1.

INTRODUCTION

Protein quantification is a vital part in biochemistry research. Scientist have

promoted and developed a wide range of methods for protein quantification; in spite
of this variety, each of these assays has its limitation due to biochemical separation
and processes and purposes of experimenters. Base on different factors of the
experiment or research, suitable assays will be applied to achieve the best outcome
with acceptable error.

In experiment 1, Hartree-Lowry is the best method for protein quantification that

can be produced in soybean because of its sensitivity. It can determine the
concentration of protein within the amount of 0.01 – 0.1 mg/mL; However, more time
is required to perform this method.

Standard protein, which has known concentration, is essential to construct

calibration curve. Albumin solution is selected as an appropriate standard, and Folin-
Ciocalteu solution is contributed to the experiment in other to develop a color, whose
intensity is measured by colorimetric method.

The principle of Hartree-Lowry assay can be apprehended through two reactions.

In the first reaction, undergone alkaline condition, the divalent copper ion forms a
complex with peptide bonds (the coordination of peptide bonds), in which it is reduced
to monovalent ion. The other reaction is the reduction of Folin-Ciocalteu reagent by
tyrosine and tryptophan residues in protein.

2. OBJECTIVES

The purpose of this practical is understanding and applying the Hartree-Lowry assay
in protein quantification and obtain knowledge about the advantages and
disadvantages of this assay. Moreover, we have to learn how to extract protein from
soybean, measure optical density (∆OD) by a spectrophotometer, calculate the amount
of protein that contained in soybean, construct the standard curve between the protein
concentration and optical density.
 3. EQUIPMENTS
- Test tubes - Motar
- Pipettes and pumps - Cylinder
- Volumetric flask (100ml) - Falcons (50ml)
- Beakers (50ml, 100ml) - Spectrophotometer
- Towel - Centrifuges

4. CHEMICALS
- 0.1 % Albumin solution
- Solution A: was prepared by getting 2g of Na2CO3 and dissolved it in 0.1M NaOH
to make 100ml
- Solution B: contain 100ml that dissolved 0.5g of CuSO4.5H2O in 1% Sodium
Citrate
- Solution C: is the mixture of solution A and B at the rate 48:2
- Folin-Ciocalteu reagent.

5. PROCEDURE

Step 1: Extraction
• 1st time:
Step 1: Get about 20mL of distilled water and put small amount of water into the
stone mortar and grind down the sample.
Step 2: Then put the rest of the water to the stone mortar and grind down the sample
carefully.
Step 3: Use a towel to squeeze the extract into a beaker. The grounds of the soybean
is still kept in the stone mortar.
• 2nd time:
Do the same as the 1st time with about 10 mL of distilled water.
• 3rd time:
Do the same as the 1st time with about 10 mL of distilled water.
Step 2: Dilution
- After extraction, we fill the solution into 2 falcons so that 2 falcons have the same
weight. Then put in the centrifuges, which 2 falcons with the same weight in the
opposite side, with 5000rs/m for 10 minutes.
- After centrifuging, the extract will be separated into 3 layers, the bottom layer
contains debris, the middle layer contains protein, the uppermost layer contains
mostly lipid.
- Use the pipet to take out 1mL from the middle layer and put into a volumetric flask.
Then, add water until it reachs the marked line on the flask → 100 times diluted
solution
- After that, pour the solution into a clean beaker. Rinse the flask with water then add
1mL of the 100-diluted solution and 99mL of water like the previous step to get
10,000-diluted solution

Step 3: Making standard curve and quantifying protein content of sample

- Prepare 6 test tubes numbered from 1 to 6
- The 0.1% albumin solution is diluted with the different amount of water to make the
protein solutions with different concentrations (0, 50, 100, 150, 200, and 250μg/mL).

Follow the table:

Tube Number 1 2 3 4 5 6
0,1% Albumin solution (mL) 0 0,5 1,0 1,5 2,0 2,5
Distilled Water (mL) 10 9,5 9,0 8,5 8,0 7,5
Shake test tube well
The concentration of the test tube 0 50 100 150 200 250
 - After that, make test tubes of standard protein solutions following the table
below by preparing 10 test tubes numbered from 1’ to 10’:
Tube Number 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’
Protein solution 0,4 0,4 0,4 0,4 0,4 0,4
Original solution
0,4 0,4
(100-diluted)
Sample solution
0,4 0,4
(10,000-diluted)
Solution C 2 2 2 2 2 2 2 2 2 2
Shake each tube well and keep them for 10 minutes
Folin-Ciocalteu 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2
Shake each tube well and keep them for 10 minutes
Distilled Water 2,4 2,4 2,4 2,4 2,4 2,4 2,4 2,4 2,4 2,4
Shake the tubes well, keep them for 5 minutes and measure the A750nm

Caution: The phenolic compounds are very sensitive when exposed to light
and/or air. So, during this reaction with folin's it needs to be put in dark place to
complete the reaction and change the color. So, we covered 10 test tubes with foil
before adding folin.

