DETERMNATON OF THE pKA VALUES OF AN AMINO ACD

Rveyda AKN, Gebze Technical University, Turkey

Expriment 2

AIM
Playing an integral role in the structure and function of proteins, amino acids are perhaps one of the
most important biomolecules as proteins themselves participate in nearly every physiological event
within a cell. In order to understand the acid-base properties of proteins and their resulting behavior
as polyionic macromolecules, we will begin by investigating the properties of their constituent amino
acids.

INTRODUCTION Simple amino acids, like glycine, have two

With each containing at least one amino and
carboxylic acid group, amino acids are
classified as amphoteric substances and react
with both acids and bases as follows:
The ionic form of the amino acid present in an
aqueous solution is dependent upon the
solutions pH. In this experiment you will
identify an unknown amino acid via an acid-
base titration. Titration curves of amino acids
are very useful for identification as you can see
in the example for glycine given below;
dissociation steps: (1) the loss of H+ from the
acidic carboxyl group at low pH; and (2) the loss
of H+ from the more basic amino group at high
pH. The pKa value for each dissociable group of
an amino acid can be determined from such a
titration curve by extrapolating the midpoint of
each buffering region (the plateau) within the
curve. Also revealed from the diagram is a
point on the curve where the amino acid
behaves as a neutral salt. Specifically, this point
is known as the isoelectric point (pI), and is
loosely defined as the pH where the amino acid
is predominantly a zwitterion. Furthermore,
the pI can be approximated as halfway
between the two points of strongest buffering
capacity and can be estimated by;
where K1 and K2 are the dissociation constants
for the deprotonation of glycines carboxylic
acid and amino groups. Charged amino acids
possess R-groups having acidic or basic side
chains giving them more than two dissociable
H+ ions. For example, glutamic acid has a
carboxylic acid side chain in addition to its -
carboxyl and -amino groups resulting in a
titration curve more complex than that
observed for glycine. Where glycines titration
curve possessed only two plateau regions and
thus two pKa values, glutamic acid possesses
threetwo in the acidic pH region, pKa1 (- Transfer Count pH Transfer Count pH
carboxyl group) = 2.2 and pKa2 (-carboxyl 0. 2,17 24. 3,19
group) = 4.3 ; and one in the basic pH region, 1. 2,17 25. 3,38
pKa3 (-amino group) = 9.7. Members of the 2. 2,19 26. 3,69
basic family of amino acids, like lysine, will also 3. 2,21 27. 4,51
exhibit three pKa values. However, due to the 4. 2,23 28. 8,6
extra amino group, they will have only one pKa 5. 2,25 29. 9,08
in the acidic region and two pKa values in the 6. 2,27 30. 9,35
basic region. Tables of pKa and pI values of each
7. 2,29 31. 9,54
amino acid are readily available and can be
8. 2,31 32. 9,72
used as standards to identify an unknown
9. 2,34 33. 9,88
amino acid. Furthermore, identification of the
10. 2,36 34. 10,1
regions of the titration curve require a
11. 2,39 35. 10,2
thorough knowledge of the protonation and
12. 2,4 36. 10,5
deprotonation process of an amino acid and an
13. 2,42 37. 10,7
understanding of the definition of an
isoelectric point. In summary, titration curves 14. 2,45 38. 11
are helpful in the identification of amino acids. 15. 2,48 39. 11,3
Nonetheless, a meticulous titration of an 16. 2,51 40. 11,5
amino acid unknown will produce a titration 17. 2,55 41. 11,6
curve that is distinctive to the amino acid. A 18. 2,61 42. 11,7
titration is a fast, inexpensive way to determine 19. 2,68 43. 11,8
the identity of an unknown sample; it would 20. 2,76
not however be a good way to determine the 21. 2,84
contents of a mixture of amino acids. To that 22. 2,95
end, you will each be assigned a single 23. 3,05
unknown amino acid to identify by titration
versus a standardized solution of NaOH.

MATERALS AND METHODS X amino acid;

X unknown amino acid, pH meter, NaOH

1)Into a 250 ml beaker, place 100 mL of one of 14 y = 0.2577x

the unknown amino acid solutions. R = 0.8067
12
2)The concentration of the solutions is 20 mM. 10
Thus, in 100 mL you will have 2.0 mmol of the
8 X
amino acid.
pH

Protein
6
3)You will be titrating with 0.10 M NaOH, 0
which will gradually increase the pH of your 4

solution. 2

4) To titrate through one buffering region (one 0

0 10 20 30 40 50
pKa) will require 20 mL of the NaOH.
NaOH quantity
RESULT
pH value of unknown protein according to
1000 l were added until transferred 17th then
NaOH exchange.
2000 l were added until 43th.
DISCUSSON

We have an unknown amino acid and. We

titrated it with NaOH and looked at pI.
Nevertheless, ph meter changed slightly until
the 18th transfer, also we transfered 1000l all
time. Therefore, we trasfered 2000 l to quick
get result. Amino acid has both amino group
and carboxyl. In this way it acts like a buffer so
as shown in the above experiment, it is difficult
to titrated amino acid. Briefly, according to
graphic and informations X unknown amino
acid is Glutamic acid. Because pK1 (-carboxyl)
=2.19 of glutamic acid and initial titration of X
amino acid is approximately 2.19 so thry are
almost similar. Also, pK1 (-amino) =9.66 of
glutamic acid and pI=3.2. It was difficult to
change its pH when the pH was 9. Therefore,
according to our datas X amino acid is Glutamic
acid.

REFERENCE
www.chem.fsu.edu
vlab.amrita.edu
Albert and E.P Serjeant The determination of
Ionization Constants, Chapman and Hall, Ltd.,
London(1971). This reference gives details of
research methods for determining pKas.
Greenstein, J.P. and M. Winitz, Chemistry of
the Amino Acids, vol I, Wiley and Sons, New
York (1961) pp482-491. The method for
determining pI is explained here.
West, E.S and W.R. Todd. Textbook of
Biochemistry, Macmillan, NY, 1955, p. 311.
Datain the table are from this reference.