Week 4 Analysis and Denaturation of Proteins PDF

lOMoARcPSD|47037285 WEEK 4 Analysis and Denaturation of Proteins biochem (Our Lady of Fatima University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Violet Kimiko (violetkimiko@...

lOMoARcPSD|47037285 WEEK 4 Analysis and Denaturation of Proteins biochem (Our Lady of Fatima University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Violet Kimiko ([email protected])  lOMoARcPSD|47037285 Biochemistry For Medical Laboratory Science Laboratory Week 4: Analysis and Denaturation of Proteins Pre-lab: 4. Heavy Metals: Heavy metals combine with Amino Acids (AA) - basic unit of proteins carboxyl groups to form free carboxylate Polypeptide - connected AAs anions (precipitation) Proteins - connected polypeptides ○ Mercury ○ Lead Functions of Proteins: ○ Silver Biological catalysts (enzymes) 5. Organic Acids: Organic acids combine with Transporters (transport proteins) basic/protonated amino groups to form Effectors (hormones) precipitation of alkaloids ○ Hormones ○ Picric Acid - antiseptic chemical for - proteins and lipids formed together burns - turn on the activity of organs - secreted from glands in the Objectives: endocrine system To be able to identify the different AAs in a protein sample through color tests Levels of Protein Structures: To observe the different ways to denature Primary (1°) - connected monomers / single proteins peptide bonds Secondary (2°) - folded 1° Procedures: Cysteine Prepare… - AA that folds peptide chains 5% albumin solution (2.5 mL egg white + - has sulfur groups 47.5 mL H2O) ○ α Helix - spiral (like DNA) Concentrated albumin (egg whites) ○ β-Pleated Sheets - wavy (like folded cardboard) 1. Color Tests for AAs and Proteins Tertiary (3°) a. Biuret Test - combination of 2° i. Add 2 mL 5% albumin on one test - protein starts to have small functions tube and 2 mL urea on the other Quaternary (4°) / Globular Protein ii. Add 1 mL 10% NaOH and 5 drops - combination of 3° CuSO4 solution on each test tube - protein has functions iii. Observe color of precipitate formed ○ Hemoglobin Results Denaturation - unfolding of the 2°, 3°, 4° structures Substance Observation of proteins Ovo Albumin - albumin in eggs 5% Albumin Violet ppt Urea Blue-green ppt Denaturation of Proteins by… 1. Heat: In high temperatures, atoms vibrate and peptide bonds are broken physically. Broken peptide bonds tend to coagulate. 2. Strong / Inorganic Acids: In adding acids, precipitation occurs, splitting salt linkages. ○ Hydrochloric Acid (HCl) ○ Sulfuric Acid 3. Organic Solvents: Forming water molecules (OH + H → H2O) disrupt H bonds Residues - proteins devoid of water ○ Alcohols (hydroxyl group OH) Downloaded by Violet Kimiko ([email protected])  lOMoARcPSD|47037285 b. Lead Acetate Test i. Add 1 mL 5% albumin on one test b. Inorganic Acids tube and 5% gelatin on the other i. Add 3 mL 5% albumin on both test ii. Add 5 drops 10% NaOH and 3 tubes drops 5% lead acetate on each ii. Add concentrated HCl drop by test tube drop to test tube 1 iii. Shake and heat to boiling point iii. Add concentrated H2SO4 drop by until precipitate is formed drop to test tube 2 iv. Observe color of precipitate formed iv. Record number of gtts that forms the precipitate Results Results Substance Observation Substance Observation 5% Albumin Black ppt HCl 1 gtt 5% Gelatin Yellow ppt H2SO4 4 gtts 2. Denaturation Tests (Coagulation) a. Heat c. Alcohols i. Add 1 mL concentrated albumin i. Add 1 mL 5% albumin on one test and 3 mL H2O on both test tubes tube and 1 mL 5% gelatin on the ii. Heat test tube 1 on boiling water for other 10 minutes ii. Add 5 mL 95% ethanol on each iii. Stir/Shake test tube 2 but do not test tube heat iii. Observe the precipitate formed iv. Filter both test tubes Results v. Test filtrates using biuret’s reagent vi. Compare intensity of color Substance Observation precipitate 5% Albumin Few white ppt Results Stable white Substance Observation 5% Gelatin ppt Clear white ppt Test tube 1 → bright pink 3. Denaturation Tests (Precipitation) a. By Heavy Metals Cloudy white i. Add 3 mL concentrated albumin Test tube 2 ppt → light on both test tubes pink ii. Add 5 drops of lead acetate on test tube 1 iii. Add 5 drops of silver nitrate on test tube 2 iv. Add excess drops then observe if precipitate is dissolved or grow more v. Record number of gtts that forms the precipitate Results Substance Observation Lead acetate 11th gtt Silver nitrate 11th gtt Downloaded by Violet Kimiko ([email protected])  lOMoARcPSD|47037285 1. Why is sterilization by heat and disinfecting b. By Alkaloidal Reagents skin using alcohol an effective way to kill i. Add 3 mL concentrated albumin microorganisms? and 2 mL picric acid on a test - Alcohols coagulate proteins. In higher tube alcohol concentrations, rapid coagulation of ii. Observe reaction surface proteins prevents effective penetration of cells. Results Substance Observation 2. Explain why egg whites and milk are used as an antidote for heavy metal poisoning. No reaction; - Egg whites and milk contain the two metallothionein. This protein has a high Picric acid chemicals did affinity for binding with heavy metals, such not mix as lead and mercury. Summary of Tests: 3. Explain why picric acid and tannic acid 1. Color Tests for AAs and Proteins are used for treatment for burns. a. Biuret Test - These acids readily form salts on contact - positive for >2 peptide ponds (>3 AAs) with many metals in order to improve - does not turn positive for dipeptides wound healing and reduce scar tissue - (+): blue to violet color formation. b. Lead Acetate / Sulfur Test - positive for sulfur AAs Sulfur-Containing AAs: i. Methionine ii. Cysteine c. Ninhydrin Test - general test for proteins - positive for all AAs - uses ninhydrin (chromogen) reagent - (+): blue to purple color d. Xanthoproteic Test - specific for AAs that have benzene rings in their structure e. Millon’s Test - positive for phenol AAs - (+) xanthoproteic = (+) Millon’s f. Hopkin’s Cole Test - positive for tryptophan Tryptophan - has an indole ring g. Sakaguchi Test - positive for arginine Arginine - has guanido group 2. Denaturation Tests a. Coagulation i. Heat ii. Strong acids iii. Alcohol b. Precipitation i. Heavy metals ii. Organic acids Downloaded by Violet Kimiko ([email protected])