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1 Biological Macromolecules & enzyme Heba Al-Tamimi

Biological Macromolecules & enzyme - JUfilesjufiles.com/wp-content/uploads/2016/05/03.Macromolecules.Lab... · 2 Macromolecules Examples: 1. Carbohydrates 2. Lipids 3. Proteins 4

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Biological Macromolecules & enzyme

Heba Al-Tamimi

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MacromoleculesExamples:

1. Carbohydrates2. Lipids3. Proteins4. Nucleic acids

All of the above are polymers exceptlipids

Polymers : formed by joining one or more building blocks called MONOMERS.

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Dehydration Synthesis

• Polymers are formed by dehydration synthesis that involves removal of water.

HO H

HO HO HH

H2O

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Hydrolysis

• Separates monomers by “adding water”

HO HO HH

HO H

H2O

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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

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CarbohydratesMonosaccharide: one sugar unit

Examples: glucose (C6H12O6)FructoseGalactose

glucose

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CarbohydratesDisaccharide: double sugar Examples:

– Sucrose (glucose+fructose)– Lactose (glucose+galactose)– Maltose (glucose+glucose)

glucoseglucose

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CarbohydratesPolysaccharides: polymers composed of many sugar building blocks

Examples: starchglycogencellulose

glucoseglucose

glucoseglucose

glucoseglucose

glucoseglucose

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

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+ CuSO4/0H

-CooH

1-

2- reducing

Maltose

Glucose + Galucose

+ Cu2O(Red –brown precipitate)

Benedict’s solution

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3- Non-reducing

4- Non-reducing

Sucrose

Lactose

Glucose + Galactose

Glucose + Fructose

Benedict’s test

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1cm solution

2cmBenedict

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

glucose maltose sucrosestarch

H2O

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)

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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

glucose sucroseglucogen

starch H2O

2 dropLugol’s

Negative Test Positive Test

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Proteins

Proteins: Structure• Proteins are made up of…

Monomer (basic unit):amino acids• 20 different kinds!

Amino acids linked by peptide bonds

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ProteinsNinhydrin test for amino acids (aas.)

aas. having free amino groups are readily detected with ninhydrin -> purple or violet

The Biuret test for polypeptideThe 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

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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

albumin glycogen milk a.aH2O

5 drop ninhydrin

+ heat

Ninhydrin test for amino acid

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The Biuret test for polypeptide

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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

albumin glycogen milk a.aH2O

2 cm Biuret

5-4 The Biuret test for polypeptide

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1% CuSO4 -> Cu2+ Complexes w at least 4-6 peptide bonds and produces violet color

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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.

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Lipids “demo”5-6 Lipid Solubility testLipids 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?

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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…

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Enzyme Substrate(s) +enzyme product(s)

We will take 2 examples1- casein (protein milk)+Rennin coagulation2- catechol +catechol oxidase benzoquinone

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Optimal

conditions

Optimal

conditionOptimal

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

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6-1 Effect of temperature on enzyme activity

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Put 3 ml Rennin

Place at 0 C for 5 min

Place at 100 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

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Put 3 ml Rennin

Add 3 ml HCl

Add 3 ml H2O

Add 3 ml NaOH

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

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

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Go To Work……

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