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Calculating Pi
Calculating Pi
The Problem
• A protein’s net charge depends on the number of
charged amino acids it contains and the pH of its
environment.
– The isoelectric point is the pH at which the net
charge is zero.
• Each amino acid has a different dissociation
constant, KD.
– The amino acids that can be ionized at
physiological pHs are: aspartate (D),
glutamate (E), lysine (K), arginine (R), and
histidine (H).
– Also, the N and C termini can be ionized.
• The important point is: most of these amino
acids are not fully ionized or fully unionized at
physiological pHs.
• It is your job to calculate the proportion of
each amino acid that is ionized at a given pH.
Once this is done, calculating the net charge is
straightforward.
– Multiply the proportion by the number of that
amino acid, taking into account the differences
between acidic (-COOH) and basic (-NH2) types.
Amino Acids Contributing to Charge
• Charged amino acids come in two forms:
• Acidic amino acids (and the C-terminal) are in the –COOH form (uncharged)
at low pH and in the –COO- form (-1 charge) at high pHs.
– These include aspartic acid (D) and glutamic acid (E)
• Basic amino acids (and the N-terminal) are in the –NH3+ form (+1 charge)
at low pH and in the –NH2 form (uncharged) at high pH.
– These include lysine (K), arginine (R) and histidine (H).
• All other amino acids do not affect charge under physiological conditions.
Dissociation Constant
• The association and dissociation of the H+ with the amino acid
is governed by its dissociation constant, KD.
• Dissociation constant is the ratio of the concentration of the
dissociated forms to the concentration of the combined form.
– Each amino acid has a separate dissociation constant.
– The equation below uses [R-] to indicate the concentration of the
amino acid when the H+ is dissociated from it.
• The pK value is analogous to pH.
– pK = -log10KD
– KD = 10-pK
[ H ][ R ]
KD
[ RH ]
Calculating the We rearrange to get:
[ H ][ R ]
[ RH ]
Proportion with H KD