VNG – International University

BIOCHEMISTRY
LAB REPORT

Lecturer: MSc. Nguyễn Hồng Lan

Group 3
Nguyễn Thị Nhất Linh BTBTIU14108
Huỳnh Ngọc Tuyết BTBTIU14108
Văn Công Thắng BTBTIU14207
Liêu Tòng Văn BTBTIU14283
Ngô Thị Thanh Thuý BTBTIU14230
Class: Friday morning

EXPERIMENT 1
 PROTEIN QUANTIFICATION APPLYING HARTREE-
LOWRY ASSAY

I. INTRODUCTION

1. Purpose of experiment:
• To understand and apply the Hartree-Lowry assay in protein quantification.
To have a basic knowledge about the advantages and disadvantage of
Hartree-Lowry assay.
• To practice extracting protein from soybean, measuring optical density
(OD) with a spectrophotometer, building the standard curve, calculating the
amount of protein contained in soybean.
• Principles:
• A reagent is added to the protein solution to develop a color whose intensity
is measured in a spectrophotometer.
• A standard protein with known concentration is treated with the same
reagent.
• Base on the OD of the standard protein at different concentrations, we can
construct a standard curve which presents the relationship between these
two values (OD and concentration).
• Biuret reaction: reactions of copper ions with peptide bonds under alkaline
conditions.
• The oxidation of aromatic protein residues (E.g. Tyrosine, Tryptophan) by
Folin’s reagent.

2. Brief procedure:
 Use test tubes of Albumin (standard protein) with known concentrations to
determine OD by spectrophotometer graph the calibration curve.
  Use the calibration curve to find the protein of the sample based on average
OD calculate protein content in 100g of soybean.

II. RESULTS AND DATA ANALYSIS

Tube number 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’

1.69 0.14
0.02 0.15 0.28 1.568 0.144
OD 0.101 0.203 0.25 8 4
7 5 9
1.633 0.144
0.12 0.22 0.26
ΔOD 0 0.074 0.176 1.606 0.117
8 3 2
Protein concentration
0 50 100 150 200 250 X Y
(µg/mL)

Standard curve of protein concentration and optical

density
0.3

0.25 f(x) = 0 x + 0.01

Optical density

R² = 0.99

0.2

0.15 ∆
O
0.1 D

0.05

0
0 50 100 150 200 250 300

Protein concentration (μg/mL)

 The x-axis indicates protein concentration and y-axis indicates ∆OD.

 The equation of ∆OD line y=0.001 x +0.0149and R2=0.9886
 The value R2 quantifies good of fit R2 >0.95 , the value R2=0.9886
 Base on the equation of ∆OD line, the concentration of standard protein is:
x = (y -0.0149)/0.001
 Calculate the gram of protein in soybean
 From the calibration curve equation: y=0.001 x +0.0149, the unknown protein
concentration of tube 7-8 and 9-10 can be determined:
1.606−0.0149
 Tube 7-8: x 1= =¿ 1591.1(μg/ml)
0.001
Base on the graph, we can see that ΔOD of tube 7-8 is out of range, so the
value will be not be used to calculate further results.
0.117−0.0149
 Tube 9-10: x 2= =¿102.1( μg/ml)
0.001
 The gram of protein in of soybean is:
102.1 x 10000
m1 = = 203792.4 (μg) = 0.204 g
5.01
The gram of protein in 100g of soybean: m=0.204 x 100=¿ 20.4 ( g )

III. DISCUSSION

Look at the result, we see that it is expected to observe the blue color
corresponding with the amount of protein in each test tubes. The blue color
in each test tube is different because of the differences level of protein
concentration. The darker solution indicates the higher protein
concentration in the solution. From the test tube 1' to 6', the blue color level
increase. In test tube 7' and 8', they are clearly show dark blue color while
the color of test tube 9' and 10' are clear blue because test tube 7’ and 8’ are
only 100 dilute in albumin (protein) solution.
 The amount protein in 100g soybean sample in this experiment is not the
same as the number in nutrition fact table of 100g soybean in
[Link] (Source: Nutrient data for this listing was provided by
USDA SR-21) might be caused by:
 The samples we extracted are not from mature seeds so that the
concentration of nutrients is not high enough.
 The samples we extracted is a different type of soybean with the type
being done by USDA (Asian soybean, Bristish soybean, America
soybean, etc…)
 During the extraction time we might have some mistake (e.g. not
filtering carefully)

