Isolation of Albumin from Chicken Liver - CHEM 41 Presentation PDF

CHEM 41 ISOLATION OF ALBUMIN FROM CHICKEN LIVER MUSA - NAPOLES - OLIVA - SAMSON Proteins  Proteins have critical function in nearly all biological processes function follows structure adopt a unique conformation due to internal bonding patterns from amino acid sequence conformational specif...

CHEM 41 ISOLATION OF ALBUMIN FROM CHICKEN LIVER MUSA - NAPOLES - OLIVA - SAMSON Proteins  Proteins have critical function in nearly all biological processes function follows structure adopt a unique conformation due to internal bonding patterns from amino acid sequence conformational specificity allows proteins to bind to specific molecules that lead to certain reactions  General Steps to Determine Protein Structure SOURCE ISOLATE PROTEIN CONFIRM IDENTITY 1. Homogenization 2. Purification techniques based on The identity of protein We must first figure out specific physical and can be detected through the source of the protein chemical attributes qualitative tests that give we aim to investigate. (dialysis, colored species. chromatography, gel electrophoresis) Albumin Albumin heterogenous group of proteins that provide colloidal osmotic effect keeps intravascular fluid inside vessels and prevent leakage synthesized in the liver and circulated via blood plasma facilitates transport of nutrients and hormones composition: 67% alpha helices 10% turns 23% random coils 0 beta sheets Significance Albumin concentration reflects physiological state of the organism. Knowing the structure of albumin may help us in understanding the mechanisms that underlie 35 its production and 2 ? regulation. HE ------- ---- Heliu m  Objectives 1 2 3 Apply fractional Determine the presence precipitation for isolating Determine the basis for of proteins using albumin from chicken the isolation of albumin. qualitative tests. liver. O₂ Methods Mg METHODS A. Separation of Protein Fraction from Chicken Liver 20 g Wash and pat Weigh 20g and Add 5mL of PBS buffer Blend until Transfer to 15ml dry liver mince (pH 7.4) per gram smooth conical tubes Centrifuge @3000 Decant and collect rpm (for 10 minutes) supernatant Phosphate Buffer (pH 7.4) RATIONALE PBS with a pH of 7.4 was used because A. Separation of Protein it provides a stable environment for albumin extraction Fraction from Chicken Liver Homogenizing solution Blender used to create a stabilized environment The sample is blended to mechanically for cell components that will be break open cells and release the released when cell are broken down. soluble proteins. Supernatant Centrifuge at 3000 rpm (for 10 mins) A liquid or medium free from precipitate Separates nuclei, unbroken cells, and obtained after centrifugation other cell components from desired protein fraction METHODS B. Isolation of Albumin 25% Measure volume Calculate and weigh Prepare 25% Centrifuge Collect of supernatant amount of buffered saturated solution @5000 rpm (for supernatant obtained ammonium sulfate 10 minutes) and discard (NH₄)₂SO₄ to be precipitate added METHODS B. Isolation of Albumin 25% Calculate and Prepare 25% weigh amount of saturated solution ammonium sulfate (NH₄)₂SO₄ to be added METHODS B. Isolation of Albumin 25% Measure volume Calculate and weigh Prepare 25% Centrifuge Collect of supernatant amount of buffered saturated solution @5000 rpm (for supernatant obtained ammonium sulfate 10 minutes) and discard (NH₄)₂SO₄ to be precipitate added METHODS