Bio Chem Lab Report 01
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Transcript of Bio Chem Lab Report 01
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BIOC 12141
PREPARATION OF CRUDE INVERTASE
EXTRACT
&
DETERMINATION OF SPECIFIC ACTIVITY
OF INVERTASE
Student Name : I.W. Chathuranga Chandrasiri
Admission Number : BS/2012/181
Perform Date : 15/08/2012
Submission Date : 22/08/2012
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Date : 2014.08.15
Experiment No : 01
Experiment title :
Preparation of crude invertase extract and
Determination of specific activity of invertase
Objectives :
To achieve the ability to prepare an enzyme extract.
To understand the enzyme units and specific activity.
Introduction :
Proteins form the class of biological macromolecules that have the most well-defined
physicochemical properties, and consequently they were generally easier to isolate and
characterize than nucleic acids, polysaccharides, or lipids. Furthermore, proteins, particularly
in the form of enzymes, have obvious biochemical functions. The central role that proteins
play in biological processes has therefore been recognized since the earliest days of
biochemistry.
Theory :
Protein extraction
Cellular location of the protein to be purified determines the procedures that can beused to obtain an extract containing the protein in soluble form. Extracellular proteins such
as those fermentation broths or in serum simple removed from insoluble material from
centrifugation or filtration. Intracellular enzymes and those found in the organelles of
eukaryotes may be released into extract by general cell disruption techniques.
Membrane bound proteins present a greater problem for isolation because they
normally cannot be released by simple disruption procedures. Protein associated with
phospholipids and protein liquid complex must be dissociated before the extraction. Extrinsic
membrane protein those bound only to the surface membrane can generally release byraising ionic concentration 1M NaCl or by freezing and thawing. Intrinsic membrane protein
those embedded in the membrane best released by treatment with either a detergent such
as SDS or organic solvents such as butanol. In the all cases precaution must be taken to
minimize protein denaturation and proteolysis.
Protein Purification
To be able to isolate a specific protein from a crude mixture the physical and/orchemical properties of the individual protein must be utilized. There is no single or simple
way to purify all kinds of proteins. Procedures and conditions used in the purification process
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of one protein may result in the inactivation of another. The final goal also has to be
considered when choosing purification method.
The purity required depends on the purpose for which the protein is needed. For an
enzyme that is to be used in a washing powder, a relatively impure sample is sufficient,
provided it does not contain any inhibiting activities. However, if the protein is aimed for
therapeutic use it must be extremely pure and purification must then be done in several
subsequent steps.
The aim of a purification process is not only removal of unwanted contaminants, but
also the concentration of the desired protein and the transfer to an environment where it is
stable and in a form ready for the intended application.
Yeast cell walls are composed of two layers of -glucan, the inner layer being insoluble
to alkaline conditions. Both of these are surrounded by an outer glycoprotein layer rich in the
carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. These types of
extracellular barrier confer shape and rigidity to the cells.
Plant cell walls are particularly strong, making them very difficult to disrupt
mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical
disruption using glass beads or sand, whereas bacterial cell walls are the easiest to break
compared to these other cell types.
Historically, physical lysis was the method of choice for cell disruption and extraction
of cellular contents; however, it often requires expensive, cumbersome equipment and
involves protocols that can be difficult to repeat due to variability in the apparatus (such as
loose-fitting compared with tight-fitting homogenization pestles). Also, traditional physical
disruption methods are not conducive for high throughput and smaller volumes typical of
modern laboratory research.
Enzyme units
The enzyme activity is frequently expressed in terms of units such that one unit is
amount of enzyme that catalysis the conversion of 1 micromole of substrate per minute
under defined conditions.
The SI unit of enzyme activity is katal which represents the transformation of 1 mole of
substrate per second.
1U = 1 mol min-1 1 katal = 1 mol s-1
1U = kat/60 = 16.67 nkat
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Specific activity
Purity of an enzyme is expressed in terms of the specific activity, which is the number of
enzyme units per milligram of protein.
The more purified an enzyme preparation is the greater is its specific activity.
