Lab 2 Cell Biomolecules Function and Detection PDF

CELL BIOMOLECULE'S FUNCTIONS & DETECTION Elective course Biochemistry Department Faculty of Pharmacy BUE  OUTLINE 1. Introduction 2. Proteins and way of detection. 3. Carbohydrates and way of detection. ...

CELL BIOMOLECULE'S FUNCTIONS & DETECTION Elective course Biochemistry Department Faculty of Pharmacy BUE  OUTLINE 1. Introduction 2. Proteins and way of detection. 3. Carbohydrates and way of detection.  Introduction Chemicals or molecules present in the living organisms known as biomolecules. These biomarkers are fundamental building blocks of living organisms as they support the biological processes essential for life such as reproduction, growth and sustainence. e.g., carbohydrates, proteins, lipids, enzymes, nucleic acids, etc. The diversity in their shape and structure provides diversity in their functions.  Proteins Proteins account for more than 50% of the dry mass of most cells. Protein functions include accelerating chemical reactions inside the cell, transportation and cellular communications.  Proteins (Receptor Protein) There are two general types of receptors: cell-surface and intracellular receptors. Examples of Cell-surface receptors:  G Protein-Coupled Receptors  Receptor Tyrosine Kinase  Notch Receptors  Ion Channel Receptors Examples of intracellular receptors:  Nuclear Hormone Receptors: Examples include the estrogen receptor and the glucocorticoid receptor.  Intracellular Protein Kinase  Intracellular Calcium Receptors Phosphorylation G Protein-Coupled Receptors Amino acid polymers  Levels of Protein structure 1-Primary structure: 2-Secondary structure:  found in most proteins, consists of coils and folds in the  the sequence of amino acids in a protein, is like the polypeptide chain. order of letters in a long word.  The coils and folds of secondary structure result from hydrogen  Primary structure is determined by inherited genetic bonds between repeating constituents of the polypeptide information. backbone.  Typical secondary structures are a coil called an  helix and a folded structure called a  pleated sheet.  Levels of Protein structure 3- Tertiary structure: 4- Quaternary structure:  results when two or more polypeptide chains form  It is determined by interactions between R groups, rather than interactions between backbone constituents. one macromolecule.  Collagen is a fibrous protein consisting of three  These interactions between R groups include hydrogen bonds, polypeptides coiled like a rope. ionic bonds, hydrophobic interactions, and van der Waals  Hemoglobin is a globular protein. consisting of four interactions. polypeptides: two alpha and two beta chains.  Strong covalent bonds called disulfide bridges may reinforce the protein’s structure. Proteins detection methods  1- Biuret test (At least 2 peptide bonds) Principle Cu+2 2 ml sample + (violet 1 ml biuret color) reagent Alk. medium Positive with: Proteins and peptides bearing at least 2 peptide bonds. Non-Proteins compounds containing at least 2 peptide bonds. (Such as Urea)  2-Ninhydrin test (Test for Alpha amino acids) Principle 1 ml sample + 0.5 Ninhydrin R. (violet blue color) ml ninhydrin B.W.B for 3 reagent mins Positive with: Proteins and peptides having α-amino acid. Negative with: ᵦ-alanine Gamma amino butyric acid (GABA)  3- Xanthoproteic reaction (Aromatic amino acids) Principle Conc. HNO3 Polypeptide Quinoid with aromatic Alk.mediu structure ring m (Orange color) Specific For: Aromatic amino acids: 1. Neutral Aromatic: (Phenylalanine-Tyrosine-Tryptophan) 2. Basic Aromatic: (Histidine)  4-Millon’s reaction (Test for Tyrosine amino acid) Principle 1 ml sample+ 3 HNO3/HgNO3 (Purple red color dps Millon's B.W.B for 5 spots) reagent mins Tyrosine a.a  5- Adamkiewicz’s reaction (Tryptophan amino acid) Principle Glacial CH3COOH/glyoxylic 2 Tryptophan acid violet red molecules Conc. compound H2SO4/heating Specific For: Tryptophan amino acid  6- Reactions for sulphur containing A.As (Cysteine, A-Fohl’s Cystine(Sulphur Reaction and Methionine) containing amino acid) Principle 2 ml Alk.medium 10% sample+ 3 NaOH Na ml 10% B.W.B for 3 sulphide NaOH min. 5 drops 10% lead Acetate, B.W.B (2 min) Lead sodium Sulphide plumbite (Black Deposit)  7- Reactions for sulphur containing A.As (Cysteine, Cystine B-Nitroprusside Reactionand(Sulphur Methionine) containing amino acid) Principle S Alk.medium 10% NaOH Na containing B.W.B for 2 sulphide a.acid min sodium violet red nitroprusside complex  Classification of proteins A-Simple proteins: Made of amino acids linked to each other through peptide bonds. B- Conjugated proteins: In addition to amino acids, it contains non protein component (prosthetic group). C- Derived proteins: They are either hydrolyzed Or denatured proteins.  Phosphoproteins-conjugated proteins Phosphoric acid forms ester linkage with OH-group of serine, threonine or tyrosine. Examples: 1.Caseine → milk 2.vitelline → egg yolk Conjugated proteins Protein part Non-protein part (prosthetic gp)  Carbohydrate e.g. proteoglycans  Lipids e.g. lipoproteins  Phosphate gp e.g.  Carbohydrates  Carbohydrates include sugars and the polymers of sugars.  The simplest carbohydrates are monosaccharides, or single sugars.  Carbohydrate macromolecules are polysaccharides, polymers composed of many sugar building blocks.  Carbohydrates The Functions of Carbohydrates in the Body 1. Energy Production 2. Energy Storage: If the body already has enough energy to support its functions, the excess glucose is stored as glycogen (the majority of which is stored in the muscles and liver).  Carbohydrates The Functions of Carbohydrates in the Body 3. Building Macromolecules: Although most absorbed glucose is used to make energy, some glucose is converted to ribose and deoxyribose, which are essential building blocks of important macromolecules, such as RNA, DNA, and ATP. 4.Carbohydrates on cell surface: Carbohydrates on cell surfaces are involved in cell recognition, adhesion, and communication.. Carbohydrates detection methods 1- Molish test (Test for carbohydrates) Principle Alpha-naphthol 2 ml CHO solution (acidic medium) + 2 ml Molisch reagent To form Furfural + 5 dps conc H2SO4 on wall Positive with all carbohydrates 2- Benedict’s test (Test for reducing carbohydrates) Anhydrous sodium carbonate (alkaline conditions which are required for the redox reaction) Copper(II) sulfate pentahydrate (reducing agent) 1 ml CHO solution + 3 ml Benedict reagent B.W.B for 5 mins +ve with (Glucose, Fructose, Lactose) 3- Ketose test (Test for Ketones): Principle resorcinol and concentrated hydrochloric acid to hydrolyze of polysaccharide and oligosaccharide 2ml CHO ketoses yields simpler sugars followed by furfural. solution +ve with + 2ml Ketose Sucrose & Fructose reagent B.W.B for 5 mins 4- Barfoed’s test (Test for monosaccharides): 2ml CHO solution copper (II) acetate in 1% acetic acid + 2ml Barfoed solution reagent +ve with Glucose B.W.B for 5 mins & Fructose 5- Iodine test (Special test for starch): Principle I2/KI 2ml starch soln + +ve with 2 drops I2 starch