NAME: Latiyah Timothy LAB PARTNER: Jasiel Mohammed

ID# 816012983 DATE: Session 3- 10th March 2020

COURSE CODE: Bioc 2169: Practical Skills in Biochemistry II

TITLE OF LAB: Characterization of Yeast Invertase using Bradford's Assay and Nelson's Assay
AIM: To characterize and determine the activity of yeast invertase from Saccharomyces cerevisiae
using Bradford's Assay and Nelson's Assay.

THEORY:
In this lab, yeast invertase from Saccharomyces cerevisiae was characterized by Bradford's assay and
Nelson's assay. Bradford's assay is a colourimetric method that is used to quantify the total protein
concentration in a sample. This method uses the dye Coomassie blue which upon binding to proteins
in an acidic environment via Van der Waals forces and hydrophobic interactions, shifts spectrally
from reddish-brown with an absorbance maximum at 470nm to blue with an absorbance maximum of
610nm. There is a great difference between the two dye forms at 595nm, which makes it the ideal
wavelength to measure the colour of the protein-Coomassie blue complex. The colour formation of
the protein-dye complex is associated with the basic amino acids such as arginine, lysine and
histidine. (Walker and Wilson 2010)
Advantages of this assay are that the reagent is simple to prepare, the colour develops rapidly and is
stable and it is not time-consuming. The detection limit for this assay is 20μg protein cm-3. The main
disadvantage of this assay is that surfactants used in a protein isolation protocol, even at low
concentrations can cause the dye to precipitate out of solution. Another disadvantage is that the
amount of binding to the protein is dependent on the content of the basic amino acids in the protein.
The fact that this assay is done in an acidic medium is another disadvantage as some proteins are not
soluble in acidic media. (Walker and Wilson 2010)

Figure 1: A reaction schematic for the Coomassie blue dye and the protein in the Bradford's Assay
(ThermoFisher Scientific n.d.)

The Nelson-Somogyi assay is a colourimetric technique used to quantify the concentration of

reducing sugars in a sample. This method is used to determine invertase activity via the formation of
reducing sugars after the hydrolysis of sucrose by yeast invertase. Reducing sugars such as glucose
has a terminal aldehyde group and can become oxidised to form a carboxylate while the oxidising
agent Cupric ions (Cu2+) in alkali can be reduced to form a cuprous ion (Cu+). Ketoses such as
fructose have to first be converted to an aldose sugar by alkaline conditions and heat to reduce the
cuprous ion. The principle of this assay is that when the reducing sugars are heated with alkaline
copper tartrate, the copper tartrate is reduced from Cu2+ to Cu+ and forms cuprous oxide. This oxide is
then treated with arsenomolybdic acid, the molybdic acid is reduced to molybdenum blue which
forms the blue colour. This colour is then measured spectrophotometrically at 510nm. The advantage
of this method is that the colour formed is stable over long periods and the optical density is
proportional to glucose concentration. A disadvantage of this method is that it is time-consuming and
cannot differentiate between types of reducing sugars. (Somogyi 1952)

(Reducing sugar + Cu2+ → Cu+ + oxidized sugar)

Figure 2: Nelson-Somogyi Assay Reaction. (Shao and Hui-Mei Lin 2018)