Specific activity =number of enzyme units (U)
Number of mg of total protein=
U
Materials :
Chemicals
o Glucose solution
o H2O
o
Nelsons reagento Arsenomolybdate reagent
o Bakers yeast
o pH 5 citrate buffer
o Sucrose solution (1%)
o BSA solution
Procedure :
Extraction of invertase
1g of bakers yeast was taken into a motor and a little amount of sand added in to
it.Citrate buffer (pH 5, 100 mM, 2.0 mL) was also added. The mixture was grinded very well
until become slurry. Then buffer (3.0 mL) was added and mixed well. The mixture was
centrifuged at 5000g for 15 min at 40 C.
Invertase assay
Sucrose (1%,0.1 mL ) and invertase crude solution(100uL)was taken and citrate buffer
(100Mm,pH 5) required amount was added, to make the final volume of the tube 2.00
mL.The mixture was incubated for 5 min at 370 C. Nelsons procedure was followed to
determine the amount of reducing sugars.
Nelsons procedure
Nelsons reagent (1.0 mL) was added to stop the reaction. The tube was boiled for 20
min. Cooled to room temperature and arsenomolybdate reagent (1.0 mL) was added. Tubes
were mixed well and allowed to stand for 5 min.Distilled water (7.0 mL) was added and mixed
and the absorbance was measured. Blank experiment was also carried out without adding
the enzyme.
Glassware
o Test tubes.
o Mortar
o
Micro pipettes.o
Droppers
o Spectrophotometer
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Tube No
1 2 3 4 5 6 7 8 9 10
Glucose(4mM)- 0.02 0.05 0.1 0.15 0.2 0.25 0.3 - -
Fructose(4mM)- - - - - - - - 0.2 -
Sucrose(4mM)- - - - - - - - - 0.2
H2O1.0 0.98 0.95 0.90 0.85 0.8 0.75 0.7 0.8 0.8
Nelsons reagent1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Cover the tubes with cotton wool. Boil for 20 min. cool tubes to room temperature
Arsenomolybdate
reagent
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Mix tubes on a Vortex mixer and allow to stand for 5 min
Water7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0
Mix on votex mixture
Dilution
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Slandered curve for the determination of proteins.
Addition 1 2 3 4 5 6
Albumin bourn serum (1 mg/mL) 1.0 2.0 3.0 4.0 5.0 -
Enzyme crude solution - - - - - 1.0
Biuret reagent 2.0 2.0 2.0 2.0 2.0 2.0
H2O 4.0 3.0 2.0 1.0 - 4.0
Result :
Absorbance of the invertase assay
Absorbance for the Blank = 0.90
Absorbance for the sample = 0.83
Absorbance of the reducing sugars
Sample Absorbance
1 0.07
2 0.20
3 0.32
4 0.58
5 0.67
6 0.79
7 0.82
8 0.85
9 0.63
10 0.64
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Absobance of the yeast invertase test
sample Absorbance
1 0.17
2 0.20
3 0.23
4 0.28
5 0.32
6 0.24
Calculation:
2 = 4.0 10
0.02 10
10 10
= 8 x 10-6 mol L-1
3 =4.0 10 0.05 10
10 10
= 20 x 10-6 mol L-1
4 = 4.0 10
0.10 10
10 10
= 40 x 10-6 mol L-1
5 =4.0 10 0.15 10
10 10
= 60 x 10-6 mol L-1
6 =
4.0 10 0.20 10
10 10
= 80 x 10-6 mol L-1
7 =4.0 10 0.25 10
10 10
= 100 x 10-6 mol L-1
8 =
4.0 10 0.30 10
10 10 = 120 x 10-6 mol L-1
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Standard curve for determination of reducing sugars
Tube number Concentration mol/L absorbance
1 0.07
2 8.0 x 10-6 0.20
3 20.0 x 10-6 0.32
4 40.0 x 10-6 0.58
5 60.0 x 10-6 0.67
6 80.0 x 10-6 0.79
7 100.0 x 10-6 0.82
8 120.0 x 10-6 0.85
1 =1.0 1.0
7.0
= 1.42 x 10-1 mg mL-1
2 =1.0 2.0
7.0
= 2.84 x 10-1 mg mL-1
3 =1.0 1.0
7.0
= 4.28 x 10-1 mg mL-1
4 =1.0 1.0
7.0
= 5.17 x 10-1 mg mL-1
5 =1.0 1.0
7.0
= 7.14 x 10-1 mg mL-1
Standard curve for concentration of total protein
Tube number Concentration mg/mL absorbance
1 1.42 x 10-1 0.17
2 2.85 x 10-1 0.20
3 4.28 x 10-1 0.23
4 5.17 x 10-1 0.28
5 7.14 x 10-1 0.326 ? 0.24
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
8.0 x 10-6 20.0 x 10-6 40.0 x 10-6 60.0 x 10-6 80.0 x 10-6 100.0 x 10-6 120.0 x 10-6
