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# Experiment #2

Objectives:

## To isolate the following proteins:

1. Casein from skimmed milk by isoelectric precipitation
2. Albumin from skimmed milk by heat denaturation
3. Gluten from wheat flour by difference in solubility
4. Myoglobin from beef hearts by salt-induced precipitation
To analyze chemical groups responsible for color reactions and explain the
principle involved in each test
To perform acid, alkaline, and enzymatic hydrolysis on the isolated proteins
To determine the amino acid components of the proteins by thin-layer
chromatography
To quantitatively determine protein concentration in a given sample through

Methodology:

## Put the needed amount of

Bovine serum albumin (BSA)
standard (100 g/mL) and
water.
reagent to each tube.

## Put 5 drops of from each tube

in a microwell plate

## Read the absorbance at 595

nm within an hour

## Plot the A595 against

concentration (g/mL)

Results:
TUBE NO.

mL
STANDAR
D
mL H2O

0
mL

0.10
mL

0.15
mL

0.20
mL

0.25
mL

0.30
mL

0.35
mL

0.40
mL

0.45
mL

1.50
mL
0.04
5
0

1.40
mL
0.48
3
3.33

1.35
mL
0.51
8
5

1.30
mL
0.59
8
6.67

1.25
mL
0.65
1
8.33

1.20
mL
0.68
4
10

1.15
mL
0.69
1
11.6
7

1.10
mL
0.70
7
13.3
3

1.05
mL
0.79
4
15

ABSORBA
NCE (A595)
Concentra
tion
(ug/mL)
(Add 1.5 mL Bradford Reagent to all the tubes to make 3mL)

## Slope (m) : 0.024527592

y-intercept (b) : 0.415923962
Line regression equation (y=mx+b): y=0.025x+0.416
r2= 0.972943768

10
(Unkno
wn)
1.25 mL

-------1.644
50

1. Why is the milk solution heated to 40 OC during the isolation of
casein?
It is because by heating the milk solution to 40 degrees celcius, it will
induce denaturation of protein. This will lead to the binding of the
added 10% acetic acid with the denatured protein making the
environment necessary for casein coagulation.
2. What are the other protein components of milk aside from
casein?
Aside from casein, milk contains also some serum (whey) protein
family. This serum consists of approximately 50% -lactoglobulin, 20%
-lactalbumin, blood serum albumin, immunoglobulins, lactoferrin,
transferrin, and many minor proteins and enzymes.
3. What are the principles involved in the isolation of proteins in
the experiment?
The principle involved in the isolation of proteins in the experiment is
isoelectric precipitaion which involves the principle on isoelectric pH of
certain solutions. Casein has an isoelectric pH of 4.6 and is insoluble in
solutions with pH lower than 4.6. The pH of milk is around 6.6 which
gives casein the negative charge and makes it a soluble salt. Once an
acid is added to the solution, the negative charge of casein becomes
neutral, precipitating the neutral protein which is casein.
enzymatic hydrolyses.
Acidic Hydrolysis
Advantage: It has a fast rate of reaction which facilitates continuous
processing
Disadvantage: It has low product yield and time consuming
Basic Hydrolysis
Advantage: It is more environment friendly compared to other processes
Disadvantage: It is costly and time consuming
Enzymatic Hydrolysis
Advantage: It is a milder process compared to other chemical
conversions. It requires less energy and produces less by-products
Disadvantage: It has low reaction rated and very rare

## 5. Provide the following for the qualitative color reactions used in

experiment: specificities, positive results, and principles
involved.
Biuret Test: 2.5 M NaOH & 0.1 M CuSO4; (+) color change
Ninhydrin Test: 0.1% Ninhydrin Solution; (+) blue-violet
coloration
Xanthoproteic Test: HNO3 & NaOH; (+) color change
Millons Test: Millons reagent; (+) color change
Hopkins-Cole Test: Hopkins-Cole reagent & H2SO4; (+) color
change at interface
Sakaguchi Test: 10% NaOH, 0.02% Naphthol solution, & 2%
NaOBr; (+) color change
Nitroprusside Test: 3M NaOH, 2% nitroprusside solution; (+)
formation of red solution
assay compared to other quantitative methods
Unlike other protein assays, the Bradford protein assay is less
susceptible to interference by various chemicals that may be present
in protein samples. However, the bradford assay is linear over a short
range often making dilutions of a sample necessary before analysis. It
is also inhibited by the presence of detergents.