Enzyme Units, Enzyme Activity, Total Protein, Specific Activity, Enzyme Yield and Fold
Purification was also determined. Enzyme units (U) refer to the ability of the enzyme to convert one
μmol of the substrate to the product in one minute under specific conditions such as 25oC and at
optimum pH. (Walker and Wilson 2010)
Enzyme Activity is the measure of the quantity of active present and refers to enzyme units
(μmol/min) multiplied by the reaction volume (ml), therefore units for enzyme activity is
μmol/min/ml. (Walker and Wilson 2010)
The specific activity of an enzyme is the number of enzyme units present (U) per milligram of
protein. It is a measure of purity, as the enzyme undergoes more purification steps the specific activity
increases. The units are (U/mg). (Walker and Wilson 2010)
Fold purification is the specific activity at a purification level divided by the specific activity at the
initial purification step. As purification of the enzyme increases the fold purification increases.
(Walker and Wilson 2010)
Enzyme Yield is defined as a percentage where the total units of enzyme (U) of a purified fraction
divided by the total units of the crude fraction. As the enzyme goes through more purification steps
the yield decreases. A yield of more than 30% is considered acceptable. (Walker and Wilson 2010)
Total Protein is the amount of protein present in a sample and can be calculated by the protein
concentration of a part of the fraction and multiplying it by the fraction's total volume. The unit for
total protein is milligrams. (Walker and Wilson 2010)
APPARATUS AND MATERIALS:
1. Nelson’s Reagents: to remove the toxic arsenic compounds from the colour reaction system.
Solution Nel A: 25g of sodium carbonate
(provided) 25g of sodium tartrate
20g of sodium bicarbonate
20g of sodium sulphate (anhydrous)
Dissolved in distilled water and made up to 1L
Solution Nel B: 15g copper sulphate. 5 H2O
(provided) 2 drops of conc. sulphuric acid
Dissolved in 100mL distilled water
Prepare Nelson’s Reagent: -mix 50mL of Solution Nel A and 2mL of Solution Nel B: 26ml
2. Arsenomolybdate Reagent: to react with cuprous oxide and form molybdenum blue for
spectroscopy. 9ml
50g of ammonium molybdate
6g of sodium arsenate
42mL of conc. sulphuric acid
Make up to 1L with distilled water
3. Bradford’s Reagent: 19ml
100mg Coomassie Brilliant Blue G-250
50mL 95% Ethanol
100mL 85% (w/v) Phosphoric Acid
Dissolve and dilute to 1L with deionized water
4. Diluted Folin Ciocalteau’s Reagent (1:1 with water) this has been provided for you.
5. Additional reagents
Standard Protein solution: BSA 400μg/mL: 2.6ml
Standard Glucose solution: 4mM: 1.5ml
Standard Fructose solution: 4mM: 0.2ml
Standard Sucrose solution: 4mM: 0.2ml
0.5M Sucrose: 2.6ml
METHOD:
Protein determination by the Bradford Method
A calibration curve was prepared by pipetting 0, 0.1, 0.2, 0.2, 0.3, 0.5, 0.5 and 0.8mL of the provided
standard protein solution (BSA, 400μg/mL). The volumes were made up to 1.0mL with distilled
water. The fractions were diluted as follows, fraction 1- 1:4 dilution ratio to prepare a 1mL sample,
fraction 2; 1:4 dilution ration to make up a 1mL volume; fraction 3; a 1:1 dilution ration to prepare a
0.5mL solution; fraction 4; no dilution.
11 sample tubes were prepared as outlined using the diluted fraction which was previously prepared,
tube 1-0.99mL of water and 0.01mL of diluted fraction, tube 2 -0.98mL of water and 0.02mL of
diluted fraction, tube 3 -0.95mL of water and 0.05mL of diluted fraction, tube 4 -0.99mL of water and
0.01mL of diluted fraction, tube 5 -0.98mL of water and 0.02mL of diluted fraction, tube 6 -0.95mL
of water and 0.05mL of diluted fraction, tube 7 -0.99mL of water and 0.01mL of diluted fraction, tube
8 -0.98mL of water and 0.02mL of diluted fraction, tube 9 -0.95mL of water and 0.05mL of diluted
fraction, tube 10 -0.7mL of water and 0.3mL of diluted fraction and tube 11 -0.5mL of water and
0.5mL of a diluted fraction.
There were 19 tubes in total 8 calibration curve tubes +11 sample tubes) each containing 1mL of
solution each. 1mL of Bradford reagent was added to each tube, the tubes were vortexed and left
standing for exactly 45 minutes.
The absorbance was read against the calibration curve blank at 595nm. A calibration curve of
absorbance against milligrams of protein was plotted.

