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In the context of polypeptide
chains, amino acids are called
Peptide bonds can link amino acids to produce long chains
An “average” polypeptide is between 100 to 1500 amino acids long. (~11-165 kD)
Titin: 34,500 amino acids!
Polypeptide sequences are written (and numbered) from the N- to Cterminus.
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Polar, uncharged amino acids of note: cysteine forms
covalent bonds in proteins
% Acid/Base chemistry review
Ka HA H+ + A- [H + ][A − ]
Ka is the acid dissociation equilibrium constant. ⎡[H + ][A − ] ⎤
pKa = − log Ka = − log ⎢
Ka Lysine side chain (free)
Ka = 2.9 x 10-11
pKa = 10.54
What is the predominant form when the pH < pKa?
What about when pH = pKa?
What about when pH ≈ pKa?
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.%$(* What you need to know about amino acid pKas
• Be able to draw out the reaction the pKa describes for each
functional group in the amino acid. • Know that pKas of the carboxyl group and amine groups that are not
part of the sidechain are about 2.0 and 9.0, respectively. • Know the predominant charge state of each functional group on
each amino acid at pH 7.0. • If you need to know the exact pKa values for a calculation, we will
give them to you, but we may not tell you which pKa corresponds to
which functional group. Example: We may ask you to do a calculation with Histidine, and
tell you that the pKa values for His are 1.8, 9.3 and 6.0.
N ! O H
O H O H H N
H &"! %$ ! O N
H N "#
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0.6 0.5 %
0 5 10
H+ + A- HA [H + ][A − ]
[HA] ⎛ [HA] ⎞
[H + ] = Ka ⎜⎜ − ⎟⎟
⎝ [A ] ⎠
- log[H + ] = − log Ka + log⎜⎜
⎛ [A − ] ⎞
pH = pKa + log⎜⎜
⎝ [HA] ⎠
! Determining the fraction of a titratable group in a particular state
What is the fraction of lysine side chain that is protonated at a
General expression for dissociation of a weak acid: HA Ka H+ A - The fraction of the protonated form is (by definition): f HA = [HA ]
[HA]+[A - ] Use the equation for Ka to get [HA]: [H + ][A - ]
Ka Combining these two equations, f HA = [HA ] [H + ][A - ]
Ka [HA]+[A - ] we get: f HA [H + ][A − ] K a
[H ][A − ] K a + [A − ] Multiplying through by Ka, this reduces to: f HA [H + ]
K a + [H + ] A similar expression can be derived for the fraction of conjugate
base f A− = Ka
K a + [H + ] These two equations allow for the calculation of
the fraction of acid or conjugate base at any pH
for an acid with a known pKa.
note that only the
numerator is different f HA [H + ]
K a + [H + ] f A− = Ka
K a + [H + ] How much of the unprotonated lysine sidechain exists at pH 9.54?
(note that this is 1.0 pH units below the pKa)
Using the Henderson-Hasselbalch equation: [A − ]
pH = pKa + log
[Lys-NH3+] [A − ]
9.54 = 10.54 + log
= 0.1 at pH 9.54
(1.0 pH unit less than pKa) or expressed as a fraction/percent: 0.1/1.1 = 9.1%
Note that we consider a titratable group to be predominantly in
only one form once the pH outside one unit of its pKa. Note
that in other cases we will ask you to calculate precisely the
ratio, concentration, or fraction of a species in solution. Significant concentrations of the acid and conjugate base are
present when pH is near the pKa
What is the pH when the fraction of unprotonated lysine is
20%? f A − = 0.2 = Ka
K a + [H + ] 10 − pKa
0.2 = − pKa
+ 10-pH pKa = −log Ka
10-pKa = Ka
pH = 9.9 Amino acids have multiple titratable functional groups
For example, glycine:
Glycine is a an important
neurotransmitter that binds to a
receptor found in the central nervous
system called GlyR.
What are the possible charge states, and what are
their concentrations at different pH values?
At what pH values does the molecule have a net
positive or negative charge?
K a1 >> K a 3
1 Ka4 << 1
&!% # ' f NH3 + $' [H + ]
[H ] + K a2 f A− = '
" K a1
K a1 + [H + ] f NH3+:A- = f NH3+ f A −
f NH3:A- K a1
[H + ]
[H ] + K a2 K a1 + [H + ]
f NH3:A- = 0.66
.*#& .'#+ ).,#( - ) K his
[H + ]
[H + ]
[H ] + K NH3 K his + [H + ] [H + ] + K COOH
/ / % / & / '
[H + ]
[H + ]
K a1 + [H + ] K a2 + [H + ] f− = K a1
K a1 + [H + ] K a2 + [H + ]
[H + ]
[H + ]
K a1 + [H + ] K a2 + [H + ] K a1 + [H + ] K a2 + [H + ]
pI = pKa1 + pKa2
2 The pI is often (but not always!) the average of the two pKas around the zero charge
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- Winter '13