Methodology
Protein Assay Using the Bradford Method
A series of test tubes were prepared as follows:
TUBE
NO.
mL
STANDA
RD
mL H2O

1
(Bla
nk)
0 mL

0.10
mL

0.15
mL

0.20
mL

0.25
mL

0.30
mL

0.35
mL

0.40
mL

0.45
mL

1.50
mL

1.40
mL

1.35
mL

1.30
mL

1.25
mL

1.20
mL

1.15
mL

1.10
mL

1.05
mL

10
(Unkno
wn)
1.25 mL

--------

## Table 1 Bradford Assay test tube samples

1.5 mL of Bradford Reagent was added to each test tube. After 5 minutes
standing, 5 drops of each sample were put into a microwell plate. The
microwell was subjected to an absorbance reading machine. The absorbance
was read at 595 nm. An albumin standard curve was constructed with a plot
of concentration (ug/mL) against absorbance (A595). Linear regression
analysis was also used to determine the concentration of the samples.
Results and Discussion:
The Bradford assay is commonly used to determine the total protein
concentration of a sample. The method is based on the binding of Coomassie
dye to proteins in acidic solution leading to an increased absorbance of the
sample at 595 nm. [1] The assay is sensitive to about 20 to 200 microgram
protein.
The dye involved in the process is the Coomassie Brilliant Blue G-250. Under
acidic conditions the red form of the dye is converted into its bluer form,
binding to the protein being assayed. During the formation of this complex,
two types of bond interaction take place: the red form of Coomassie dye first
donates its free electron to the ionizable groups on the protein, which causes
a disruption of the protein's native state, consequently exposing its
hydrophobic pockets. These pockets in the protein's tertiary structure bind
non-covalently to the non-polar region of the dye via van der Waals forces,
positioning the positive amine groups in proximity with the negative charge
of the dye. [2] The bond is further strengthened by the ionic interaction

between the two. The binding of the protein stabilizes the blue form of the
Coomassie dye; thus the amount of the complex present in solution is a
measure for the protein concentration, and can be estimated by use of an
computed amount of concentration can be seen on Table 2.
TUBE NO.

ABSORBA
NCE (A595)
Concentra
tion
(ug/mL)

1
(Bla
nk)
0.045
0

0.48
3
3.33

0.51
8
5

0.59
8
6.67

0.65
1
8.33

0.68
4
10

0.69
1
11.6
7

0.70
7
13.3
3

0.79
4
15

10
(Unkno
wn)
1.644
?????

## The concentration of the standards were calculated using the formula

C1V1=C2V2, wherein
C1=concentration of the sample
V1=amount of BSA
C2= concentration of the unknown
V2=total volume including water
When a large number of samples are being run, it is preferable to
construct a calibration or standard curve. Standard curves may be obtained
by measuring the absorbance of a series of standard solutions whose
concentration is in the range of the solution being measured. Each standard
solution must be prepared in exactly the same manner as the solution being
measured, the absorbance values of the standard solution is read directly
from the graph. The albumin standard curve for test tubes 2 to 9 can be seen
as follow.

## In Figure 1, x is concentration and y is absorbance. It is likely that the

unknown will have absorbance numbers outside the range of the standard.
These should not be included calculations, as the equation given cannot
apply to numbers outside of its limitations. Same also applies for the blank
test tube. Using the obtained results from the absorbance and concentration,
we then get the linear equation (y=mx+b) and compute for the r2 value.
Ideally, the R2 value will be as close to 1 as possible. R represents the sum
of the square values of the fit subtracted from each data point. Therefore, if
r2 is much less than one, consider redoing the experiment to get one with
more reliable data. [4]

## The equation of the standard curve is then used to determine the

concentration of the unknown protein. Manipulating the equation y=mx+b, it
will result in x = (y-b)/m. Thus in finding the concentration (x) of the unknown,
changing the values of the unknowns absorbance (y) which is 1.644, yintercept (b) of 0.416, and a slope (m) of 0.025, the result will be 50.07.

References