B. Isolation of Albumin 46% P1 Measure volume Calculate and Prepare 46% Centrifuge Collect of supernatant weigh amount of saturated solution @5000 rpm (for supernatant obtained ammonium sulfate 10 minutes) and precipitate (NH₄)₂SO₄ added (P1) METHODS B. Isolation of Albumin 46% Calculate and Prepare 46% weigh amount of saturated solution ammonium sulfate (NH₄)₂SO₄ added METHODS B. Isolation of Albumin 46% P1 Measure volume Calculate and Prepare 46% Centrifuge Collect of supernatant weigh amount of saturated solution @5000 rpm (for supernatant obtained ammonium sulfate 10 minutes) and precipitate (NH₄)₂SO₄ added (P1) METHODS B. Isolation of Albumin 64% P1 Measure volume Calculate and Prepare 64% Centrifuge Collect of supernatant weigh amount of saturated solution @5000 rpm (for supernatant obtained ammonium sulfate 10 minutes) and precipitate (NH₄)₂SO₄ added (P2) METHODS B. Isolation of Albumin 64% Calculate and Prepare 64% weigh amount of saturated solution ammonium sulfate (NH₄)₂SO₄ added METHODS B. Isolation of Albumin 64% P1 Measure volume Calculate and Prepare 64% Centrifuge Collect of supernatant weigh amount of saturated solution @5000 rpm (for supernatant obtained ammonium sulfate 10 minutes) and precipitate (NH₄)₂SO₄ added (P2) METHODS B. Isolation of Albumin P2 100% Measure volume Weigh amount of Centrifuge Collect of supernatant solid (NH₄)₂SO₄ to @5000 rpm (for precipitate and obtained obtain 100% 10 minutes) combine with concentration P2 METHODS B. Isolation of Albumin 100% Weigh amount of solid (NH₄)₂SO₄ to obtain 100% concentration METHODS B. Isolation of Albumin P2 100% Measure volume Weigh amount of Centrifuge Collect of supernatant solid (NH₄)₂SO₄ to @5000 rpm (for precipitate and obtained obtain 100% 10 minutes) combine with concentration P2 METHODS B. Isolation of Albumin Dissolve with 6mL of 2% NaCl P1 P2 P1 P2 Fractional Precipitation RATIONALE B. Isolation of Albumin “Salting in/Salting out” Protein molecules initially have hydration Stepwise concentrations shells (water molecules) allow for maximized Addition of salt increases ionic strength recovery and purity until it competes with the proteins for the 25% - 46%: Removing water and exposes proteins’ hydrophobic impurities (like other proteins core with lower solubilities) Causes proteins to aggregate 64%: Albumin is precipitated Proteins have different solubilities and while other proteins with aggregate at different salt concentrations higher solubilities remain  Buffered ammonium sulfate (NH₄)₂SO₄ RATIONALE High solubility in water (NH₄)₂SO₄ Minimal effect on temperature B. Isolation of Albumin No harmful effect on most proteins Why buffered? Addition of ammonium sulfate acidifies solution - can lead to denaturation of the protein Buffered (NH4)2SO4 improves the stability Addition of NaCl of the protein being isolated 2% NaCl Allows greater solubility of proteins Centrifugation at 5000RPM for 10mins. Separates protein of interest from rest of substance through difference in solubility (aggregated = higher density) METHODS C. Hydrolysis +NH4OH +HCl P1 P2 HP1 HP2 30 mins HP1 HP2 HP1 HP2 HP1 HP2 Transfer 3mL of P1 Add 3mL Cover & place in Cool test Add NH4OH 1 & P2 to test tubes 2 3 hot water (30 mins) 4 tubes 5 until neutralized of 6M HCl Repeat 5 & 6 until 6 Check pH using litmus paper 7 samples are neutralized  Protein hydrolysis RATIONALE C. Hydrolysis Follows the mechanism of amide acid- catalyzed hydrolysis Klein (2017)  RATIONALE +HCl 6M HCl C. Hydrolysis HCl breaks down peptide bonds which allows for the isolation of amino acids. HP1 HP2 What are hydrolysates? Significance of hot water bath (30 mins) Products derived from hydrolysis, Increasing the temperature increases typically consisting of smaller HP1 HP2 30 the reaction rate of hydrolysis. To peptides and free amino acids. mins complete the reaction we would need at least 24 hours even at 100 °C  QUALITATIVE TESTS Biuret Test O₂ Tests for the presence of proteins, specifically peptide bonds Mg QUALITATIVE TESTS 1. Biuret Test Preparation of negative Preparation of samples and positive control Distilled 1% P1 P2 HP1 HP2* water ovalbumin QUALITATIVE TESTS 1. Biuret Test + 1mL 1% NaOH + 3 drops CuSO4 Distilled 1% P1 P2 HP1 HP2* water ovalbumin QUALITATIVE TESTS 1. Biuret Test - + QUALITATIVE TESTS 1. Biuret Test Tests for the presence of proteins, specifically peptide bonds Copper ions provided by CuSO4 react + 1mL 1% NaOH with peptide bonds (specifically nitrogen atoms) Added before CuSO4 Form a purple-colored chelate complex Makes medium basic Intensity of purple color - associated (allows formation of with concentration of protein in sample complex) Absorbance can be analyzed QUALITATIVE TESTS 1. Biuret Test Mechanism for biuret test (Karki, 2018)  QUALITATIVE TESTS Ninhydrin Test O₂ Tests for the presence of amines, α- amino acids, free amino acids Mg QUALITATIVE TESTS 2. Ninhydrin Test Preparation of negative Preparation of samples and positive control Distilled 1% P1 P2 HP1 HP2* water tryptophan QUALITATIVE TESTS 2. Ninhydrin Test + 3 drops Ninhydrin reagent Distilled 1% P1 P2 HP1 HP2* water ovalbumin QUALITATIVE TESTS 2. Ninhydrin Test Vortex mix samples Distilled 1% P1 P2 HP1 HP2* water ovalbumin QUALITATIVE TESTS 2. Ninhydrin Test Place in a boiling water bath for 5 minutes Cool to room temperature QUALITATIVE TESTS 2. Ninhydrin Test - + QUALITATIVE TESTS 2. Ninhydrin Test Tests for the presence of primary/secondary amines and α- amino acids Ninhydrin reagent Acts as an oxidizing agent Facilitates oxidative deamination: Removes the carboxyl group and Ninhydrin with amino complexes amino group of the amino acid with another ninhydrin molecule Releases CO2 + an aldehyde to form a diketohydrin called a Transfers amino group to Ruhemann’s complex which has ninhydrin a deep purple color QUALITATIVE TESTS 2. Ninhydrin Test Tests for the presence of primary/secondary amines and α- amino acids Mechanism of Ninhydrin Test (Sapkota, 2022)  QUALITATIVE TESTS Xanthoproteic Test O₂ Tests for the presence of aromatic amino acids (- phenylalanine) Mg QUALITATIVE TESTS 3. Xanthoproteic test Preparation of negative Preparation of samples and positive control Distilled 1% P1 P2 HP1 HP2* water tryptophan  QUALITATIVE TESTS 3. Xanthoproteic test + 1mL of conc. HNO3 Vortex mix samples Distilled 1% P1 P2 HP1 HP2* water tryptophan QUALITATIVE TESTS 3. Xanthoproteic test Place in a boiling water bath for 3 minutes Cool to room temperature  QUALITATIVE TESTS 3. Xanthoproteic test + conc. NH4OH dropwise Check pH & repeat until alkaline Distilled 1% P1 P2 HP1 HP2* water tryptophan QUALITATIVE TESTS 3. Xanthoproteic Test - + QUALITATIVE TESTS 3. Xanthoproteic Test Tests for the presence of aromatic acids (specifically tryptophan and tyrosine, as phenylalanine typically negative) + HNO3 Added to yield intensely yellow-colored nitro derivatives Induces nitration reaction with the aromatic ring (Karki, 2018) QUALITATIVE TESTS 3. Xanthoproteic Test Tests for the presence of aromatic acids (specifically tryptophan and tyrosine, as phenylalanine typically negative) + NH4OH Alkali turns the nitro derivative residue into orange due to the formation of a salt (Basnet, 2020)  QUALITATIVE TESTS Sakaguchi Test O₂ Tests for the presence of arginine and guanidine Mg QUALITATIVE TESTS 4. Sakaguchi test Preparation of negative Preparation of samples and positive control Distilled 1% arginine P1 P2 HP1 HP2* water QUALITATIVE TESTS 4. Sakaguchi Test + 20 drops of 10% NaOH Distilled 1% arginine P1 P2 HP1 HP2* water  QUALITATIVE TESTS 4. Sakaguchi test +4 drops 1% napthol in 95% ethanol Vortex samples Distilled 1% arginine P1 P2 HP1 HP2* water QUALITATIVE TESTS 4. Sakaguchi test Incubate 3 mins + bromine water dropwise until color change Distilled 1% arginine P1 P2 HP1 HP2* room temperature water QUALITATIVE TESTS 4. Sakaguchi Test - + QUALITATIVE TESTS 4. Sakaguchi Test Tests for the presence of arginine and guanidine (Sapkota, 2022) QUALITATIVE TESTS 4. Sakaguchi Test Tests for the presence of arginine and guanidine + NaOH 1% α-naphthol in 95% ethanol + Br2 water Needed to make the In the presence of an Acts as an oxidizing solution alkaline for oxidizing agent, the agent (it can react the reaction with the guanidinium group in with NaOH to form Sakaguchi reagents arginine reacts with α- NaBrO or sodium to proceed naphthol hypobromite) (Sapkota, 2022)  Results and Discussion O₂ Mg  RESULTS AND DISCUSSION Isolation of Albumin O₂ This step utilized fractional precipitation to isolate the albumin from other soluble proteins and substances. Mg  Protein fraction if the correct calculations were used... AFTER HOMOGENIZATION 25% SATURATION 46% SATURATION 64% SATURATION contains little to no primarily composed of proteins are mostly globulin or albumin globulin (heavier almost purely albumin and globulin (Herbert, Pelham, & proteins precipitate composed of albumin Pittman, 1973) first) Actual Concentrations 1st Concentration [Supposedly 25%] (100%)(17.141) = (20+17.141)x 17.141 = 37.141x x = 46.151% 2nd Concentration [Supposedly 46%] (100%)(51.34) + (46.151%)(20) = (20+51.34)x 60.5702 = 71.34x x = 84.9% 3rd Concentration [Supposedly 64%] (100%)(55.05) + (84.9%)(20) = (20+55.05)x 72.031 = 75.05x x = 96%  RESULTS AND DISCUSSION Hydrolysis O₂ “Until now, no hydrolysis method can completely liberate all amino acids from a protein substrate and recover them with 100 % yield.” - (Mustățea et al., n.d.) Mg  RESULTS AND DISCUSSION Qualitative Tests O₂ This step detects and confirms the identity of albumin based on the detected amino acids present. Mg  Test Sample Results P1 + Biuret Qualitative HP1 +* Tests P1 -* Ninhydrin HP1 -* Note: those with “*” P1 + have the opposite Xanthoproteic theoretical results HP1 -* P1 + Sakaguchi HP1 -*  Test Sample Results P1 + HP1 +* Ninhydrin + P2 Biuret Test (theoretical) - HP2 (theoretical) detects presence of at Note: * should show the opposite result. least 2 peptide bonds control P1 HP1 For HP1, hydrolysis did not completely occur, not all peptide bonds were cleaved.  