Reference protein value (gram per 100g of soybean) 13g

Experimental protein value (grams per 100 gram soybean) 20.4 g
Absolute error (%) 56.9%

IV. CONCLUSION

This class give the chance to practice how to extract from raw materials,
how to construct a standard curve and how to use a spectrometer. Moreover,
we can acknowlege that the Hatree-Lowry assay is highly recommend for
protein quantification bacause of its sensitivity.

V. REFERENCE

[Link]
products/2621/2#ixzz4Nrtoboq8
 EXPERIMENT 2
EXPLORING THE ENZYMATIC ACTIVITY OF
BROMELAIN
I. INTRODUCTION

The purpose of this experiment :

 Ascertaining how the enzyme Bromelain catalyze in a reaction
 Have acknowlegement about Tyrosine production from Casein

Principles:
 Use 3 kind of test tube which are Tyrosine, samples, and water
 Tyrosine tubes are use as control function while water tubes are blank
 Use Tricloroacetic acid to different function with each tube to inhibit or stop
the reaction in the approriate time
 Applying of colormetricall quantification by Folin reagent
 Use spectrophotometer to measure OD

II. RESULTS AND DATA ANALYSIS

Tubes of water Tubes of sample Tubes of Tyrosine

Tube number 1 2 3 1 2 3 1 2 3
0.10
OD 0.086 0.097 0.214 0.229 0.218 0.257 0.239 0.260
9
Average OD 0.097 0.220 0.252
∆OD 0 0.123 0.155
The activity is described by the formula:

450 × ∆OD M 1 x 1 UI
∆ ODT
× ×
10 x 2 mL ( )
Where:
 ΔODM = Average ODS – Average ODW
 ΔODT = Average ODT – Average ODW
 x1 : Tyrosine solution (ml)
 x2 : Enzyme solution (ml)
We have ΔODM= 0.123 and ΔODT=0.155 and the amount of Tyrosine solution (x 1) is
0.5 mL and the amount of sample solution (x2) is 0.2mL.
Bromelain enzymatic activity in this experiment:
450 ×(0.123) 1 0.5
× × =89.27 (UI /ml)
0.155 10 0.2

III. DISCUSSION

In this assay, casein acts as a substrate. When the protease we are testing
digests casein, the amino acid tyrosine is liberated along with other amino acids
and peptide fragments. Folin & Ciocalteus Phenol, or Folin’s reagent primarily
reacts with free tyrosine to produce a blue colored chromophore, which is
 quantifiable and measured as an absorbance value on the spectrophotometer.
The more tyrosine that is released from casein, the more the chromophores are
generated and the stronger the activity of the protease.
From the steps in lab manual, there are 3 tubes: Tube of tyrosine, sample
and water respectively. After putting 2.5mL 2% casein in the phosphate buffer
in each tube and adding 5 mL of 5% trichloroacetic acid in tube of tyrosine and
tube of water, white precipitation forms in each tube beacause of reaction
between the trichloroacetic acid and casein to make sure that casein solution is
reacted completely and bromelain extracted from pineapple soluttion,which is
added later, can not catalyze the casein to form tyrosin while with respect to
tube of sample, trichloroacetic acid is added at the end of the process after
addition of bromelain in order to inhibit bromelain to stop the catalyzed
reaction . During the process, we do add 0.5 mL of 0.2M HCl to create acidic
environment.

