ACTIVITY 2BPRECIPITATION TEST FOR PROTEIN

GROUP 6 BMLS 2HMARIMON, Dave Michael C. April 21, 2014MACION, Ayn Rand Louise V.TANDUYAN, Maria CristaliaRUIZO, Roichinne DATUMANONG, Madatu Datudick Jr.BAID, Ivy ZeaMALIRONG, 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.

RESULTS AND OBSERVATIONS:

NAME OF TESTSUBSTANCE TO BE TESTEDREAGENTS USEDVOLUME OF REAGENTS USEDOBSERVATIONS/RESULTS

A. Precipitation reaction by concentrated mineral and organic acidsProteinsA. Concentrated Sulfuric AcidB. Concentrated HClC. Concentrated HNO3D. Glacial Acetic Acid5 drops5 drops5 drops5 dropsA. Whitish solution with precipitate at the bottomB. Whitish solution with precipitate at the bottomC. Clear solution ; NO PRECIPITATED. Little cloudy solution ; NO PRECIPITATE

B. Precipitation by metallic saltsProteinsA. Dilute Mercuric ChlorideB. Dilute Lead AcetateC. Dilute Copper SulfateD. Dilute Ferric ChlorideF. Dilute Barium Chloride1 mL1 mL1 mL1 mL1 mLA. (NOT PERFORMED)B. Whitish solution ; NO PRECIPITATEC. Light Bluish solution ; NO PRECIPITATED. Clear Orange solution ; NO PRECIPITATEE. Clear solution ; NO PRECIPITATE

C. Precipitation by alkaloidal reagentsProteinsA. Picric Acid SolutionB. Trichloroacetic AcidC. Tannic AcidD. Phosphotungstic Acid1 mL1 mL1 mL1 mLA. Yellowish solution with precipitate at the bottomB. Whitish solution with precipitate at the topC. Brownish solution with precipitate at the bottomD. Whitish solution with precipitate at the top

D. Precipitation by alcoholProteinsA. 95% alcohol + Dilute HClB. 95% alcohol + 10% NaOHC. 95% alcohol5 mL + 2 drops5 mL + 2 drops5 mLA. Clear solution ; NO PRECIPITATEB. Clear solution ; NO PRECIPITATEC. Little cloudy solution ; NO PRECIPITATE

E. Coagulation by heatProteinsAcetic Acid

2 dropsCoagulated whitish substance

F. Test for denatured/Coagulated Proteins

ProteinsMILLONS TESTMillons ReagentXANTHOPROTEIC TESTConcentrated Nitric AcidAmmonium HydroxideBIURET TEST10% Sodium Hydroxide0.5% Copper SulfateHOPKINS COLE TESTHopkins Cole ReagentPure Concentrated Sulfuric Acid

2 drops

5 drops5 drops

1 mL1 mL

1 mL10 dropsCloudy faded flesh solution ; NO PRECIPITATE

Yellow precipitate at the top

Cloudy light bluish solution ; NO PRECIPITATE

Cloudy whitish solution ; NO PRECIPITATE

PROOF/DOCUMENTATION:

A. PRECIPITATION REACTION B. PRECIPITATION BY C. PRECIPITATION BY ALKALOIDAL BY CONCENTRATED MINERAL METALLIC SALTS REAGENTS AND ORGANIC ACIDS

FROM LEFT TO RIGHT: FROM LEFT TO RIGHT: FROM LEFT TO RIGHT:

A. CONCENTRATED SULFURIC ACID A. DILUTE LEAD ACETATE A. PICRIC ACID SOLUTION B. CONCENTRATED HCl B. DILUTE COPPER SULFATE B. TRICHLOROACETIC ACID C. CONCENTRATED HNO3 C. DILUTE FERRIC CHLORIDE C. TANNIC ACID D. GLACIAL ACETIC ACID D. DILUTE BARRIUM CHLORIDE D. PHOSPHOTUNGSTIC ACID

D. PRECIPITATION BY E. COAGULATION BY HEAT F. TEST FOR DENATURED/ ALCOHOL COAGULATED PROTEINS

FROM LEFT TO RIGHT: FROM LEFT TO RIGHT:

A. 95% ALCOHOL + DILUTE HCl A. MILLONS TEST B. 95% ALCOHOL + 10% NaOH B. XANTHOPROTEIC TEST C. 95% ALCOHOL C. BIURET TEST D. HOPKINS COLE TEST

ANALYSIS AND CONCLUSION: GUIDE QUESTIONS

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

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? 3rd 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.

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- purple3. 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 TOPINK-PURPLE CONFIRMS PRESENCE OF PROTEIN