Assay of Fractions for Invertase Activity using Nelson’s Procedure for Reducing Sugars
Preparation of the Calibration Curve for reducing sugars
A calibration curve was prepared using 4mM glucose. 0, 0.05, 0.05, 0.1, 0.15, 0.2, 0.2, 0.25 and
0.3mL of the standard glucose solution was pipetted into 9 test tubes respectively. 0.2mL of 4mM
Fructose and 4mM Sucrose was also pipetted into 2 additional test tube, respectively. All volumes
were made up to 1mL with distilled water.
1 mL of Nelson’s Reagent was added to EACH tube, the samples were VORTEX and place in a
boiling water bath for EXACTLY 20 minutes. The tubes were allowed to cool to room temperature
and then 1mL of the Arsenomolybdate reagent was added using a mechanical pipette. The samples
were mixed well on a VORTEX mixer and left standing at room temperature for 5 minutes. 7mL of
distilled water was added to ALL tubes, the samples were mixed well on the VORTEX mixer. The
absorbance readings were taken at 510nm using the 0 tube from the calibration curve as the blank. A
calibration curve for reducing sugars of Absorbance vs. micromoles of reducing sugar was then plot.
Assay of fractions for Invertase Activity
Dilutions of the fractions were made with cold deionized water (0 – 4°C) as follows:
Fraction 1 - 1 in 2000 (Prepare 20mL)
Fraction 2 - 1 in 1500 (Prepare 15mL)
Fraction 3 - 1 in 3000 (Prepare 30mL)
Fraction 4 - 1 in 1000 (Prepare 10mL)
A series of tubes for incubation as shown in Table 1 was set up
 RESULTS:
Table 1: Results obtained for Protein Calibration Curve:

Tube # Volume of Volume of Milligrams of Absorbance (595 nm)

Standard BSA Water Added Protein (mg)
(ml) (ml)
1 0 1 0 0
2 0.1 0.9 0.01 0.074
3 0.2 0.8 0.02 0.214
4 0.2 0.8 0.02 0.215
5 0.3 0.7 0.03 0.384
6 0.5 0.5 0.05 0.663
7 0.5 0.5 0.05 0.665
8 0.8 0.2 0.08 0.786

Table 2: Results obtained for the Protein Determination by Bradford's Assay for the Four Diluted
Fractions:
Fraction 1 2 3 4
Tube # 9 10 11 12 13 14 15 16 17 18 19
Volume of 0.01 0.02 0.05 0.01 0.02 0.05 0.01 0.02 0.05 0.3 0.5
Fraction (ml)
Volume of 0.99 0.98 0.95 0.99 0.98 0.95 0.99 0.98 0.95 0.7 0.5
water added
(ml)
Dilution 1:4 1:4 1:4 1:4 1:4 1:4 1:1 1:1 1:1 Undiluted Undiluted
Absorbance 0.401 0.756 0.929 0.121 0.187 0.514 0.078 0.169 0.383 0.046 0.057
(595nm)
Milligrams of 0.0353 0.0665 0.0817 0.0106 0.0164 0.0452 0.0069 0.0149 0.0337 0.0040 0.0050
Protein (mg)
Concentration 3.53 3.325 1.634 1.06 0.82 0.904 0.69 0.745 0.674 0.0133 0.01
of Protein
(mg/ml)
Average 2.83 0.928 0.703 0.0183
concentration
of Protein
(mg/ml)

Table 3: Summary of Results for Protein Determination:

Fraction Average Protein Protein Total Protein (mg)

Concentration Concentration in
(mg/ml) Stock (mg/ml)
Crude (F1) 2.83 14.15 346.68
Heat (F2) 0.928 4.64 64.50
Alcohol (F3) 0.703 1.406 7.03
Column (F4) 0.0183 0.0183 0.183
 Table 4: Results Obtained for the Calibration Curve for Reducing Sugars:

Tube Volume of Glucose Standard Absorbance (510 nm) μmol of Reducing

# (ml) sugar
1 0.00 0.00 0
2 0.05 0.088 0.2
3 0.05 0.095 0.2
4 0.10 0.162 0.4
5 0.15 0.185 0.6
6 0.20 0.185 0.8
7 0.20 0.197 0.8
8 0.25 0.226 1.0
9 0.30 0.417 1.2
10 Fructose (4mM, 0.2) 0.659 0.8
11 Sucrose (4mM, 0.2) 0.027 0.8

Table 5: Results obtained for Reducing Sugars Test using Nelson's Assay for the Four diluted
fractions
Tube Fraction Dilution Absorbance Corrected μmol of Reducing
#1 (510nm) Absorbance Sugars
1 Sucrose Blank - 0.001 0 0
Crude 2 0.02ml 1:2000 0.014 0.013 0.0451
3 0.06ml 1:2000 0.089 0.088 0.305
4 0.1ml 1:2000 0.245 0.244 0.847
Heat 5 0.02ml 1:1500 0.035 0.034 0.118
6 0.06ml 1:1500 0.049 0.048 0.167
7 0.1ml 1:1500 0.213 0.212 0.736
Alcohol 8 0.02ml 1:3000 0.069 0.069 0.240
9 0.06ml 1:3000 0.040 0.039 0.135
10 0.1ml 1:3000 0.038 0.037 0.128
Column 11 0.02ml 1:1000 0.004 0.003 0.0104
12 0.06ml 1:1000 0.006 0.005 0.0174
13 0.1ml 1:1000 0.003 0.002 0.00694
14 Glucose Blank - 0.005 0 0
15 Glucose Standard - 0.519 0.514 1.784
 Table 6: Summary of Results for Nelson's Assay for the Four Diluted Fractions

Tube Fraction μmol of μmol/min/ml Units/ml Average Units/ml

#1 Reducing Units/ml Of Stock
Sugars Fraction
Crude 2 0.02ml 0.0451 0.226 0.11275 0.263 526.94
3 0.06ml 0.305 0.508 0.25417
4 0.1ml 0.847 0.847 0.42350
Heat 5 0.02ml 0.118 0.590 0.295 0.267 401.08
6 0.06ml 0.167 0.278 0.13917
7 0.1ml 0.736 0.736 0.368
Alcohol 8 0.02ml 0.240 1.2 0.60 0.259 776.50
9 0.06ml 0.135 0.225 0.1125
10 0.1ml 0.128 0.128 0.064
Column 11 0.02ml 0.0104 0.052 0.026 0.015 14.66
12 0.06ml 0.0174 0.029 0.0145
13 0.1ml 0.00694 0.007 0.00347

Table 7: Purification Table for the enzyme Yeast Invertase:

Purification Total Protein Total Specific Enzyme Fold
Step (mg) Enzyme Activity (U/mg) Yield (%) Purification
Activity (U)
Crude (F1) 346.68 12910.1 37.239 100 1
Heat (F2) 64.50 5575.1 86.435 43.2 2.32
Alcohol (F3) 7.03 3882.5 552.276 30.2 14.8
Column (F4) 0.183 146.6 800.911 1.14 21.5

CALCULATIONS:
1) Determining mg of Protein for calibration curve: Using Tube 8
Concentration of BSA = 100μg/ml
(0.8 × 100)
∴ in 0.8 ml of BSA = = 80μg
1
1μg = 10−3 mg

