Department of Chemical & Biomolecular Engineering

THE NATIONAL UNIVERSITY

of SINGAPORE

Chemical Engineering Laboratory I

Experiment B1
Protein Quantification, Activity and Specific Binding Constant

Name

Matric No.

Group

Date of Expt.

GRADE :

A. Learning objectives
1.
2.
3.
4.

Identify the parts of an HPLC system.

Obtain practical experience in the use of HPLC as a means for protein quantification.
Determine protein concentration in unknown samples.
Appreciate the importance of and difference between protein activity vis--vis protein
concentration.
5. Determine specific binding constant for a protein-receptor pair.

B. Introduction
I. High Performance Liquid Chromatography (HPLC)
HPLC has become the unsurpassed technique for isolating and purifying proteins and
peptides. It is the workhorse for any protein downstream processing train, that no laboratory
is ever complete without a number of HPLC systems.
In liquid chromatography, a protein mixture is sent through a chromatographic column and
the mixture components are chased by a mobile phase. Based on differential migration
through the column (which is a function of the chemistry between the packing materials and
the proteins), the proteins are separated, eluted, quantified and/or collected. The basic
components of an HPLC system are shown in Figure 1. This consists of a high pressure
pump (mostly two), a supply of mobile phase(s), a column containing the stationary phase,
an injection unit for introducing the samples onto the column, an in-line detector and some
method of displaying the detector signal. The choice of the stationary and the mobile phases
depends on the proteins in the mixture, and the sequence of the chromatographic technique
in the downstream processing train. Identify the parts mentioned above in the HPLC system
in the laboratory.

Figure 1. Block Diagram of an HPLC System

II. Specific Binding Constants
In the association reaction between a ligand and a receptor, like the interaction of a cell
receptor and a drug, the binding constant provides a quantitative indication of the affinity of

the ligand for the receptor. This equilibrium constant is an important parameter for
applications in drug design, biomolecular interactions, protein purification studies, and so on.
In general the association reaction can be represented by the enzyme-substrate reaction:

ES EP

where E is the enzyme, S is the substrate and P is the product concentrations, all measured
at equilibrium.
Using a Michaelis-Menten approach, the reaction kinetics for this reaction can be
represented by:

qm S
Km S

where q is the reaction rate, qm is the maximum reaction rate, and Km is the binding
constant, in units consistent with that of S.
This equation can be linearised in a number of ways to obtain the kinetic parameters:
a.
Lineweaver-Burke

1
1 Km 1

q qm qm S
b.

Langmuir

S Km
1
S

q qm qm
c.

Eadie-Hofstee

q qm K m

q
S

C. Experimental
I. Protein Quantification
HPLC can be used in the analytical mode to determine the protein concentration in an
unknown sample. To do this, a calibration curve which correlates the area under the eluted
peak with protein concentration is first obtained using standard solutions of the protein of
interest. A typical chromatogram for two well resolved proteins, thyroglobulin and albumin,
is shown in Figure 2. Note the maximum absorbance (A280) in the chromatogram. Read up
on Beer-Lamberts law.

Eluent Volume, mL
Figure 2. Typical Chromatogram
You will determine the protein concentrations of the unknown samples provided based on
the calibration curve built in the HPLC system.

You are provided with the following for this experiment:

1.
A HPLC system which has been properly set up and equilibrated. You only need
to inject 20L of each of your samples, and your data will be recorded and
analysed by the computer.
2.
The model protein you will use is the enzyme -amylase. This protein is
extracted from soya beans. -amylase hydrolyses -1,4-glucose linkages in
starch by cleaving successive maltose units from the non-reducing ends of the
starch chains.
A stock solution (10mL of 0.2mg/mL) of commercial -amylase is available for
your use. Commercial -amylase is used as a standard to characterize the
activity of natural and non-stable -amylase.
3.
Two unknown samples containing -amylase (20mL each), the concentrations of
which you are to determine. One sample, labeled S25, has been stored at 250C,
and the other, labeled S60, has been stored at 600C, each for 2 hours.
II. Protein Activity
You are to determine the protein activity of the two samples of -amylase provided. One unit
of -amylase activity has been arbitrarily defined in this experiment as the amount of enzyme
needed to hydrolyse 10mg of starch per 30 minutes.
In addition to that provided in the previous experiment, you are also provided with the
following:
1.
A UV-visible spectrophotometer with wavelength set at 600nm.
2.
100mL of ~0.4% starch solution (actual concentration needs to be measured)
3.
200mL of 0.5mM iodine solution.
4.
100mL of 0.1M CaCl2 and 0.15M KCl mixture solution
Protocol:
1.
2.
3.

4.

5.

Pipette 5mL of the iodine solution into each of the 10-20 test tubes.
Place 3mL of the starch solution (0.4% starch) in a test tube
Prepare a control to be used as blank for the absorbance measurements by
pipetting 1mL of the sample -amylase solution into 3mL of CaCl2 and KCl
mixture solution (in place of the starch solution). Add 0.2mL of this to a test
tube containing iodine.
At t=0, add 1mL of sample S25 to the starch solution. At 0.5 minute intervals,
add 0.2mL of this reaction mixture into the iodine solutions. Measure the
absorbance against the control. The end-point of starch hydrolysis is defined
as the time to reach A600 = 0.4. This time should be in the range of between 3
and 10 mins.
Repeat steps 1 to 4 using sample S60.

III. Specific Binding Constants

Choose one of the linearised forms of the Michaelis-Menten relation, and design an
experiment to determine the specific binding constant for starch and -amylase. You may
use the following reaction protocol:
1.
Pipette 5mL of the iodine solution into each of 5 test tubes.
2.
Prepare five pre-determined starch concentrations in the range of 0.5-2%
from the 3% starch provided (dilute with deionized water).
3.
Place 3mL of the starch solutions prepared into each of 5 test tubes.
4.
At t=0, pipette 1mL of sample S25 into each of the 5 test tubes containing
starch.

5.

After 5 minutes, pipette 0.2mL of the reaction mixture into the iodine solution.
Measure the absorbance at 600nm against a suitable control.

D. Discussion
1.

Briefly describe the experiment that you designed in CI. What are the protein
concentrations in S25 and S60 in mg/mL?

2.

Based on the end-points of starch hydrolysis in CII, determine the protein activity
present in S25 and S60. Account for any differences, and discuss the significance of
protein activity vis--vis protein concentration (answers in Q1) with regards to
pharmaceutical drug assay.

3.

Explain your choice of the linearised form of the Michaelis-Menten relation in CIII.
Briefly explain the experiment that you designed in reference to this choice.
Determine the binding constant between starch and -amylase.

References:
Bailey, J. E. and Ollis, D. F. Biochemical Engineering Fundamentals, 2nd Ed., Chapter 3,
McGraw-Hill, 1986.