Lab 3 Protein Chemistry PDF

Faculty of Medicine Academic Year: 2024-2025 Year: 1 Semester: 1 Module: Human Body Function (HBF) 102 Lab 3 – proteins & amino acids color reactions By: Dr. Hanaa B. Atya Lecturer of Biochemistry & Molecular Biology Department: Biochemist...

Faculty of Medicine Academic Year: 2024-2025 Year: 1 Semester: 1 Module: Human Body Function (HBF) 102 Lab 3 – proteins & amino acids color reactions By: Dr. Hanaa B. Atya Lecturer of Biochemistry & Molecular Biology Department: Biochemistry & Molecular Biology 12/12/2024 22  Objectives  Understand Protein Structure: Investigate how the structure of proteins affects their reactivity in color tests.  Identify Proteins and Amino Acids: Learn to differentiate between various proteins and amino acids using specific color reactions.  Learn Color Reaction Mechanisms: Explore the chemical mechanisms behind color changes in protein and amino acid tests.  Data Interpretation: Enhance skills in interpreting results and understanding potential limitations of color tests. 12/14/2024 HBF - 102 33  Introduction Proteins are macromolecules composed of amino acids linked together through peptide bonds. All amino acids found in proteins have this basic structure, differing only in the structure of the R-group or the side chain. 12/14/2024 HBF - 102 44  Introduction Structure of proteins can be divided into 4 orders: 1- Primary Structure 2 - Secondary structure 3 - Tertiary structure 4 - Quaternary structure 12/14/2024 HBF - 102 55  Protein denaturation  Denaturation is a process in which a protein loses its genetic shape due to the disruption of weak chemical bonds and interactions so becomes biologically inactive.  The loss of secondary, tertiary, and quaternary structure of protein sufficiently to cause loss of function. Example of denaturating agents: 1- Physical agents (Heat, vigorous shaking, UV radiation). 2- Chemical agents (acids and alkali, organic solvents, PH). 12/14/2024 HBF - 102 6 Protein denaturation Effect of heat (physical agent) Effect of Acid (chemical agent) 12/14/2024 HBF - 102 7  Physical Properties of Proteins 1- Proteins are colorless and tasteless. 2- Protein structures are of two patterns - Globular proteins and fibrous proteins. 3- In general proteins have large molecular weights. 4- Proteins like the amino acids exhibit amphoteric property i.e., they can act as acids and alkalis. 5-The solubility of proteins depends upon the pH. Lowest solubility is seen at isoelectric point. The solubility increases with increase in acidity or alkalinity. 12/14/2024 HBF - 102 8 Chemical properties of Proteins 1- Hydrolysis of proteins: Proteins can be hydrolyzed by acid, alkali and proteases and broken down to peptides and mixture of amino acids. 2- Esterification: Proteins with reaction with alcohols gives its corresponding esters. 3- Amino acids reacts with amines to form amides. 12/14/2024 HBF - 102 9  Detection of proteins 1. Precipitation by Organic Solvents Principle: Proteins in solution form hydrogen bonds with water. Organic solvents like acetone, ether or ethanol when added to a protein solution in water, reduce the number of hydrogen bonds causing aggregation, precipitation and denaturation of proteins. Method: 1- Take 1 ml of protein solution in test tube. 2- Add 2 ml of ethanol. 3- Mix and let it stand for 5 minutes. Observation: A white precipitate is formed. Comment: Protein is precipitated by organic solvents. 12/14/2024 HBF - 102 10 2. Biuret test (specific for peptide bond) Principle: The nitrogen atoms in the peptide bonds (C-N bonds) in proteins bind with copper(II) ions Cu 2+ in Biuret reagent and produce a violet color 12/14/2024 HBF - 102 11 3. Ninhydrin test (specific for α amino acids) Principle: Ninhydrin reacts with free α amino acids to give + - + a bluish-purple colored complex.  Ninhydrin is a powerful oxidizing agent and causes oxidative decarboxylation of α amino acids producing an aldehyde.  Give positive with all α amino acids (free α amino group).  Give Negative with proline (no free α amino group). 12/14/2024 HBF - 102 12 4. Xanthoproteic Test (aromatic amino acids) Principle: The benzene ring in tyrosine and tryptophan undergo nitration on treatment with strong nitric acid at elevated temperature forming a yellow precipitate. The yellow precipitate turns orange due to ionization, in alkaline medium. Method: 1- Take 2 ml of protein solution in a test tube +1ml of conc. Nitric acid and mix. 2- Heat the solution for one minute then cool it. 3- then add 2 ml of 40% sodium hydroxide. NaOH Observation: In acid medium yellow color is formed. In alkaline medium orange color is formed. Comment: Indicates the presence of aromatic amino acids. (Tyrosine and tryptophan). 12/14/2024 HBF - 102 13 5. Lead Acetate Test (presence of Sulfur group). Used for detecting the presence of Sulfur group in: Cysteine, Cystine Principle: 1. Free SH group (Cysteine amino acids): Lead acetate +free sulfide ions lead sulfide PPT (a brown to black color). 2. Protein or Cystine: heat Proteins or Cystine + NaOH free sulfide group Lead acetate +free sulfide ions lead sulfide PPT (a brown to black color). 12/14/2024 HBF - 102 14  5. Lead Acetate Test (presence of Sulfur group). Methionine doesn’t give a positive result in this test as the sulfur in the methionine is not released by the treatment with NaOH. 12/14/2024 HBF - 102 15 Practical work You are provided with two samples ( A & B ). Determine the presence of protein in each sample after addition of biuret reagent. Sample Sample (A) (B) 12/14/2024 HBF - 102 16 Practical work You are provided with two biological samples ( A & B). Determine the presence of protein in each sample. Samples Observation Comment ………………………………… ………………………………… Sample (A) ……………………… ……………..……… ………………………………… ………………………………… Sample (B) …………..………….……………………. 12/14/2024 HBF - 102 17  Interactive Question Which of the following reagents is commonly used in the Biuret test for proteins? A) Ninhydrin B) Alkaline CuSO4 C) Benedict's solution D) iodine solution What color indicates a positive result in the Biuret test? A) Blue B) Yellow C) Purple D) Green 12/14/2024 HBF - 102 18 18  Interactive Question Which of the following tests is used to detect the presence of proteins specifically? A) Ninhydrin test B) Biuret test C) Millon’s test D) Xanthoproteic test What color change occurs in the ninhydrin test when amino acids are present? A) Blue to purple B) Yellow to red C) Purple to yellow D) Colorless to blue 12/14/2024 HBF - 102 19 19  Interactive Question What does a positive ninhydrin test indicate? A) Presence of carbohydrates B) Presence of proteins C) Presence of free amino acids D) Presence of lipids In the xanthoproteic test, which amino acid is primarily detected? A) Glycine B) Tyrosine C) Alanine D) Methionine 12/14/2024 HBF - 102 20 20  Interactive Question  Why proline gives a yellow color in ninhydrin test?  Discuss the reasons of which aromatic amino acids give positive result but not aliphatic ones in Xanthoproteic test?  Which of the amino acids contain (-SH) group?  What is the difference between cysteine and cystine ? 12/14/2024 HBF - 102 21  References “Lippincott's Illustrated Reviews: Biochemistry." This textbook provides a comprehensive overview of biochemical concepts, including the biochemical properties of proteins and the methods used to analyze them, such as colorimetric assays. 12/14/2024 HBF - 102 22 22 12/14/2024 HBF - 102 23