6. RESULTS
RESULT TABLE

Tube number 1' 2' 3' 4' 7' 5'8' 9' 6'10'
OD 0.159 0.16 0.069 0.066
0.067 0.099 0.138 0.156 0.177 0.2
0.1575 0.0675
ΔOD 0 0.032 0.071 0.089 0.11 0.133 0.0905 0.0005
Protein concentration
(µg/mL) 0 50 100 150 200 250 x y
 STANDARD CURVE
0.16

0.14 y = 0.0005x + 0.007

R² = 0.9816
0.12

0.1
OD

0.08

0.06

0.04

0.02

0
0 50 100 150 200 250 300

Protein concentration (µg/ml)

CALCULATE THE AMOUNT OF PROTEIN IN 100 GRAM OF SOYBEAN

• We have display equation: y = 0.0005x + 0.007

• With solution X, we have ΔOD = 0.0905
→ 0.0905 = 0.0005x + 0.007
→ x = 167
➔ Protein concentration of X is : 167 (µg/mL) = 1.67x10-4 (g/mL)
• With solution Y, we have ΔOD = 0.0005
→ 0.0005 = 0.0005x + 0.007
→ x = -13 (we ruled out this result because it doesn’t make sense, the value for
protein concentration can never be a negative number)
• The protein concentration in 100-diluted sample solution: 1.67x10-4 (g/mL)
The protein concentration in 10,000-diluted sample solution:
concentration100-diluted /100=1.67x10-4x10-2= 1.67x10-6 (g/mL)
• The gram of soybean protein in 100mL of 100-diluted sample solution:
mprotein= 100xconcentration100-diluted = 100x1.67x10-4 =1.67x10-2 (g)
• The protein concentration of the original soybean extract:
concentration100-diluted x100=1.67x10-4 x 100=1.67x10-2 (g/mL)
The grams of protein in 40ml of soybean extract:
1.67x10-2x40=0.668 (g)
0.668
The ratio of protein in 2g of soybean: = 0.334 = 33.4%
2
➔ There is approximately 33.4g of protein in 100g of soybean
 7. DISCUSSION

- Looking at these images, we can easily see that the color of the tubes from 1’ to 6’ gets
more and more intense due to the increase in protein concentration (from 50µg/mL
to 250µg/mL). The color in 2 pairs of test tubes 7’-8’ and 9’-10’ are the same because
they have the same protein concentration.

- The given concentration of albumin in 6 test tubes helped us to build the standard
curve. However, looking into the graph, we can see that 1 point has drifted far away
from the line, this indicates a greater OD value (compared to the other points). The
propable reason for this to happen can be a mistake during the process of making the
solution, we may have put too much of albumin and Folin-Ciocalteu reagent into the
test tube or we didn’t put in enough water; contaminants from test tubes also
contribute to the problem

- Using the curve, we can approximate the protein concentration in the 100 times
diluted and 10000 times dilluted soybean extract then figure out the amount of
protein containing in 2 grams of soybean. But these are just close approximation,
there are many reasons preventing us from getting the true value of protein quantity.
The first reason we can think of is that we can never be sure if we had everything
extracted from the soybean. Second, Folin-Ciocalteu reagent (or some similar
chemicals) easily gets denatured when contact with light, we can not guarantee that
no light has contacted with Folin-Ciocalteu reagent.

- During calculation, solution Y’s OD gave us a negative protein concentration, we

can deduce that solution Y is over-diluted, we cannot use it to figure out the protein
concentration in the soybean extract.
 8. CONCLUSION
• The experiment mainly focuses on measuring the protein concentration in
soybean in order to figure out how much protein can be extracted from soybean.
The result is roughly 33.4g of protein from 100g of soybean
• The experiment also helped us understand more thoroughly about Hartree-Lowry
assay. After seeing the result, we can now improve our result by cleaning
equipments carefully, practice using the pipette to get the exact volume of
chemicals needed to perform the experiment.
9. REFERENCE
• Biochemistry Lab Manual