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Experiment No.

12 PROTEINS

Objectives: 1. To extract proteins from corn 2. To establish their physical and chemical properties Materials: Corn grains 10% NaOH solution Ethanol 0.1 % CuSO4 NaCI 0.1% KOH solution NHO3 NH4OH H2SO4 Procedures: A. Extraction of Protein Soluble in Water 1. Grind about 50 g of corn grains in a coffee mill. 2. Add 125 ml of water and macerate for about 4 hours. 3. Filter off first in muslin cloth, then through a filter paper. 4. Test the filtrate for the presence of protein. B. Extraction of Globulin 1. Treat the residue in procedure A after water extraction with about 125 mL of 10% NaCI and macerate for 12-24 hours. 2. Filter first through muslin cloth, then through a filter paper 3. Dialize the extract for 24 hours 4. Filter off the precipitate of globulin which will have separated out and dissolved in 10% NaCI. 5. Test the solution for the presence of protein. C. Extraction of the Prolamine Zein 1. Extract residue after the salt extraction with 75 mL of hot 95% alcohol Coffee mill Hopkins cole reagent Millons reagent 10% NaOH Muslin cloth Erlenmeyer Funnel Bunsen burner Water bath

2. Filter and concentrate the filtrate containing the zein in a water bath. 3. Pour a few drops concentrated extract into absolute alcohol then in distilled water. 4. Evaporate to smaller volume; pour all the extract into the excess of distilled water 5. All a little of solid NaCI. 6. Filter off the precipitate of zein which will settle slowly 7. Test the precipitate for protein D. Extraction of Glutenin 1. Take about half of the residue after alcohol extraction 2. Extract the residue with 0.1% caustic potash solution 3. Filter off the extract containing the glutenin 4. Test the filtrate for the presence of protein Identification Tests: A. Organoleptic Evaluation. Determine the color, appearance, and physical state of proteins. B. Physical test. Determine the solubility of protein in water, alcohol, and ether. C. Chemical test. 1. Buret reaction. To about 2 mL of the protein solution, add 1 mL of 10% NaOH solution and add a drop of 0.1% CuSO4 solution. Mix well. Note the color formed. 2. Xanthoproteic reaction. To 3 mL of the protein solution, add 1 mL of concentrated HNO3. Observed the formation of a precipitate. Boil the mixture. Note the color change. Then add concentrated NH3 to make the solution alkaline. Note the color change. 3. Millons reaction. To 2 mL of the protein solution, add 1 mL of Millons reagent. Note the formation of a precipitate. Warm the precipitate. Note the color change. 4. Hopkins cole reaction. To about 2 mL of protein solution, add an equal amount of Hopkins cole reagent. Allow 1 mL of pure concentrated H 2SO to flow down the side of the inclined tube to form a layer of acid beneath the protein mixture. Note the color formed.

Results of identification tests: A. Organoleptic Evaluation Test Color appearance Physical state B. Physical test: Solubility tests. Test Water Alcohol Ether C. Chemical tests Test Buret test Xanthoprotelic Physical state Conclusions: A B C D A B C D A B C D

Questions: 1. What are proteins? Give the fundamental units of protein.

2. Identify the essential amino acids. Why are they called essential amoni acids?

3. What plant parts are rich sources of protein?

4. What is the consequences of protein deficiency in man?

5. Give the different classification of proteins.