ACTIVITY 2B - PRECIPITATION TEST FOR PROTEIN

ACTIVITY 2B PRECIPITATION TEST FOR PROTEIN

GROUP 6 MARIMON, Dave Michael C. MACION, Ayn Rand Louise V. TANDUYAN, Maria Cristalia RUIZO, Roichinne DATUMANONG, Madatu Datudick Jr. BAID, Ivy Zea MALIRONG, Chiezyl

OBJECTIVES:   

To observe the reactivity of reagent and solutions in each test. To know the practical reactions in each test. To identify and observe the precipitate in each test which yield a positive result.

BMLS – 2H April 21, 2014

RESULTS AND OBSERVATIONS: NAME OF TEST A. Precipitation reaction by concentrated mineral and organic acids

SUBSTANCE TO BE TESTED Proteins

B. Precipitation by metallic salts

Proteins

C. Precipitation by alkaloidal reagents

Proteins

D. Precipitation by alcohol

Proteins

E. Coagulation by heat

Proteins

F. Test for denatured/Coagulated Proteins

Proteins

REAGENTS USED A. Concentrated Sulfuric Acid B. Concentrated HCl C. Concentrated HNO3 D. Glacial Acetic Acid A. Dilute Mercuric Chloride B. Dilute Lead Acetate C. Dilute Copper Sulfate D. Dilute Ferric Chloride F. Dilute Barium Chloride A. Picric Acid Solution B. Trichloroacetic Acid C. Tannic Acid D. Phosphotungstic Acid A. 95% alcohol + Dilute HCl B. 95% alcohol + 10% NaOH C. 95% alcohol Acetic Acid MILLON’S TEST Millon’s Reagent XANTHOPROTEIC TEST Concentrated Nitric Acid Ammonium Hydroxide BIURET TEST 10% Sodium Hydroxide 0.5% Copper Sulfate HOPKIN’S – COLE TEST Hopkin’s Cole Reagent Pure Concentrated Sulfuric Acid

VOLUME OF REAGENTS USED 5 drops 5 drops 5 drops 5 drops 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 5 mL + 2 drops 5 mL + 2 drops 5 mL 2 drops

OBSERVATIONS/RESULTS A. B. C. D. A. B. C. D. E. A. B. C. D. A. B. C.

Whitish solution with precipitate at the bottom Whitish solution with precipitate at the bottom Clear solution ; NO PRECIPITATE Little cloudy solution ; NO PRECIPITATE (NOT PERFORMED) Whitish solution ; NO PRECIPITATE Light Bluish solution ; NO PRECIPITATE Clear Orange solution ; NO PRECIPITATE Clear solution ; NO PRECIPITATE Yellowish solution with precipitate at the bottom Whitish solution with precipitate at the top Brownish solution with precipitate at the bottom Whitish solution with precipitate at the top Clear solution ; NO PRECIPITATE Clear solution ; NO PRECIPITATE Little cloudy solution ; NO PRECIPITATE Coagulated whitish substance

Cloudy faded flesh solution ; NO PRECIPITATE 2 drops Yellow precipitate at the top 5 drops 5 drops Cloudy light bluish solution ; NO PRECIPITATE 1 mL 1 mL Cloudy whitish solution ; NO PRECIPITATE 1 mL 10 drops

PROOF/DOCUMENTATION:

A. PRECIPITATION REACTION BY CONCENTRATED MINERAL AND ORGANIC ACIDS FROM LEFT TO RIGHT: A. CONCENTRATED SULFURIC ACID B. CONCENTRATED HCl C. CONCENTRATED HNO3 D. GLACIAL ACETIC ACID

B. PRECIPITATION BY METALLIC SALTS FROM LEFT TO RIGHT: A. DILUTE LEAD ACETATE B. DILUTE COPPER SULFATE C. DILUTE FERRIC CHLORIDE D. DILUTE BARRIUM CHLORIDE

C. PRECIPITATION BY ALKALOIDAL REAGENTS FROM LEFT TO RIGHT: A. PICRIC ACID SOLUTION B. TRICHLOROACETIC ACID C. TANNIC ACID D. PHOSPHOTUNGSTIC ACID

D. PRECIPITATION BY ALCOHOL

E. COAGULATION BY HEAT

F. TEST FOR DENATURED/ COAGULATED PROTEINS

FROM LEFT TO RIGHT: A. 95% ALCOHOL + DILUTE HCl B. 95% ALCOHOL + 10% NaOH C. 95% ALCOHOL

ANALYSIS AND CONCLUSION: GUIDE QUESTIONS

FROM LEFT TO RIGHT: A. B. C. D.

MILLON’S TEST XANTHOPROTEIC TEST BIURET TEST HOPKIN’S – COLE TEST

1. GIVE THE PRINCIPLES INVOLVED IN EACH OF THE TEST. 

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Precipitation reaction by concentrated mineral and organic acids. Salt bridges results from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of various acidic or amine amino acid side chains will have this effect. As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salts change partners with the positive and negative ions in the new acid or base added. Precipitation by metallic salts. Proteins are precipitated by salts of heavy metals, such as mercuric chloride, zinc sulfate, etc. in weak alkaline solution, protein molecules carry negative charges and combine with positively charged metal ions to form insoluble salts which precipitate from the solution. The precipitated proteins are denatured and this process is irreversible. Precipitation by alkaloidal reagents. Alkaloidal reagents (e.g. tannic acid and trichloroacetic acid) are high molecular weight anions. The negative charge of these anions counteracts the positive charge of the amino group in proteins in giving a precipitate. Precipitation by Alcohol. Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bond is formed instead between the new alcohol molecule and the protein side chains. Where does the maximum precipitation occur? 3 rd tube. Coagulation by Heat. The result was coagulation by albumin solution. Egg- white is faintly alkaline. Complete precipitation takes place only in faintly acid solution. The temperature in which coagulation takes place depends on a large extent on the amount of acid and of salts present. Test for denatured/Coagulated Proteins. The term denaturation is used more frequently than coagulation by scientific investigators at the present time to denote certain changes in proteins. Definite characteristics of the proteins are changed when they are coagulated, among which is loss of solubility in water and dilute salt solutions. In some instances and under certain conditions the coagulation process may be reversible. Manner in which denaturation may be bought about. Coagulation of proteins may be brought about by a variety of processes. But in addition to heat the action of acids, akalies, salts, alcohol, mechanical agitation, radiation, and ultra-sonic vibrations may denature the protein and convert it from soluble into insoluble form weight anions. The negative charge of these anions counteracts the positive charge of the amino group in proteins giving a precipitate. Therefore, even a protein is denatured it will still give a positive result from the Qualitative of proteins.

2. USING MILK AS TEST SOLUTION, HOW WILL YOU PROVE THAT MILK IS A PROTEIN.

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Biuret solution is used to identify the presence of proteins. Biuret reagent is a blue that, when it reacts to protein, will change color to pink- purple.

Procedure:     

To a test tube, add 40 drops milk solution Add 3 drops of Biuret reagent Shake gently to mix Note color change Proteins will turn the solution into pink- purple

3. SHOW A SCHEMATIC DIAGRAM TO ARRIVE AT YOUR ANSWER. TO A TEST TUBE, ADD 40 DROPS OF MILK SOLUTION

ADD 3 DROPS OF BIURET REAGENT (SODIUM HYDROXIDE 10% AND COPPER SULFATE 0.5%

SHAKE GENTLY TO MIX

CHANGE IN COLOR OF SOLUTION TO PINK-PURPLE CONFIRMS PRESENCE OF PROTEIN