Report biochemistry lab 2

Bài phúc trình Thực tập môn Sinh hóa 2. Ngành Công nghệ sinh học Trường Đại học Cần Thơ. Trình bày toàn bộ các thao tác và cũng như giải đáp các câu hỏi trong quá trình tham gia môn học của nhóm bằng tiếng anh.

CAN THO UNIVERSITY

PRACTICE REPORT BIOCHEMISTRY LABORATORY II

CODE BT231

TABLE OF CONTENTS

2 The principle of a spectroscopic method 2

3 Extraction of crude bromelain from pineapple 2

4 Precipitation of protein by ammonium sulphate 4

5 Determining protein content by the Bradford method 7

6 Determining specific activity of enzymes by the Kunitz method 15

7 Determining molecular weight of protein by SDS-PAGE 21

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2 The principle of a spectroscopic method

- Introduction: Spectrophotometry is a general analytical method in analytical laboratories that can be used for identification or measurement This measurement can also

be used to measure the amount of a known chemical substance

- Principle:

+ The basic principle is that each compound absorbs or transmits light over a certain range

of wavelengths In other words, is to shine a beam of electromagnetic radiation onto a sample and observe how it responds to such a stimulus The response is usually recorded

as a function of radiation wavelength

+ Includes absorption of ultraviolet, normal light, or infrared radiation used in quantification

+ Absorption of light usually occurs with substances:

Organic molecules, Metal, and Metal-organic complex

- UV (170nm to 380nm): use ultraviolet light to determine the absorbency of a substance

As matter absorbs light, it generates a spectrum

- VIS (380nm to 780nm): based on the absorption of visible light by chemical compounds, which results in the production of distinct spectra The UV-VIS spectroscopic method is used to quantify the amount of DNA or protein in a sample, for water analysis, and as a detector for many types of chromatography

3 Extraction of crude bromelain from pineapple

- Purpose: Extract the crude bromelain from unripe pineapple

- Principle: The crude bromelain is extracted from pineapple through centrifugation The enzymatic extract is subjected to centrifugation for eliminating cellular debris, organelles, and other molecular aggregates, thereby leading to partial purification

of enzymes

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It also assists towards the enzymatic characterization as depending on the mass as well as shape, the enzyme will travel over solution with a definite velocity and occupy a distinctive position in the centrifuge tube

- Material: unripe pineapple

- Consumable: eppendorf tube, plastic graduated cylinder

- Equipment: micropipette, pipette tip

3.1 A protocol for extraction of crude bromelain from pineapple

To describe steps

- The unripe pineapple is cut into three parts: skin, flesh, and core

- The blender is used to grind Then, the liquid is filtered

- The liquid is poured into centrifuge tube The tube needs to be measured carefully before putted into centrifuge

- After centrifugation, a micropipette is used to transfer the liquid slowly into

a plastic graduated cylinder The volume is noted

- The liquid is continued to be centrifuged again

- After centrifugation, a micropipette is used to transfer the liquid slowly into

a plastic graduated cylinder The volume is noted

- The final liquid is crude bromelain It must be stored in eppendorf tubes at low temperature (about 4oC)

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3.2 Statistics about amount of pineapple fruit (Tons)

Province Group Species of

pineapple

2020 2019 2018 2017

Long An (tons) A Queen 14,076 13,145 14,152 13,215 Tien Giang (tons) B Queen 250,000 300,000 244,000 123,500 Hau Giang (tons) C Queen 28,327 27,319 22,880 16,240 Kien Giang (tons) D Queen 115,000 91,050 75,780 51,750

4 Precipitation of protein by ammonium sulphate

4.1 Introduction

- Purpose: The main purpose of protein precipitation is to separate the protein from the solution either to eliminate interferences or to purify them Depending on the solubility and molecular structure of the protein, the efficacy of various precipitation methods can be different

- Principle of protein precipitation by Ammonium Sulphate: When high concentrations of small, highly charged ions such as Ammonium Sulfate are added, these groups compete with the proteins to bind to the water molecules This removes the water molecules from the protein and decreases its solubility, resulting in precipitation

Ammonium Sulfate including other salts are exploited towards precipitation in a process known as “salting out”

- Principle of dialysis: Dialysis works on the principles of the diffusion of solutes and ultrafiltration of fluid across a semi-permeable membrane Diffusion is a property of substances in water; substances in water tend to move from an area of high concentration to an area of low concentration

