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Biological Macromolecules

Practical Biology Biol 101

Lab 4

University of HailFaculty of Sciences

Department of Biology

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All living organisms are composed of four classes of macromolecules:

Carbohydrates

Lipids

Proteins

Nucleic acid

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Carbon containing compounds are called organic compounds.

Carbon is the backbone of organic molecules, but its only one of several important bioelements.

These bioelements are hydrogen, nitrogen, oxygen, phosphorous and sulfur.

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The chemical properties of the different classes depend on the presence of specific functional groups:

Aldehyde group

Ketone group

Carboxyl group

Amino group

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Identification of CarbohydratesCarbohydrates are the main energy storing molecules.

Monosaccharide : monomers (glucose, ribose, galactose).

Disaccharides: two monomers connected with glycosidic

bond (sucrose, lactose).

Polysaccharides: polymers; more than three monomers

(starch).

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Identification of carbohydratesA. Benedict’s test for reducing sugars: The test depends on the presence of free Aldehyde or

ketone group. Monosaccharides and some disaccharides have these groups

free are called reducing sugars. Free groups found in reducing sugars will react with

Benedict’s reagent (CuSo4 which is blue in color with NaOH).

The test is both qualitative and quantitative test; Small amount of reducing sugar green color. Large amount of reducing sugar red- orange color.

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Procedure: Label 7 clean test tubes (1-7)

Fill the test tubes with 1 ml of one of the following solutions.

D.W

gluc

ose

sucr

ose

starc

h

milk

appl

e jui

ce

mal

tose

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Fill each test tube with 3 ml of Benedict’s reagent Put the test tubes in boiling water for 5 minutes

If color change to:

Record your results in a proper table

Yellow Orange Red

Positive test for reducing.

If color remains blue (the color of Benedict’s reagent)

Negative result.

Green

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D.W gluc

ose

sucr

ose

starc

h

milk

appl

e jui

ce

malt

ose

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B. Lugol’s iodine test for starch

Starch is a polysaccharide consisting of many glucose monomers linked together into long branching chains.

It is the primary storage carbohydrate in plants.

In the presence of iodine (I2-KI), a solution containing starch will turn blue-black in color.

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Procedure: Label 7 clean test tubes (1-7)

Fill the test tubes with 2 ml of one of the following solutions.

D.W

gluc

ose

sucr

ose

starc

h

milk

appl

e jui

ce

Potat

o ju

ice

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A blue-black

Add two drops of lugol’s iodine to each test tube.

Record the result in the report.

If color change to:

Positive test for starch

If color remains yellow - brown (the color of the iodine)

Negative result.

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D. W gluco

se

sucr

ose

starc

h

milk

appl

e jui

ce

Potat

o ju

ice

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Identification of ProteinsProteins are the key substances in the structural and

physiological function of living things.

Proteins are polymers of amino acids in which the carboxyl group of one amino acid is linked with the amino group of the next amino acid in a covalent bond called the peptide bond.

A. Ninhydrin test for amino acids Ninhydrin reagent reacts with free amino groups i.e free

amino acids to form a purple or violet colored substance.

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Ninhydrin reagent can also be used to detect proteins, but they must be heated or digested to hydrolyze the protein into free amino acids.

Procedure label 4 clean test tubes (1-4)Fill the test tubes with 2 ml of one of the following solutions:

1) Albumin solution.2) Milk. 3) Amino acid solution (Lysine).4) Distilled water.

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To each of the test tubes, add 10 drops of ninhydrin reagent and heat the test tubes in boiling water for 5 minutes (avoid inhaling, poisonous fumes).

A purple color is the +ve result.

Record the color of the tubes content in the lab report.

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B. Biruet test for polypeptides

The test reveals the presence of peptide bond i.e. proteins

Biuret reagent CuSo4 reacts with the peptide bonds between the amino acids changing in color from light blue to violet under alkaline conditions.

The intensity of the violet color is proportional to the protein concentration.

In the test, Cu+2 must complex with at least four to six peptide bonds to

produce a color.

Biuret reagent does not react with free amino acids. Short peptides don’t react positively.

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Procedure: Label 5 clean test tubes (1-5)

Fill the test tubes with 2 ml of one of the following solutions.

D.W

Lysin

e

starc

h

album

ine

milk

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Fill each test tube with 2 ml of Biuret reagent

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If color change to:

Record your results in a proper table.

Violet Positive test for Biuret test

If color remains blue (the color of Biuret reagent).

Negative result.

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D.W

milk

Lysin

e

starch

album

ine

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Identification of lipidsLipids are a heterogeneous group of compounds that are

insoluble in water, but are soluble in organic solvents such as ether and acetone.

There are three major classes of lipids: Neutral fats (triglycerides). Phospholipids Steroids

A. Sudan red test for fats Sudan red is a lipid soluble dye, when added to a mixture of

lipids and water, The dye will move into the lipid layer giving the lipid a red color.

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Procedure

Fill the test tube with 2ml of Water .Add 10 drops of oil and mix.The two liquids do not mix after shaking the test tube,

because the oil molecules are hydrophobic (insoluble in water) to produce an emulsion.

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Add 10 drops of Sudan IV solution to the test tube and mix.

Record your observations.

Two layers are formed, the upper one is the lipid stained with Sudan red IV

The water remains in the bottom layer

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Thank You