Practice Problems on Peptide Ionization Properties (solutions on next 2 pages)
From learning objectives for Lecture 4 (Peptides):
Be able to estimate the approximate net charge on a short peptide at any given pH. This requires being
given (e.g., on cover sheets of exams) or knowing the approximate pKa values of the ionizable groups
in peptides and proteins (the single α-amino group and single α-carboxyl group on the peptide, and
any ionizable R groups) as well as the chemistry/charge properties of those groups in their conjugate
acid and conjugate base forms.
Approximate ("generic") pKa
Approximate ("generic") pKa
Ionizable
Ionizable
in
peptides
&
proteins
(from
peptides & proteins (from
group in peptides
group in peptides inBerg,
Berg,
Tymoczko
&
Stryer,
Tymoczko & Stryer,
and proteins
and proteins
Biochemistry, 6th ed., 2007)
Biochemistry, 6th ed., 2007)
3.1
thiol
8.3
α-carboxyl
side chain carboxyl
4.1
aromatic hydroxyl
10.9
imidazole
6.0
10.8
ε-amino
8.0
guanidino
12.5
α-amino
•To answer questions about peptide ionization properties, you need to know
a) the pKa values of the 9 ionizable groups in peptides (and proteins),
b) the structures of those ionizable groups (or in the case of arginine and histidine, at least
approximate structures)
c) the acid-base properties of the group, how the group ionizes: Is the conjugate acid (protonated
form) + charged, in which case its conjugate base (the unprotonated form) is neutral, or is the
conjugate acid neutral, in which case its conjugate base is – charged?
•In any question about the charge properties of a peptide, focus just on the ionizable groups -- make notes
on the sequence. If you need to write out shorthand structures of the groups, do that for practice while
you're learning the structures and properties of the various groups, but on an exam, the more "shorthand"
you can use in your analysis, the less time it will take you.
Study questions/problems:
1. What would be the approximate net charge for each of the following peptides at pH 1? at pH 5? at pH
7? at pH 9? at pH 11.5? at pH 13? (You can consult the table on the cover of the 2005 or 2006
Exam 1 for pKa values.) [Why is this useful? You're not likely to be electrophoresing short peptides
and needing to figure out toward which pole they would move, right? But thinking about net charges
on peptides helps you to learn the charge properties of the ionizable groups in proteins, which are very
important in enzyme catalytic mechanisms, binding interactions between biomolecules, etc.]
Approx. net charge on whole peptide at each pH
pH 1
pH 5
pH 7
pH 9 pH 11.5 pH 13
A. Phe-Glu-Asn-Cys-Arg
B. Met-Tyr-Leu-Lys-Asp
C. Cys-Arg-Gln-His-Lys-Trp
These aren't that complicated -- an exam question might just ask for the net charge at one pH, but it's
instructive to look for practice at what happens to the charge at a number of different pHs, so that's what
we're doing here.
You can also make up your own peptide sequences for practice; they're more interesting if they have some
ionizable side chains, not just non-ionizable R groups, since such peptides would only have the α-amino
and α-carboxyl groups for ionizable groups.)
2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis
required the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean
the Cys residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys
residue in that protein had a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme
molecules would have the Cys R group in its ACTIVE form at pH 7.0?
Bioc460 2008, Dr. Ziegler, © Summer 2008
Peptide ionization practice problems, p. 1
SOLUTIONS
1.A. Phe-Glu-Asn-Cys-Arg
List all ionizable groups (I myself make handwritten notes right on/above/below the sequence):
“+” or “–“ indicates the predominant (“majority”) charge state (more than half in this charge state); if
pH is less than 1 unit away from pKa, so group is not 90% in that state as it would be if the pH were a
full pH unit above or below the pKa, the predominant charge at that pH is in parentheses: (+) or (–).
Approx. net charge on individual GROUP at each pH
ionizable functional group
pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13
α-NH2 (pKa ~8) (N-terminal amino group)
+
+
+
0
0
0
Glu R carboxyl (pKa ~4.1)
0
–
–
–
–
–
Cys R-SH (pKa ~8.3)
0
0
0
(–)
–
–
Arg R-guanidino (pKa ~12.5)
+
+
+
+
+
(0)
α-COOH (pKa~3.1) (C-terminal carboxyl group)
0
–
–
–
–
–
APPROX. NET CHARGE ON WHOLE
+2
0
0
–2
–2
–3
PEPTIDE AT THIS pH (sum of individual
groups' charges)
1.B. Met-Tyr-Leu-Lys-Asp
List ionizable groups.
Approx. net charge on individual GROUP at each pH
ionizable functional group
pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13
α-NH2 (pKa ~8) (N-terminal amino group)
+
+
+
0
0
0
Tyr R aromatic OH (pKa ~10.9)
0
0
0
0
(–)
–
Lys R-ε -NH2 (pKa ~10.8)
+
+
+
+
(0)
0
Asp R-carboxyl (pKa ~4.1)
0
–
–
–
–
–
α-COOH (pKa~3.1) (C-terminal carboxyl group)
0
–
–
–
–
–
APPROX. NET CHARGE ON WHOLE
+2
0
0
–1
–3
–3
PEPTIDE AT THIS pH (sum of individual
groups' charges)
1. C. Cys-Arg-Gln-His-Lys-Trp
List ionizable groups.
Approx. net charge on individual GROUP at each pH
ionizable functional group
pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13
α-NH2 (pKa ~8) (N-terminal amino group)
+
+
+
0
0
0
Cys R-SH (pKa ~8.3)
0
0
0
(–)
–
–
Arg R-guanidino (pKa ~12.5)
+
+
+
+
+
(0)
His R-imidazole (pKa ~6)
+
+
0
0
0
0
Lys R-ε -NH2 (pKa ~10.8)
+
+
+
+
(0)
0
α-COOH (pKa~3.1) (C-terminal carboxyl group)
0
–
–
–
–
–
APPROX. NET CHARGE ON WHOLE
+4
+3
+2
0
–1
–2
PEPTIDE AT THIS pH (sum of individual
groups' charges)
Bioc460 2008, Dr. Ziegler, © Summer 2008
Peptide ionization practice problems, p. 2
2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis
required the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean
the Cys residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys
residue in that protein had a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme
molecules would have the Cys R group in its ACTIVE form at pH 7.0?
Whenever a question asks about either the ratio of base/acid (or acid/base), or the proportion of a
functional group that's in one charge state or the other, you know you'll need the HendersonHasselbalch Equation, which relates 3 parameters: pH, pKa, and base/acid ratio (but it's a
logarithmic relationship). You know the pH (given: pH = 7.0) and the pKa of this Cys-SH (GIVEN
as 7.5, so don't use the "generic" pKa value). Solve for base/acid ratio, and convert to fraction or
per cent that's in base form, as in the example at the end of the posted general chemistry review
PDF.
pH = pKa + log
[base]
[acid]
"$ [base] R ! S ! %
=
# [acid] R ! SH &
log
[base]
= pH ! pKa = 7.0 ! 7.5 = !0.5
[acid]
[base]
0.316 R ! S!
= 10!0.5 =
=
[acid ]
1
R ! SH
0.316
0.316
Fraction base =
=
= 0.24
1 + 0.316 1.316
or as a per cent, 24% of the Cys thiol would be in the form of the good nucleophile, the conjugate
base, i.e., the thiolate anion, R-S–.
Bioc460 2008, Dr. Ziegler, © Summer 2008
Peptide ionization practice problems, p. 3