how we calculate net charges

   Calculating the NET charge on a polypeptide: example from lecture

Glu-Gly-Ala-Lys

Step 1: Write out the structure with the charges that you would expect at pH 0

How do you know what charges would be expected at pH 0?

Based on the pKa values for all the ionizable groups.

What are ionizable - should be able to recognize these based on

amino acid structures.

In this example - we have four ionizable groups (write out in order of

increasing pKa values).

Ionizable Group pKa

Carboxy-terminus (COOH) ~2.0

Glutamic acid carboxyl group (COOH) ~4.2

Amino-terminus (NH3

+) ~9.7

Lysine ε-amino group (NH3

+) ~10

Recall that below a pH represented by a particular pKa value, that group will

be protonated! What is the definition of pKa? Recall that it is based on the

dissociation of an acid and is the point at which 50% of the groups are ionized

This means that at pH 0, all the titratable groups will be protonated, as drawn

below.

Now calculate the NET charge on the molecule at pH 0.0

peptide Nterminus

Glutamic Acid

carboxyl group

Lysine

ε-amino group

peptide

C-terminus

NET CHARGE

ON MOLECULE

+1 0 +1 0 +2

Step 2: At what pH will the NET charge on the peptide change by 1 charge unit?

Based on the pKa values: if you increase the pH above 2.0, but keep it below 4.2,

you will change the charge by 1. (So, you can pick any pH from 2.01 to 4.19).

Assume pH 3.0: (Above pH 2.0, you deprotonate the peptide C-terminus.)

peptide Nterminus

Glutamic Acid

carboxyl group

Lysine

ε-amino group

peptide

C-terminus

NET CHARGE

ON MOLECULE

+1 0 +1 -1 +1

Step 3: At what pH will the NET charge on the peptide change by 1 charge unit?

Based on the pKa values: if you increase the pH above 4.2, but keep it below 9.7,

you will change the charge by 1. (So, you can pick any pH value from 4.21 to 9.69)

Assume pH 7.0 (Above pH 4.2, you deprotonate the glutamic acid carboxyl group)

peptide Nterminus

Glutamic Acid

carboxyl group

Lysine

ε-amino group

peptide

C-terminus

NET CHARGE

ON MOLECULE

+1 -1 +1 -1 0

Step 4: At what pH will the NET charge on the peptide change by 1 charge unit?

Based on the pKa values: if you increase the pH above 9.7, but keep it below 10,

you will change the charge by 1. (So, you can pick any pH value from 9.71 to 9.99)