Test Sample Results P1 -* HP1 -* Ninhydrin Ninhydrin + P2 (theoretical) Test HP2 + (theoretical) Note: * should show the opposite result. detects amines, α- control P1 HP1 amino groups, and free amino acids Both samples exhibited false negative results which may be a result of Ninhydrin’s poor sensitivity to proteins and/or lapses in methods  Test Sample Results P1 + HP1 -* Xanthoproteic Xanthoproteic P2 + (theoretical) Test HP2 + (theoretical) detects presence of Note: * should show the opposite result. aromatic amino acid except Phe control P1 HP1 The method of hydrolysis conducted in the experiment was not effective enough. Hydrolysis using 6M HCl reportedly shows destruction of Tryptophan and low recovery of Tyrosine.  Test Sample Results P1 + HP1 -* Sakaguchi Sakaguchi P2 + (theoretical) Test HP2 + (theoretical) Note: * should show the opposite result. detects arginine or guanidinium + control P1 HP1 compounds P1 showed a (+) results while HP1 a false negative results which may be due to the loss of yield in hydrolysis  Conclusion 1 2 3 Due to Using qualitative The experiment was miscalculations, an results, the presence successful in isolating excess of ammonium and absence of albumin from chicken sulfate was used in specific compounds liver salting out were detected  Recommendation 1 2 3 Implementation of Utilization of ideal Exploration of other methodology more conditions for protein isolation and carefully hydrolysis purification methods References Barampouti, E. M., Mai, S., Moustakas, K., Dimitris Malamis, & Loizidou, M. (2021). Status and perspectives of agricultural residues in a circular and resource-efficient context. Elsevier EBooks, 49–102. https://doi.org/10.1016/b978-0-323-85223-4.00018-x Basnet, A. (2020, April 4). Xanthoproteic test: Principle, Reaction, Reagents, Procedure and Result Interpretation | Online Biochemistry Notes. Online Biochemistry Notes. http://biocheminfo.com/2020/04/04/xanthoproteic-test-principle-reaction-reagents-procedure-and- result-interpretation/ Introduction to Hydrolysis | Waters. (2024). Waters.com. https://www.waters.com/nextgen/en/education/primers/comprehensive-guide- to-hydrolysis-and-analysis-of-amino-acids/introduction-to- hydrolysis.html#:~:text=Furthermore%2C%20the%20versatility%20of%20HCl,amino%20acids%20(Figure%201) O₂ Karki, G. (2018, April 20). Biuret test-Principle, Requirements, Reagents, Preparation, Procedure and Result. Online Biology Notes. https://www.onlinebiologynotes.com/biuret-test-principle-requirements-reagents-preparation-procedure-and-result/ Karki, G. (2018, April 23). Xanthoproteic test: Objective, Principle, Reagents, Procedure and Result - Online Biology Notes. Online Biology Notes. https://www.onlinebiologynotes.com/xanthoproteic-test-objective-principle-reagents-procedure-and-result/ Klein, D. (2017). Carboxylic acids and their derivatives. In Organic Chemistry (3rd ed.). Hoboken, NJ: John Wiley & Sons, Inc. Mustățea, G., Ungureanu, E., & Iorga, E. (n.d.). PROTEIN ACIDIC HYDROLYSIS FOR AMINO ACIDS ANALYSIS IN FOOD -PROGRESS OVER TIME: A SHORT REVIEW. Retrieved October 3, 2024, from https://keypublishing.org/jhed/wp-content/uploads/2020/07/03.-Full-paper- Gabriel-Must%C4%83%C8%9Bea.pdf?fbclid=IwZXh0bgNhZW0CMTEAAR2wdqQSp7DgE5b- _WleDgoOJxnQ6225DkVe2jCtCuZOIz3fu9NBcXaNhN4_aem_-YQyGXYNu9UaS3KjbUPFCg Penke, B., Ferenczi, R., & Kovács, K. (1974). A new acid hydrolysis method for determining tryptophan in peptides and proteins. Analytical Biochemistry, 60(1), 45–50. doi:10.1016/0003-2697(74)90129-8 ‌ Mg Sapkota, A. (2022). Ninhydrin Test- Definition, Principle, Procedure, Result, Uses. Microbe Notes. https://microbenotes.com/ninhydrin-test/ Thank you for listening!  TRY THIS BACKGROUND FOR ONLINE O₂ CLASS. *Please delete this section before downloading. Mg