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 The principle of a spectroscopic method Extraction of crude bromelain from pineapple Precipitation of protein by ammonium sulphate Determining protein content by the Bradford method Determining specific activity of enzymes by the Kunitz method 15 Determining molecular weight of protein by SDS-PAGE 21 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 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 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 3.1 - Material: unripe pineapple - Consumable: eppendorf tube, plastic graduated cylinder - Equipment: micropipette, pipette tip 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) 3.2 Parameter Weight (gram) % Volume (mL) A pineapple fruit 1291 Part (skin) 307,97 76.63% 236 Part (flesh) 706,1 51.55% 364 Part (core) 24,81 604.59% 150 3.2 Statistics about amount of pineapple fruit (Tons) Province Group Species of 2020 pineapple 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 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 solution Chemical: Ammonium Sulfate, Ammonium Acetate Buffer 0.1M pH Consumable: beaker, plastic graduated cylinder, glass rod, plastic transfer pipettes, eppendorf tube, dialysis-tubing bag - Equipment: analytical balance 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 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 5.61g Ammonium Sulfate The mixture is stirred by a glass rod until 5.61g Ammonium Sulfate is dissolved in 10mL Crude Bromelain 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 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 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 The mixture is stirred by a glass rod until 3.14g Ammonium Sulfate is dissolved in 10mL Crude Bromelain 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 for 30 minutes After 30 minutes, centrifuge tube is taken out Then, plastic transfer pipettes are used to transfer the liquid into a plastic graduated cylinder The volume is noted to prepare for fraction precipitation of protein by 75% AS Fraction precipitation of protein by 75% AS 1.892g Ammonium Sulfate is measured by analytical balance 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 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 5mL of Ammonium Acetate Buffer 0.1M pH solution is measured by a plastic graduated cylinder Then, it is added into each bag bags are put into a beaker containing Ammonium Acetate Buffer 0.1M pH 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 4oC) 4.5 Result and storage Volume (Before precipitation) (mL) Volume (After dialysis) (mL) AS 80% 5.2 mL 4.5 mL AS 50% mL mL AS 75% mL 8.2 mL - 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) 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 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 5.1 Coomassie Brilliant Blue structure Figure 5.2 Coomassie Brilliant Blue reaction Material: Crude Bromelain, AS 80%, AS 50%, AS 75% - Chemical: BSA 1mg/mL solution, CBB solution, Ammonium Acetate Buffer 0.1M pH solution, distilled water - Consumable: test tube, eppendorf tube - Equipment: spectrophotometer, micropipette, pipette tip 5.1 The BSA standard curve - Making the BSA standard curve BSA concentration (µg/mL) Volume of BSA (µL) 30 60 120 180 240 300 30 60 120 180 240 300 1000 970 940 880 820 760 700 1000 1000 1000 1000 1000 1000 Volume of distilled water (µL) Total volume (µL) * Notes: BSA Bovine serum albumin solution 1000 - Reaction BSA 30 Volume of BSA (µL) 100 Volume of CBB (µL) 2000 concentration (µg/mL) 60 120 180 240 300 100 100 100 100 100 100 2000 2000 2000 2000 2000 2000 10 OD1 0.,483 0,732 0,7575 0,801 0,937 0,995 1,120 OD2 0,474 0,662 0,7744 0,805 0,922 0,960 1,085 OD3 0,498 0,659 0,7646 0,823 0,913 0,946 1,158 Delta OD1 0,249 0,2745 0,318 0,454 0,512 0,637 Delta OD2 0,188 0,3004 0,331 0,448 0,486 0,611 Delta OD3 0,161 0,2666 0,325 0,415 0,448 0,66 Delta ODEverage 0,1993 0,2805 0,324 0,439 0,482 0,636 * Note: CBB: Coomassie brilliant blue solution 10 - Drawing a standard curve - Equation : Y = 0,0015X + 0,1635 (Y = aX + b) 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 11 5.2 The reaction between protein and Coomassie brilliant blue - Preparation of sample Sample Total volume for the Dilution of time Dilution of time experiment (mL) Crude bromelain x x AS80 4.5 x x AS50 x x AS75 8.2 x x - Making if dilution + 24 test tubes are prepared + Dilution of time (k = 2): the sample is diluted by Ammonium Acetate Buffer 0.1M pH solution with the ratio 1:1 (300mL:300mL) The experiment is repeated times + Dilution of time (k = 5): the sample is diluted by Ammonium Acetate Buffer 0.1M pH solution with the ratio 1:4 (100mL:400mL) The experiment is repeated times - Reaction Sample Crude bromelain AS80 AS50 Volume of sample (µL) 100 Volume of CBB (µL) 2000 AS75 10 12 k OD1 0.718 0.777 5 1.022 1.081 1.055 0.829 0.772 0.626 5.3 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 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 Sample k Delta ODEverage Amount of protein (µg/mL) Crude bromelain AS80 0.322 0.212 0.557 105.667 32.333 262.333 AS50 0.393 0.585 153 281 AS75 5 0.305 0.190 0.142 94.333 17.667 0.000 Amount of protein 0.211334 0.161665 0.524666 0.765 0.562 0.001525 0.035334 0.000 (mg/mL) 13 Total volume for the experiment 3 63.4002 48.4995 2.25 2.25 2.5 2.5 4.1 4.1 0.4575 10.6002 0.000 (mL) Total protein for the experiment 157.3998 229.5 168.6 (mg) Discussion Theoretically, delta OD value must be decreased from the twofold diluted sample to the fivefold diluted sample, that means the concentration of protein decreases from the twofold dilution to the fivefold one so that after being multiplied by dilution coefficient As we observe in our results, the ΔOD value and the concentration of protein after dilution (x value) is slightly decreased 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 14 - Total protein of a pineapple fruit (mg) Sample Crude bromelain AS80 AS50 AS75 Total volume of a pineapple fruit (mL) 4.5 8.2 Amount of protein 4.6 (mg/mL) 18.45956 15.0136 0.08146 Total protein in a 27.6 pineapple fruit (mg) 83.068 75.068 0.668 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 15 6.1 The tyrosine standard curve - Making the tyrosine standard curve Tyrosine concentration (µmol/mL) 0,2 0,4 0,6 0,8 300 600 900 1200 1500 Volume of HCl 0,1N (µL) 1500 1200 900 600 300 Total volume (µL) 1500 1500 1500 1500 1500 1500 Volume of Tyrosine (µL) - Reaction Tyrosine concentration (µmol/mL) 0,2 0,4 0,6 0,8 OD1 0.049 0.304 0.552 0.804 1.081 1.327 OD2 0.051 0.296 0.587 0.801 1.068 1.337 OD3 0.049 0.04 0.569 0.815 1.074 1.326 Delta OD1 0.255 0.503 0.755 1.032 1.278 Delta OD2 0.245 0.536 0.75 1.017 1.286 Delta OD3 0.255 0.52 0.766 1.025 1.277 Delta ODEverage 0.252 0.52 0.757 1.025 1.28 16 - Drawing a standard curve - Equation : Y = 1,2794X - 0,0007, R2 = 0,9998 (Y=aX + b) The equation has R2 = 0.9886 The bigger R2 is, the more accurately equation expresses The chart leads to R2 = 0.9998 (> 95%), so we can conclude that the equation is highly accurate 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 Preparing Samples Control samples 17 Enzyme samples Step Add to the Eppendorf tube: 160 µL buffer solution, 20 µL enzyme, and 20 µL cysteine Incubating at 37 minutes Step Step Incubating and shaking by orbital shaker at orbital in 10 minutes 37℃ Adding 600 µL casein 1% Centrifugating 6000 rpm at 10 minutes Incubating and shaking by shaker at in 10 minutes Adding 600 µL TCA 15% Stabilizing at in 10 minutes in Step Centrifugating 6000 rpm at in 10 minutes Measure the absorbance at 280 nm wavelength * The experiment is repeated times 18 in 20 Adding 600 µL casein 1% Stabilizing at in 10 minutes Step Incubating at 37 minutes in 20 Adding 600 µL TCA 15% 37℃ Add to the Eppendorf tube: 160 µL buffer solution, 20 µL enzyme, and 20 µL cysteine Measure the absorbance at 280 nm wavelength 6.3 Calculation of enzyme activity - Meaning: U: activity in 1mL enzyme solution (U/mL) a: tyrosine concentration in hydrolyzed solution (µM/mL) 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 not need to be diluted, thus k=1 - Result Crude bromelin Unit of enzyme activity (µg/mL) 4.361 6.4 AS50 AS75 AS80 2.898 1.043 1.638 Result Sample Crude bromelain AS50 AS75 AS80 Volume of sample (µL) 20 20 20 20 OD (Control) 0.427 0.397 0.445 0.348 OD1 1.247 0.915 0.617 0.675 OD2 1.213 0.95 0.645 0.642 OD3 1.209 0.913 0.62 0.625 19 Delta OD (Control) Delta OD1 0,82 0,518 0,172 0,327 Delta OD2 0,786 0,553 0,2 0,294 Delta OD3 0,782 0,516 0,197 0,277 Delta ODAverage 0,796 0,529 0,19 0,299 Discussion Theoretically, the higher the concentration of enzyme, the stronger its activity expresses However, our results are the opposite This error can be explained by our mistakes when measuring the absorbance of the samples and maybe storing the samples Some samples may be put outside at room temperature for a long time, so the activity of the enzyme may be altered Conclusion Bromelain or papain break peptide bonds and release free amino acids after hydrolysis with protein In this case, tyrosine is released from casein after hydrolysis with bromelain or papain enzyme Figure Casein structure 20