∴ 80μg = 80 × 1 × 10−3 = 0.8mg of BSA

2) Determining mg of Protein in Diluted Fraction tube: Using tube 9

From the equation of the line: y = 11.373 x
Where y = Absorbance, and x = concentration of protein in mg
0.401
∴X= = 0.0353 mg of Protein
11.373
3) Determining μmol of Reducing Sugars for calibration curve: Using Tube 8
Concentration of Glucose = 4mM
4
in 1000ml = 𝑚𝑚𝑜𝑙
1000
(0.25 × 4)
∴ in 0.25 ml of Glucose = = 0.001mmol
1000
1mmol = 103 μmol

∴ 0.001mmol = 0.001 × 1 × 103 = 1.0μmol of glucose

4) Determining μmol of Reducing Sugars in Diluted Fraction tube: Using tube 9

From the equation of the line: y = 0.2881 x
Where y = Absorbance, and x = concentration of protein in mg
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 0.040 − 0.001 = 0.039
0.039
∴X= = 0.135μmol of Glucose
0.2881
5) Flow Chart of Calculations for Invertase Activity Using the Crude Fraction (Fraction 1)

Units/ml
Using tube 2: 0.0451μmol
0.0451
umol/min = 10
= 0.00451μmol/min

∴ in 0.02 ml = 0.00451U
0.00451
∴ 1ml = 0.02
× 1 = 0.226 U/ml

1unit of enzyme = 2μmol of reducing sugar per minute

1
∴ 0.226 = × 0.226 = 0.11275U
2
0.11275+0.25147+0.42350
Tube 3: 0.25417 U AVERAGE: 3
= 0.263 U/ml

Tube 4: 0.42350 U

Units/ml (activity) of Stock

U × dilution Factor
∴ 0.263 × 2000 = 526.94 U/ml

Total Units (U)

Activity of Stock × Total Volume of Stock
∴ 526.94 × 24.5 = 12910.1 U
Protein Concentration in Diluted Fraction (fraction saved in tube):
Using Tube 9: 0.0353 mg
0.0353
mg/aliquot = = 3.53 mg/ml
0.01
size of aliquot
Tube 10: 3.325 mg/ml
Tube 11: 1.634 mg/ml
3.53+3.325+1.634
AVERAGE = 3
=2.83 mg/ml

Protein Concentration in Stock Fraction (mg/mL stock)

Average (mg/ml) × Dilution factor
2.83 × 5 = 14.15 mg/ml

Total Protein (mg):

Protein conc. in Stock Fraction × Total Volume of stock
14.15 × 24.5 = 346.68 mg

Specific Activity:
12910.1
Units/mL in stock = =37.239 mg/ml
346.68
Protein (mg/mL) in stock
 Enzyme yield:
12910.1
total Units in purified fractions =12910.1 × 100 = 100%
total Units in crude extract

Fold Purification:
37.239
Specific activity of purified fraction = =1
37.239
Specific activity of crude extract
Graph 1:

Graph of Absorbance (595nm) vs Milligrams (mg) of Standdard

Protein
1
0.9
0.8
Absorbance at 595 nm

0.7
y = 11.373x
0.6 R² = 0.9398
0.5
0.4
0.3
0.2
0.1
0
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
Standard Protein (mg)

Scale: Y axis: 0.5 cm = Absorbance of 0.1 X axis:1.5cm =0.01mg of protein

Graph 2:

Graph of Absorbance (510 nm) vs Micromoles (μmol) of

Reducing Sugar
0.45
y = 0.2881x
0.4 R² = 0.8425
0.35
Absorbance at 510nm

0.3

0.25

0.2

0.15

0.1

0.05

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Reducing Sugar (μmol)