- Material: Crude Bromelain

- Equipment: analytical balance 3 digits, centrifuge tube, centrifuge

4.2 Saturated precipitation of protein

To describe steps for saturated precipitation of protein by 80% AS

5.61g Ammonium Sulfate is measured by analytical balance 3 digits and the value

The mixture is poured into centrifuge tube The tube needs to be

measured carefully before putted into centrifuge The mixture is centrifuged for 30 minutes

After 30 minutes, centrifuge tube is taken out Then, plastic transfer pipettes are used to remove the liquid, the saturated precipitation of protein by 80% AS is saved

4.3 Fraction precipitation of protein

To describe steps for fraction precipitation of protein by 50% and 75% AS

Fraction precipitation of protein by 50% AS 3.14g Ammonium Sulfate is measured by analytical balance 3 digits and the value

is noted

10mL Crude Bromelain is measures by a plastic graduated cylinder Then, Crude Bromelain is poured into a beaker containing 3.14g Ammonium Sulfate

Fraction precipitation of protein by 75% AS 1.892g Ammonium Sulfate is measured by analytical balance 3 digits and the value

is noted

11mL liquid of protein by 50% AS in the previous experiment is measures by a plastic graduated cylinder Then, protein by 50% AS is poured into a beaker containing 3.14g Ammonium Sulfate

The mixture is stirred by a glass rod until 1.892g Ammonium Sulfate is dissolved

in 11mL liquid of protein by 50% AS completely Wait 30 minutes until protein is precipitated

The mixture is poured into centrifuge tube The tube needs to be measured carefully before putted into centrifuge The mixture is centrifuged in 30 minutes

After 30 minutes, centrifuge tube is taken out Then, a plastic transfer pipettes a plastic transfer pipette is used to remove the liquid, the fraction precipitation of protein by 75% AS is saved

4.4 Dialysis

To describe steps for

3 dialysis-tubing bags are prepared The volumes of precipitation of protein

by 80% AS, 50% AS and 75% AS are measured by a plasic graduated cylinder and noted Then, they are put into each different bag

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5mL of Ammonium Acetate Buffer 0.1M pH 4 solution is measured by a plastic graduated cylinder Then, it is added into each bag

3 bags are put into a beaker containing Ammonium Acetate Buffer 0.1M pH

4 solution The beaker needs to be saved at low temperatures The solution must be changed after 4-5 hours Repeat at least three times

The volumes of precipitation of protein by 80% AS, 50% AS and 75% AS are measured again after dialysis and stored in eppendorf tubes at low temperature (about

- Storage: The volumes of precipitation of protein by 80% AS, 50% AS and 75%

AS are stored in eppendorf tubes at low temperature (about 4oC)

5 Determining protein content by the Bradford method

- Purpose: The Bradford assay is a dye-binding assay used to measure the protein concentration of a solution The aim of following experiment is to determine the protein content by the Bradford method using bovine serum albumin (BSA) in the solution (crude bromelain, AS 80%, AS 50%, AS 75% are used in these experiments)

- Principle: The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue (CBB) shifts from 465 nm to

595 nm when binding to protein occurs Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change

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In acidic solution, when not bound to proteins, the red dye has a maximum absorption wavelength of 465 nm and when combined with proteins, the dye turns blue and absorbs maximum at the maximum level is at 595 nm The absorbance at 595 nm is directly related to the protein concentration

Figure 5.2 Coomassie Brilliant Blue reaction

- Material: Crude Bromelain, AS 80%, AS 50%, AS 75%

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- Chemical: BSA 1mg/mL solution, CBB solution, Ammonium Acetate Buffer 0.1M pH 4 solution, distilled water

- Consumable: test tube, eppendorf tube

- Equipment: spectrophotometer, micropipette, pipette tip

5.1 The BSA standard curve

- Making the BSA standard curve

The equation obtained from the BSA standard curve is Y = 0,0015X + 0,1635, which

is X is the BSA concentration (µg/mL) and Y is the average of delta OD This equation obtained from the BSA standard curve (Y = 0,0015X + 0,1635) is able to be accurate because the equation has R2 = 0.9726 (> 95%)

R2 in the chart is not nearly about 1, that meanss the errors still occur because of some manipulation mistakes when using micropipette, diluting the samples, and recording the results

+ 24 test tubes are prepared

+ Dilution of 2 time (k = 2): the sample is diluted by Ammonium Acetate Buffer 0.1M pH

4 solution with the ratio 1:1 (300mL:300mL) The experiment is repeated 3 times

+ Dilution of 5 time (k = 5): the sample is diluted by Ammonium Acetate Buffer 0.1M pH