After transfer 2mL of filtered solution period, 1 mL of 0.5M NaOH and 1mL of Folin-
Ciocalteu reagent were added to 3 sample, which will react primarily with free
tyrosines. Thus, the standards have a gradation of color correlating with the amount of
tyrosine added and the highest concentrations of tyrosine appearing darkest. The
absorbance of our samples is measured by a spectrophotometer using a wavelength of
578nm – 620nm. Record the absorbance results.
In the first class, we do this experiment in the wrong way. Therefore, the result we
received is not correct. The number of Tyrosin after we calculated is lower than the
number of water. And, this lead to the result fail.
There are few reasons that we can predict why the experiment could be fail
 Wrong techniques while performing experiment that leads to the Tyrosin
become denaturated.
 Fail in preparing the chemicals.

When we did this experiment again in the make-up class, the result at that time was
correct. The standard curve is almost 100%.
We prepare the chemicals in the right way and each step in this experiments was
performed carefully.

IV. CONCLUSION

In this experiment, it introduces the method and technique to determine the activity of
bromelain by quantifying tyrosine produced from casein degradation under catalysis of
enzyme in 1mL of solution of bromelain mixture for 1 minute
After doing this experiment, it provided us the way to determine the enzymatic
activity, how to perform this experiment, and I have also known what is enzymatic
activity.
 EXPERIMENT 3
SOLUBLE CARBOHYDRATE QUANTIFICATION
APPLYING ANTHRONE ASSAY
I. INTRODUCTION

Anthrone dissolved in sulphuric acid may be used for the quantitative determination of
different carbohydrates. Quantitative determination is only possible where the identity
of sugar components is known because colour development varies with the different
sugars. Generally sugars and carbohydrates are extracted from dried and ground plant
material.
 The aim of the experiment:
To estimate the amount of carbohydrate present in the given sample
(banana) by using Anthrone method

 Principles:
Carbohydrates are dehydrated with concentrated H2SO4 to form “Furfural”,
which condenses with anthrone to form a green color complex which can be
measured by using colorimetrically at 620-630 nm (or) by using a red filter.
Anthrone react with dextrins, monosaccharides, disaccharides,
polysaccharides, starch, gums and glycosides. But they yields of color where
is to form carbohydrate to carbohydrate.

II. RESULTS AND DATA ANALYSIS

Tube number 1 2 3 4 5 6 7 8 9 10 11
OD 0.185 0.290 3.260 3.262 0.541 0.486
0.442 0.718 1.09 1.344 1.585
0.238 3.261 0.514
ΔOD 0 0.204 0.480 0.852 1.106 1.347 3.023 0.276
Carbohydrate
concentration 0 0 20 40 60 80 100 x y
(μg/mL)
Standard curve of carbohydrate concentration and optical
density
1.6
∆Optimal density

1.4
f(x) = 0.01 x − 0.04
1.2 R² = 0.99
1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120

Soluble carbohydrate Concentration (mg/mL)

 The x-axis shows carbohydrate concentration and y-axis shows ∆OD.

 The equation of ∆OD line: y = 0.014x – 0.0361 and R2 = 0.9943.
  Base on this equation, we have the concentration of standard carbohydrate:
x = (y + 0.0361)/0.014
 As results, we can see that all the tubes after adding the Anthrone reagent has
bluish- green color except the both of tube 1 and 2. Its observation
corresponding with the amount of carbohydrate of each test tubes. The green
color in each test tube is different due to the differences level of carbohydrate
concentration:
The higher carbohydrate concentration in the solution show how darker its
color is. From the test tube 1 to 7, the green color level increase.
In test tube 8 and 9, they are clearly show dark green color while the color
of test tube 10 and 11 has blue -green.

Calculate the gram of carbohydrate in banana:

 From the calibration curve equation, the concentration of soluble carbohydrate
in tube 8-9 and 10-11 can be measured:
3.023+ 0.0361
 Tube8-9: x 1= 0.014
=218.5( μg/ml)The OD value of tube 8 and tube 9 is
out of the range of the graph; therefore the carbohydrate concentration
contained is these tube cannot be determined.
0.276+0.0361
 Tube 10-11: x 2= 0.014
=¿ 22.29(μg /ml)

 The amount of carbohydrate in 100mL of 10,000 diluted solution is:

m = 22.29 × 10000 = 222900 (μg) = 0.2229 (g)
 The amount of carbohydrate in 100g of banana is:
0.2229 x 100
m= =11.145 ( g)
2