Scale: X axis: 1.89 cm= 0.2 μmol Y axis: 0.6cm =Absorbance of 0.05
DISCUSSION:
In this lab, yeast invertase was characterized, with the use of the Nelson-Somogyi assay and
Bradford's assay. Bradford's assay is a colourimetric technique used for protein quantification in a
sample. (Walker and Wilson 2010) From Bradford's assay, the concentrations of protein for each
diluted fraction was determined to be 2.83mg/ml 0.928mg/ml 0.703mg/ml and 0.0183mg/ml for
fractions 1, 2, 3 and 4 respectively. The total protein in each stock fraction was determined to be
346.68mg, 64.50mg, 7.03mg, 0.183mg for fractions 1,2, 3, and 4 respectively.
The Nelson-Somogyi assay is another colourimetric method which quantifies reducing sugars. The
invertase activity was determined using this method in the formation of glucose from the hydrolysis of
sucrose was quantified. (Somogyi 1952) A sucrose blank was used as a zero-time tube. The nelson's
reagent was added before the sucrose, therefore, no glucose should be present, however, if there were
reducing sugars present, the sucrose blank was subtracted from the reaction tubes for accurate
quantification of glucose. From Nelson-Somogyi assay, the U/ml for each diluted fraction was
determined to be 0.263U/ml, 0.267U/ml, 0.259U/ml and 0.015U/ml for fractions 1, 2, 3 and 4. The
U/ml for the stock fractions were 526.94U/ml, 401.08U/ml, 776.50U/ml and 14.66U/ml for fractions
1, 2, 3 and 4 respectively. The total units of enzyme activity for each fraction was determined to be
12910.1U, 5575.1U, 3882.5U and 146.6U for fractions 1, 2, 3 and 4 respectively.
The specific activity for fractions 1, 2, and 4 were 37.239U/mg, 86.435U/mg, 552.276U/mg and
800.911U/mg respectively. The enzyme yield for each fraction was determined to be 100%, 43.2%,
30.2% and 1.14% for fractions 1, 2, 3 and 4 respectively. Finally, the fold purification for fractions 1,
2, 3 and 4 was 1, 2.32, 14.8 and 21.5 respectively.
From the purification table, it can be seen that as the enzyme went through the purification steps, from
crude to heat to alcohol and column the total protein, the total enzyme activity and the enzyme yield
decreased as the enzyme became more purified. However, as expected the specific activity and fold
purification increased. Based on specific activity, the best isolation technique for invertase seems to
be alcohol solvation as it had a 6.4-fold increase, compared to the 2.32-fold increase and 1.5-fold
increase using heat extraction and column chromatography respectively. This same conclusion was
also drawn in a similar lab in which the specific activity increased 3.4-fold via ethanol precipitation
compared to that of the 2.3- and 2-2-fold obtained by anion exchange and gel filtration respectively.
(Timmerman 2012)Based on fold purification it can also be concluded that yeast invertase accounts
for 1 out of the 21.5 mg of total protein contained in the initial 24.5ml of the extract prepared.

SOURCES OF ERROR AND PRECAUTIONS:

-Ensure that the tubes containing Nelson's Reagent are foiled as it is light sensitive.
-Ensure that the Arsenomolybdate is foiled or stored away in a dark cupboard as it is light sensitive.
-Ensure that gloves are worn when using the Arsenomolybdate as it contains arsenic and can be toxic
if it gets in contact with skin.
References
Shao, Yijing, and Amy Hui-Mei Lin. 2018. "Improvement in the quantification of reducing sugars by
miniaturizing the Somogyi-Nelson assay using a microtiter plate." Food Chemistry 240 898-
903.
Somogyi, M. 1952. "Determination of reducing sugars by Nelson-Somogyi method." Journal of
Biological Chemistry 245.
n.d. ThermoFisher Scientific. Accessed 03 21, 2020. https://www.thermofisher.com/tt/en/home/life-
science/protein-biology/protein-assays-analysis/protein-assays/bradford-assays.html.
Timmerman, Anthony P. 2012. "The Isolation of Invertase from Baker's Yeast- An introduction to
purification tecniques." Protein Purification 29-52.
Walker, John, and Keith Wilson. 2010. Principles and Techniques of Biochemistry and Molecular
Biology Seventh edition. New York: Cambridge University Press.