Absoption
Concentration (mol/L)
graph of Concentration of Glucose vs Absorption at 540nm
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0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
1.42 x 10-1 2.85 x 10-1 4.28 x 10-1 5.17 x 10-1 7.14 x 10-1
Absorbance
Concentration (mg/mL)
Graph of Concentration of Invertase vs absorbance at 540nm
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Absorption of the sample = 0.24
Concentration of protein = 4.5 x 10-1mg/mL
Total protein content = CV
= 4.5 x 10-1mg/mL x 5.0 mL
= 2.25 mg
Enzyme activity of invertase =
= 1 x 10-4U
Specific activity =number of enzyme units (U)
Number of mg of total protein=
U
Specific activity =1 10U
2.25 mg
Specific activity = 4.445 10
Discussion :
This experiment was done to determine the enzyme activity of invertase enzyme. Forthe extraction of invertase enzyme, backers yeast was taken because backers yeast contains
the invertase enzyme in its cytoplasm.
For the extraction, the yeast sample was ground with a little amount of sand to break
the cell walls of the yeast for the whole amount of enzyme to be taken out.
A buffer was added to keep the mixture in a constant pH value because the enzymes
are denatured due to changes of the pH value and so that the enzyme activity of invertase
cannot be determined.
Then the mixture was centrifuged to separate the crude invertase enzyme form the
cell debris. The extracted enzyme is called a crude enzyme because the supernatant contains
not only the pure enzyme but also the other cell components as well.
Nelson method is an indirect colorimetric procedure that can be used to determine
the concentration of reducing sugars. Standard series of D-glucose series is used to establish
a standard curve and the amount of reducing sugars present in unknown solution is
determined from the standard curve. When glucose is heated in an alkaline solution
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containing Cu2+(Nelson reagent) glucose is oxidized and cupric ions reduced quantitatively
to cuprous ions. The tubes are covered during heating to prevent evaporation.
The blue color appears after adding arsenomolybdate is caused by the reduction of
hexavalent molybdenum atom in H3AsO4(MoO4)12 to the pentavalent state by the Cu+ ion.
This reaction is quantitative, therefore the intensity of the blue color formed is directly
proportional to the reducing sugar concentration.
The metals present in Nelson reagent and arsenomolybdate can inactive enzymes.
This property is used in the procedure to break enzymes activity. This is important since if
the enzymes activity doesnt stop it keeps hydrolyzing sucrose. Therefore cannot get a
correct value of (glucose) after a given time because the colorimeter doesnt show a proper
value.
Purity of an enzyme is expressed in terms of the specific activity, which is the number
of enzyme units per milligram of protein.
In order to measure the amount of total amount of protein present in the extracted
enzyme sample, a known amount of enzyme was reacted with biuret reagent and the
absorbance of the formed purple coloured solution was measured.
In order to calibrate the standard curve a series of standard Albumin Bovine serum was
made and the series was reacted with biuret and the absorbance of the purple coloured
series was measured using a colorimeter. With the use of the standard graph the total
amount of protein in the extracted sample can be calculated.
So that the specificity of invertase enzyme can be calculated.
Conclusion :
Specific activity of the extacted yeast invertase 4.445 10
References :
Stryer,L., Biochemistry, 4thedition,W.H. Freeman company, New York, 1999
Vote,D., Vote,J.G., Biochemistry, 4thedition, john wiley and sons inc, USA, 2011