Biological Macromolecules & enzyme Heba Al-Tamimi 

Macromolecules Examples: 1. Carbohydrates 2. Lipids 3. Proteins 4. Nucleic acids All of the above are polymers except lipids Polymers : formed by joining one or more building blocks called MONOMERS.

Dehydration Synthesis Polymers are formed by dehydration synthesis that involves removal of water. HO H H2O HO H

Hydrolysis Separates monomers by “adding water” HO H H2O HO H

Carbohydrates

Carbohydrates: Structure Classfication Monosaccharides: simple sugars (monosaccharides) Ex.: glucose Polymer (chain of units): complex carbohydrates (or polysaccharides) Ex.: starch, cellulose, chitin, glycogen Disaccharide: 2 simple sugars bonded together

Carbohydrates Monosaccharide: one sugar unit Examples: glucose (C6H12O6) Fructose Galactose glucose

Carbohydrates Disaccharide: double sugar Examples: Sucrose (glucose+fructose) Lactose (glucose+galactose) Maltose (glucose+glucose) glucose

Carbohydrates Polysaccharides: polymers composed of many sugar building blocks Examples: starch glycogen cellulose glucose cellulose

Carbohydrates: Indicator Tests Reducing sugars : Benedict’s test is used as a simple test for  detecting reducing sugars Complex Sugars(Polysaccharides) Lugol’s test /Iodine

5-1 Benedict’s solution + Cu2O 1- + CuSO4/0H Maltose 2- reducing -CooH + Cu2O (Red –brown precipitate) 1- + CuSO4/0H Maltose 2- reducing Glucose + Galucose

Benedict’s solution Sucrose Non-reducing Lactose Non-reducing 3- 4- Glucose + Fructose Lactose 4- Non-reducing Glucose + Galactose

Benedict’s test starch glucose maltose sucrose H2O 2cm Benedict 1cm solution Fill 5 of test tubes to I cm with following solution: {glucose, maltose, sucrose, starch & water} Fill each of the test tube to the 3 cm mark with Benedict’s reagent Place test tube in hot water bath (5 min) Observe color change

5-2 The iodine test(lugol’s)for starch Detect the presence of starch Starch :glucose polymer Glycogen: glucose polymer Iodine potassium iodide, I2KI, interacts w coiled starch and becomes bluish black color So ,form is important

iodine test(lugol’s) glucogen 2 drop Lugol’s glucose sucrose starch H2O 2cm solution Fill 5 of test tubes to I cm with following solution: {glucose, sucrose, starch ,glucogen & water} Add 2 drops of Iodine Observe color change

Positive Test Negative Test

Proteins

Proteins: Structure Proteins are made up of… Monomer (basic unit): amino acids 20 different kinds! Amino acids linked by peptide bonds

Proteins Ninhydrin test for amino acids (aas.) aas. having free amino groups are readily detected with ninhydrin -> purple or violet The Biuret test for polypeptide The amino group in one aas. is linked to carboxyl group on an adjacent aas. by a peptide bond this band is the site of action for the Biuret test for protein , Cu 2+must complex with at least four to six peptide bounds to produce violet color

5-3 Ninhydrin test for amino acid a.a 5 drop ninhydrin albumin glycogen milk H2O + heat 2 cm solution Fill 5 of test tubes to I cm with following solution: {albumin ,glycogen, milk ,a.a (glycine) & water} Add drops of ninhydrin reagent Place test tube in hot water bath (5 min) Observe color change @ sink Skin irritation

The Biuret test for polypeptide a.a albumin glycogen milk H2O 2 cm Biuret 2 cm solution Fill 5 of test tubes to 3 cm with following solution: {albumin ,glycogen, milk ,a.a (glycine) & water} Fill each test tube to the 4 cm with Biuret solution and mix Observe color change

5-4 The Biuret test for polypeptide 1% CuSO4 -> Cu2+ Complexes w at least 4-6 peptide bonds and produces violet color

Lipids

Lipids: Structure Fat is constructed from 2 kinds of smaller molecules : 1-glycerol 2-fatty acid Lipids are not soluble in water, but soluble in organic solvents.

Lipids “demo” 5-6 Lipid Solubility test Lipids are insoluble in polar solvent and soluble in nonpolar solvent fill 2 tubes to the 2 cm mark with corn oil In First tube, add 5 ml of tap water In second tube, add 5 ml of mineral oil Detect solubility 5-5 Sudan red for fat (book) Sudan red is a lipid soluble dye Add 10 drops of Sudan red to tube number 1 in above experiment Observe color , where is the red color found?

Nucleic Acids

Nucleic Acids: Structure Nucleic Acids are made up of… Monomers (basic units): nucleotides Two types: Deoxyribonucleic acid (DNA-double helix) Ribonucleic acid (RNA-single strand)

5-9 Dische diphenylamine test “demo” In this test ,acidic condition convert deoxyribose to a molecule that binds with diphenylamine to form a blue complex. Number 3 test tube 1-3 Into tube 1,place10 drops of banana DNA Into tube 2,place 10 drops of standard DNA Into tube 3,place 10 drops of distilled water Add 1 ml of diphenylamine regent Place in a boiling water bath for 10 min Transfer the tubes to ice Observe color change. Banana DNA Standard DNA water

Enzymes…

Enzyme Substrate(s) +enzyme product(s) We will take 2 examples 1- casein (protein milk)+Rennin coagulation 2- catechol +catechol oxidase benzoquinone Optimal conditions Optimal condition Optimal condition

Enzyme (Rennin) Rennin is found in the stomach of young mammals The enzyme coagulate the casein ,a protein found in milk Optimal condition pH = ~7 (neutral) Temperate = 37 C

6-1 Effect of temperature on enzyme activity Put 3 ml Rennin Place at 100 C for 5 min Place at 0 C for 5 min Place at 37 C for 5 min Add milk (2/3) full ,then incubate to 37 C for 15 min “Accurate measures”

6-2 Effect of pH on enzyme activity Put 3 ml Rennin Add 3 ml HCl Add 3 ml NaOH Add 3 ml H2O Add milk to each tube (2/3) full ,then incubate at 37 C for 15 min

6-4 Enzyme (Catechol oxidase) “demo” Catechole oxidase is an enzyme that turns bruised or cut fruits and vegetables brown Catechol is a clear chemical ,when it reacts with oxygen in presence of the enzyme catechol oxidase ,it forms the dark brown product ,benzoquinone

Catechol oxidase “Demo” Prepare 3 test tubes using the following table

Go To Work……