III. DISCUSSION

 100g of raw banana contains as much as 23g of carbohydrate; however, out of

23g of carbohydrate, 3g belongs to dietary fiber , and 12g belongs to sugar. In
this experiment, as we are trying to quantify the amount of soluble
carbohydrate in banana sample, it is rational to neglect the amount of
carbohydrate belonging to fiber. As for starch, sulfuric acid can hydrolyze
starch into sugar; however, the pulverized banana sample was prepared using
alcohol 90º and 80º, not sulfuric acid, so all the soluble carbohydrate used to
make the 100-diluted stock solution must have come from the original soluble
carbohydrate in the sample, which is sugar. Therefore, according to the
reference data from the USDA, we accept the amount of 12g soluble
carbohydrate in 100g raw banana.
 Reference carbonhydrate value (gram per 100g of banana) 12g
Experimental carbohydrate value (grams per 100 gram banana) 11.145 g
Absolute error (%) 7.125%

 Anthrone has very strong dehydration and release heat because its solvent is
concentrated sunfuric acid. Therefore, it need to be keep cold in order to avoid
moisture and cool down the bottle. (keep for using up to 48hr in approriate
condition e.g in frigde and is wrapped with Aluminium foil)
 The result in the following image shows that different concentration of Glucose
in each tube gives different colors. The lightest color belongs to 2 first test tube
which did not have Glucose and the darkest colors is 2 test tubes with 5mL of
100 dilution sample. Thus, we can assume that higher Glucose concentration
gives darker colors and the rise of OD values also.

IV. CONCLUSION

From this experiment, we can conclude that Carbohydrates are dehydrated with
concentrated H2SO4 to form “Furfural”, which condenses with anthrone to form a
green color complex which can be measured by using colorimetrically at 620nm.
Moreover, anthrone conclude acid sulfuric so we need to be carefully when we do
experiment with it.
After this experiment, the knowledge we can gain is the anthrone assay and
how carbonhydrate is dehydrated. Futhermore, the purity also can influence the
result important as well.

V. REFERENCE

[Link]
 EXPERIMENT 4
QUANTITATIVE DETERMINATION OF CALCIUM IN
POWDERED MILK
I. INTRODUCTION

 Purpose of the experiment:

 In this experiment, we will know how to determine the quantitive of
calcium in not only milk but also in blood sample, urine, food, etc. So,
the aim of doing this experiment is to find out which method can be
used to check the quantitive of calcium.
 General principles:
 Based on the results of a comparative study of the determination of
Calcium in milk powder using the potassium permanganate titration
methods.
 Oxalate ammonium will precipitate all Calcium ion in the solution.
Ca2+ + (COONH4)2  2NH4+ + Ca(COO)2
 Sulfuric acid added will dilute the Calcium oxalate precipitate.
Ca(COO)2 + H2SO4  CaSO4 + (COOH)2
 Titrate the solution with potassium permanganate solution to determine
the concentration of (COO)22- ion in the solution.
5(COOH)2 + 2KMnO4 + 3H2SO4  K2SO4 + 2MnSO4 + 10CO2 + 8H2O
 II. RESULTS AND DATA ANALYSIS

 Data table

Volume of Potassium Permanganate

V1(mL) 7.3

V2(mL) 7.6

V3(mL) 7.4

Average V
7.43
(mL)

 Calculation
The volume of Potassium Permanganate is determined until solutions become
light-pink color.
Step 1: Base on the reaction (3): Mn7+ + 5e →Mn2+
 The relation between the concentration (M) and the concentration (N) is:
N
M=
ne

 Where ne is quantity of electron that is used to transfer from reductive substance

to oxidative substance in reaction.
Step 2:
0.02
 The moles of 0.02N KMnO4 that contains in 1 liter of solution is: 5 mol
 The moles of 0.02N KMnO4 that contains in V liter of solution is:
V × 4 × 10-3 (mol)
 Base on the reactions (1), (2), (3), we have:
n 5 5
Ca 2+¿ ×nKMnO = ×V ×4 × 10−3 ( mol ) =0.01 V ( mol ) ¿
2 2 4

