Charge on Proteins
The charge on proteins arises from some of the amino acid side
chains, as well as the carboxy- and amino-termini, some prosthetic
groups and bound ions. This application is designed to calculate
charge based only on the side chains and carboxy - and amino-termini.
The charge on amino acid side chains depends on the pH of the
solution and the pKA of the side chains. It is also affected by
the localized environment around a side chain. We
assume the following pKA values for ionizable groups on the protein
and that the side chains will have these pKA values regardless of
their environment within the protein. We also assume
that the separation is based on the total charge on the protein,
not the mass-to-charge ratios. Therefore, a protein with a charge
of +15 will bind more tightly to a cation exchange stationary phase
(CM-Sephadex, for example) than a protein with a charge of +10,
regardless of size.
The charge on these proteins can be calculated using the method
described in Relationship of Charge to pH.
In brief, when the pH is less than the pKA of a
group, the protonated
form of the group predominates. This leaves the acidic side chains with
a charge approaching 0 and the basic side chains with a charge
approaching a limiting value of +1. Conversely, when the pH is
greater than the pKA of a group, the
deprotonated form predominates,
giving acidic side chains a charge approaching -1 and basic side
chains a charge approaching 0.
The charge on the protein is the sum of the charges on the
individual amino acid side chains. However, the charge on individual
amino acid side chains can vary when they are near a group of non-polar
or highly charged side chains. For example the normal pKA for glutamic
acid is about 4.3. In lysozyme, two glutamic acid residues are in the
active site. One is in a polar environment and has a normal pKA value.
The other glutamate side chain is in a hydrophobic environment, where
a negative charge is energetically unfavorable. Therefore the pKA value
for this glutamate side chain increases, which then decreases the extent
of the deprotonation of that side chain. This is very important in the
mechanism of lysozyme, which requires that one of the side chains be
charged (deprotonated) and the other be uncharged (protonated) at the