BIO

103 Laboratory class: 2019

Experiment 4: Buffering action of amino acids and calculating pKa & pI

Aim:

1. To understand the buffering nature of amino acids because they can act as proton
acceptor and donor at a given pH called isoelectric point (pI) or zwitter ion state (dipolar
ion state).
2. Titration of glycine or given amino acids to determine the pKa (dissociation constant) of
the amino group and the carbonyl groups and calculating the isoelectric point (pI).

Principle & Theory:

What is buffering action?

Amino acids can act as a buffer because it can react with acids and bases to keep the pH
constant. This is possible because amino acids have both an acidic group (COOH) and a basic
group (NH2), and this allows it to act as both an acid and a base. In very acidic media, the NH2
group is in its protonated form, and in the very basic media, the COOH group is in its
deprotonated form. At intermediate pH (neutral i.e.) both the amino and carboxyl groups are in
their ionic form and the net charge of the entire molecule is zero and this represents the
isoelectric point (pI). The neutral ionic state of amino acids is called zwitterion. In short, the
point at which zwitter ions exist is called the isoelectric point (pI) of a given amino acid. And,
zwitterions are compounds which are both acids and bases, so their chemical structure evolves
between three forms (Figure 1 below), depending on the pH of the environment.

Figure 1: Three forms of Glycine based on the pH of the environment.

In glycine, the COOH group has pKa1=2.34, and the H3N+ group has Ka2=9.60.

At the isoelectric point,pI = pKa1+pKa2 = 5.97

2
Why should we know about buffering action of amino acids?

Living organisms are water-based systems and they require an acid-base equilibria for optimal
functioning. Maintenance of pH is important in biological systems because the cell environment
is critical to be buffered at ~pH7, and experiments such as biological enzymatic assays require a
certain pH.

1
BIO 103 Laboratory class: 2019

Table 1: Major buffer systems of the human body:

Bicarbonate buffer CO2 + H2O ⇋ H2CO3 ⇋ H+ + HCO3- In blood plasma

Hemoglobin Hb-H ⇋ Hb- + H+ Interior of red blood cells
Phosphate buffer H2PO4- ⇋ H+ + HPO42- Most important in urine
Protein Pr-H ⇋ Pr- + H+ Intracellular fluid

Most of the amino acids are present in serum (amino acid pool) and cytoplasm of a given cell
and they maintain the desired buffering action by the above principle of acting as a proton donor
or a proton acceptor. When an amino acid dissolves in water, the zwitterion interacts with H2O
molecules – acting as both as an acid and a base. But, unlike simple amphoteric compounds that
may only form either a cationic or anionic species, a zwitterion simultaneously has both ionic
states.

The method you will learn today in the practial will be useful to calculate pKa1, pKa2....
pKan and pI of any amino acid or protein.

How does one measure pKa or pI?

The pKa value of any amino acid having dissociable groups can be calculated by acid base
titration curve by extrapolating the midpoint of each buffering region within the curve.

*pI can be calculated as halfway point between strongest buffering region and estimated as

*pI = ½ (pKa1 + pKa2) where k1 and k2 are dissociation constants for the deprotonation of
amino and carboxyl groups of an amino acid.

In today’s experiment we use Glycine to study the buffer action of amino acids, however any
amino acid that behaves as a zwitter ion at near neutral pH (at which its pI is located) can be
represented as below:

At acidic conditions (ex: pH = 2), the carboxyl group is unionized (-COOH) and the amino group
is ionized (-NH3+), this is pKa1

At alkaline conditions (ex: pH = 10), the carboxyl group is ionized (-COO-) and the amino group
is unionized (-NH2), this is pKa2

If you recall the *Bold sentences above pI = (pKa1 + pKa2) / 2

2
BIO 103 Laboratory class: 2019

Usually acid dissociates as : HA H+ + A -

pH = -log10 [H+]

pKa = - log10 Ka (equilibrium constant or Keq)

Therefore Ka = Keq

Ka = [H+][ A-] /[HA]

Taking log on both sides log10 Ka = log10 [H+][ A-] /[HA]

log10 Ka = log10 [H+] + log10 [ A-] /[HA]

= log10 [H+] = - log10 Ka + log10 [ A-] /[HA]

pH = - log10 Ka + log10 [ A-] /[HA]

Therefore pH = pKa + log [ A-] / [HA]

When [A] = [HA]; pH = pKa + log10 (is 1)

pH = pKa

3
BIO 103 Laboratory class: 2019

Calculations:

To determine the isoelectric point (pI), plot a graph taking pH values on Y axis and the volume
of NaOH added on the X axis as shown in Figure 2a and 2b (below, see points A, C are pKa1
and pKa2 respectively). Calculate the midpoint B should be calculated as per the equation given
below. pI = (pKa1 + pKa2) / 2

pI = .........+........ / 2 = ...........

2a 2b

1 A


C
B
B

C
A
0

Figure 2a: Plot showing pKa1 and pKa2 calculations and zwitter ion calculations respectively.
1b. Plot showing the calculation of pKa1, pKa2, and pI calculation in Glycine.

4
BIO 103 Laboratory class: 2019

Aim:

1. To understand the buffering nature of amino acids because they can act as proton
acceptors and donors at a given pH called isoelectric point (pI) or zwitter ion state
(dipolar ion state).
2. Titration of glycine or given amino acids to determine the pKa (dissociation constant) of
the amino group and the carbonyl groups and calculating the isoelectric point (pI).

Caution: Do not play around with solutions especially acids and bases.
You should be very careful while pipetting these acids
Materials

Glycine (0.1 M), NaOH (0.1 M), HCL (0.1 M), pH meter, measuring cylinders, pipette, Distilled
water, tissue paper

Procedure

1. Switch on the pH meter set it and calibrate the pH meter using the three standards (4, 7
and 10).
2. Once calibrated, switch the pH to the MEASUREMENT MODE
3. Take 20 ml of the 0.1M glycine in a beaker mix well / stir, adjust to pH 2.00 or lower by
adding few drops of 0.1M HCL
4. Add 5ml of 0.1M NaOH stir well and check the pH. Note down the pH value.
5. Repeat the step 4 till you reach pH 11
6. Calculate your pKa and isoelectric point, plot a graph taking pH value on Y axis and
volume of NaOH added in the X axis.

Observation Table:

Volume of NaOH added (ml) pH

5 ~2
10
… till pH 11 is reached 11

Points to wonder:

1. Why is this experiment biologically or evolutionarily important?

2. What other factor is important in this experiment which you have not consciously measured?
(Hint: refer to pH class, Lab2).

Source: Lehninger: Principles of Biochemistry 4th edition

https://groups.chem.ubc.ca/courseware/pH/section19/index.html
https://www.chem.purdue.edu/.../Spring%202013%20Lecture%205.pdf
5
BIO 103 Laboratory class: 2019