 The gram of Calcium that contains in (m)g of milk powder:

m Ca 2+ ¿
=0.01V ×40 ( g ) =0.4 V ( g ) ¿

 The gram of Calcium contains in 100g of milk powder:

m 100
Ca2+ ¿ =0.4 V × =80 V ( g) ¿
0.5

 Where:
V is the volume (L) of 0.02N KMnO4 used in determining the quantitative
measurement.
m: the weight of the powdered milk (m = 0.5g).
 The gram of Calcium contains in 100g milk powder with Average volume = 7.43
mL is:
m=80 xV ( g )=80 x 7.43=594.4 ( mg )

III. DISCUSSION

 Explanation of each step in Particial produres and Calcium quantification

1. Particial produres
 In step 1 we pretreat the milk sample by burning them in order to
prevent spattering during ashing.
 In step 3 the samples into the muffle furnace to heat with temperature of
500ºC ( this step is also called Dry ashing), water and other volatile
materials are vaporized and organic substances are burned in the
presence of the oxygen in air to CO2, H2O and N2. Most minerals are
converted to oxides, sulfates, phosphates, chlorides or silicates.
Therefore, after this step most of the left moisture in the samples will be
removed and the risk of errort in quantification also might be reduced.
2. Calcium quantification
 In step 3 we use methyl violet as an indicator to signalize a specific
range of number in the pH scale with the corresponding solution. When
the methyl violet change to yellow color means that the solution has
different pH number from the beginning.

 In some next step we heat the solution in 30 seconds to speed up the

reaction and then put these beaker in cool water to form the precipitation
formular of (COOH)2 faster prepare for the next collecting all
precipitation step. (Step 11)
  In step 12 we put acid in the Erlenmeyer flask as a enviroment to doing
titration with KMnO4. Then heat these in the water bath in order to
speed up the titration reaction.
5(COOH)2 + 2KMnO4 + 3H2SO4 → K2SO4 + 2MnSO4 +10CO2 +8H2O

 Dicussing about the result and error

Amount of Calcium in 100g milk powder (mg) 594.4g
Reference amount of Calcium in 100g milk 600g
powder (mg)
Absolute error (%) 0.93%

In our experiment, the average amount of Calcium ions in 100.0g powdered milk is
594.4mg while the original amount of Calcium ions labeled on the milk can was
600.0mg per 100.0g powdered milk. This means that the average amount of Calcium
ions in our experiment was approximately accurate comparing with the original
amount of Calcium ions labeled on the milk can.
However, there still was some error that might be occurred during the practices.
 The titration technique was not skillful: KMnO4 was dropped too slow
and the solution in the volumetric flask was cooled down. Moreover,
some drops of KMnO4 were remained in the wall of the flash and it is
hard for calculating the volume of KMnO4 used. Also, the flask was not
erratic vibrated. Therefore the accuracy of the reaction was affected.
 The solution was not stirred well while being heated in the water bath.
The time heating the flask before titration was not enough.
 Pipettes, beakers, flasks were not cleaned enough.
 The Calcium precipitate in filtering papers were not cleaned properly,
(COO)22- still existed.
Therefore, there are some attentions for improving the accuracy of experiment:

 All factors that affect the result mentioned above should be considered.
 Take the amount of chemical required accurately. For example, we use
the 1ml pipette to take out 0.5ml of chemical required. Depending on the
amount of chemical required, we need be considered to choose the
suitable pipette.
 Check the equipment, chemicals carefully and clean it clearly before
used. Do not use the paper tissue to swab it because there are still some
 paper pulp which contaminates the [Link] data recording should
be done carefully.

 Compare the results with the given data or other results for checking the
accuracy.

IV. CONCLUSION

This lab has shown how the amount of calcium present in milk can be found by
some practical steps. Throughout this experiment, we learnt that the Oxalate
Ammonium has an important role in quantitative determination Calcium in
milk powder because the calcium ions content in milk was precipitated and
filtered to determine the mass.

V. REFERENCE