of
amino acids when pH = pKa?
• When pH = pKa there are equal amounts of protonated
and deprotonated molecules in the solution
• But, uncharged amino acids have two ionisable groups
e.g. glycine

pKa1 @ pH = 2.3
= 9.6

pKa2
=
= 2.3
50 : 50
“zwitterion”

In this example, at pH 2.3, 50% of glycine are

neutral (0) and 50% are positively charged (+1);
meaning the average charge is +0.5

Slide 12
Isoelectric point, pI

• pI or isoelectric point is the pH at which a molecule on

average carries no net electrical charge pKa1
• E.g. Glycine 9.6 2.3
= 9.6

pI = pKa1 + pKa2 pKa2

= 2.3
2
pI = (9.6 + 2.3) / 2 = 6
At solution pH of 6 Glycine is neutral (zwitterion)

pH: 1 2 3 4 5 6 7 8 9 10 11 12 13 14

pH < pI Buffering pH > pI

Molecule = positively charged region Molecule = negatively charged
Slide 13
What about the charged amino acids?

• While most uncharged amino acids have a pI ~6, the R-group of

the charged amino acids affects their pI
Charged
amino acids pI pH scale
pI

Asp 2.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI

Glu 3.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI
Lys 9.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI
Arg 10.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI
His 7.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Positively-charged Negatively-charged
pH < pI Molecule “wins” H+; becomes protonated
pH > pI Solution “wins” H+; molecule is deprotonated

Slide 14
Worked example: what is the net
charge of arginine at pH 9.6?
pKa1 = 9.6 pKa2 = 2.3
Chemical pKa pKa vs. pH Charge?
group
12.5 pKa > pH Molecule protonated (+1)

2.3 pKa < pH Molecule deprotonated (-1)

pKa2 = 12.5 9.6 pKa = pH Molecules 50:50 mix of
protonated (+1) and
deprotonated (0). Charge =
average of +1 & 0 = +0.5

Net charge of arginine

at pH 9.6
Arginine
= +0.5 +1 -1
= +0.5 Slide 15
Importance of pH and Biological
buffer systems

• Maintenance of intracellular pH is vital to all cells

• Enzyme-catalyzed reactions have optimal pH
• Solubility of polar molecules depends on H-bond donors and
acceptors (these can be amino acids or proteins)
• Equilibrium between CO2 gas and dissolved HCO3– depends on pH

• Buffer systems in vivo are mainly based on

• phosphate, concentration in millimolar range
• bicarbonate, important for blood plasma
• histidine, efficient buffer at neutral pH
Physiological pH and the charged
amino acids
Physiological
Charged pH ~ 7.4
amino acids pI
pI

Asp 2.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI

Glu 3.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI
Lys 9.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI
Arg 10.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pI
His 7.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Positively-charged Negatively-charged

Lys, Arg = positively charged

Asp, Glu = negatively charged
His = ??
Slide 17
Histidine

• Unique R-group properties make it a versatile

catalytic amino acid in many enzymes
• Imidazole side chain gives rise to aromatic properties
(i.e. hydrophobic)

• But is also amphoteric – meaning it is both an acid &

base at physiological pH (i.e more hydrophilic)

• The activities of many proteins are therefore

regulated by pH through the protonation of
histidine side chains
Slide 18
Summary

• At physiological pH a generic amino acid is in a zwitterion

state: both positively and negatively charged, but overall
having a net neutral charge.
• By contrast, the amino acids Arg, Lys, Glu, Asp and His have
different charged states at physiological pH, due to the
unique chemical properties of their R-groups
• The charge state of an amino acid backbone and its R-group
is dependent on the solution pH.
• The pH at which an amino acid is neutral (zwitterion) is its
isoelectric point, or pI.
• In biology, the charge state of amino acids such as Histidine
plays an important role in enzyme catalysis

Slide 19
Resources

• Lehninger Principles of Biochemistry Seventh

Edition (2017), W. H. Freeman and Company
• Chapter 2.1, 2.2, 2.3
• Chapter 3.1

Slide 20