4 solution with the ratio 1:4 (100mL:400mL) The experiment is repeated 3 times

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OD2 0.771 0.646 1.089 0.818 1.192 0.829 0.586 0.688 OD3 0.933 0.667 1.014 0.736 1.013 0.712 0.668 0.567 Delta OD1 0.298 0.294 0.539 0.598 0.522 0.346 0.289 0.143 Delta OD2 0.297 0.172 0.615 0.344 0.718 0.355 0.112 0.214 Delta OD3 0.435 0.169 0.516 0.238 0.515 0.214 0.17 0.069 Delta ODEverage 0.322 0.212 0.557 0.393 0.585 0.305 0.190 0.142 5.3 Result

- Protein content of the experiment

- Equation : Y = 0,0015X + 0,1635 (Y = aX + b)

From the equation: Y = 0,0015X + 0,1635 obtained from the BSA standard curve (where

Y is the OD value and X is the concentration of protein (µg/mL)), we calculate the concentration of protein in the enzyme

Delta

ODEverage 0.322 0.212 0.557 0.393 0.585 0.305 0.190 0.142 Amount of

k=2, the total protein of concentration of 50% AS is highest Next, 80% AS, crude bromelain and 75 % AS is the smallest

k=5, the total protein of concentration of 80% AS is highest Next, crude bromelain, 50% AS and 75 % AS is the smallest

Conclusion

We can conclude that the higher the diluted coefficient, the smaller the concentration

of protein

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- Total protein of a pineapple fruit (mg)

6 Determining specific activity of enzymes by the Kunitz method

- Purpose: The Kunitz assay is to identify the presence or quantity of a specific enzyme in an organism, tissue, or sample

- Principle: TCA (Tricarboxylic Acid) acts as an inhibitor that inhibits the activity of enzymes At the end of the incubation period, TCA is added This stops the enzyme reaction and denatures the casein, rendering it insoluble The insoluble casein can then be removed

by centrifugation or filtration to yield a clear solution

Casein is a kind of protein, which is made of amino acids with C-N linkages In hydrolysis reaction, enzymes will hydrolyze the C-N linkages of casein 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 The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles From the standard curve the activity of protease samples can be determined

in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute

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6.1 The tyrosine standard curve

- Making the tyrosine standard curve

0,2

However, the concentration of tyrosine in the solution is just accurate at the rate of 99.98% The errors still occur because of some manipulation mistakes when using micropipette, diluting the samples, and recording the results

6.2 Hydrolysis of casein

- Reaction OD 280nm

Table 1 Preparing Samples

Step 2 Adding 600 µL TCA 15% Adding 600 µL casein 1%

Incubating and shaking by orbital shaker at 37℃ in 10 minutes

Incubating and shaking by

Vt: total volume of the reaction solution (1400µL)

T: time of reaction (10 minutes)

VE: Volume of the enzyme used in reaction (20µL)

k: dilution coefficient In this experiment, samples do not need to be diluted, thus

the enzyme may be altered

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7 Determining molecular weight of protein by SDS-PAGE

- Introduction of SDS-PAGE: SDS-PAGE (sodium dodecyl sulphate – polyacrylamide gel electrophoresis), is an electrophoresis method that allows protein separation by mass The combined use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight

- Purpose: The combined use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight

- Principle: The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the opposite sign when placed in an electric field The separation of the charged molecules depends upon the relative mobility of charged species The smaller molecules migrate faster due to less resistance during electrophoresis

The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel Polyacrylamide-gel is typically sandwiched between two glass plates in a slab gel SDS acts as a surfactant, masking the proteins' intrinsic charge and conferring very similar charge-to-mass ratios The intrinsic charges of the proteins are negligible in comparison to the SDS loading, and the positive charges are also greatly reduced in the basic pH range of

a separating gel Upon application of a constant electric field, the protein migrates towards the anode, each at a different speed, depending on its mass This simple procedure allows precise protein separation by mass

- Material: Crude Bromelain, AS 80%, AS 50%, AS 75%

- Chemical: Sample Buffer (Tris-HCl pH 6.8, SDS, Glycerol, β-mercaptoethanol, Bromophenol blue, distilled water), distilled water

- Consumable: beaker

- Equipment: electrophoresis polyacrylamide gel (4% stacking gel, 10% running gel), micropipette, pipette tip, SDS-PAGE, laboratory water bath