exp 2 formal report

Color Reactions of Intact Protein Casein and Acid/Base Hydrolyzate

Color Reactions of Intact Protein Casein and Acid/Base Hydrolyzate Ante, John MichaelCinco, Josemaria AntonioCruz, Albert Kevin De la Paz, Fallen GraceGubatan, GlennINTRODUCTIONProteinsformed by two or more amino acids bonded by peptide bondsRoles: structure, enzymes, and hormones etcProteins can be broken down by chemical or mechanical forces in a process called denaturation

Proteins can be based on structurePrimary, amino acids covalently linked togetherSecondary, arrangement in space of the atoms in the peptide backbone Tertiary, includes 3D arrangement of all atoms in the protein including the side chainsQuaternary, arrangement and interaction between subunits in a proteinNot sure about this4Amino AcidsAmino Acids are biological molecules composed of an amino group and a carboxyl groupOnly 20 are usually found in proteinsAll amino acids (except glycine) exhibit optical activity due to the presence of an asymmetric Carbon atom.----- Meeting Notes (7/11/13 08:25) -----delete asymmetric.... 5Amino acids have R-Groups and are classified according to these groupsThey are based mainly on two criteria namely:Polarity of side chains orAcidic or Basic side chainsAside from glycine, most of the amino acids are chiral but are usually found in their L-form

CASEINMost important protein in milkmaking up 80% of the proteins in cow milk and between 20% and 45% of the proteins in human milk.Hydrophobic= poorly soluble in water (H2O)

----- Meeting Notes (7/11/13 08:25) -----delete poorly7Acid HydrolyzateTryptophan is destroyedSerine and threonine are decomposed to some extentBase Hydrolyzate

Racemization Decomposition of Threonine, Serine, Arginine and Cysteine Color Reactionsfunctional groups in amino acids and proteins react to produce colored product.

color intensity varies in proportion to the number of reacting functional or free groups and their accessibility to reagent.OBJECTIVETo characterize acid/base hydrolyzate and intact protein casein using various chemical testsMETHODOLOGYColor Reaction TestDried casein was cut into small pcs.; placed in a mortar 10 ml dist. Water addedGround until fine soln was obtainedPROTEIN SUSPENSIONHYDROLYZATESakaguchi Test5 drops of Protein Susp.

5 drops hydrolyzate

+1 drop 10% NaOH+1 drop 0.02% naphthol solution

Mix

+1 drop 2% NaOBr after 3 min.Note color

Biuret Test+1 drop 2.5M NaOH+0.01M CuSO4

Mix

3 drops protein suspension3 drops hydrolyzateNote color----- Meeting Notes (7/11/13 08:25) -----change 15NINHYDRIN TEST+ 0.5 ml 1% ninhyrdin soln to bothHeated in water bath for 2-3 mins. Note Color10 drops of Protein Susp.

1 ml of undiluted hydrolyzate

+1 ml H2OXANTHOPROTEIC TEST10 drops protein susp. + 1ml H20MixNote color Heat 1 min. in water bathAlkaline SolutionTest with Litmus paperNote Color+ 3 drops HNO3Add NaOH dropwise 1 ml hydrolyzate----- Meeting Notes (7/11/13 08:25) -----edit17+ 2mL of Hopkins-Cole reagent and mixIncline the tube + 2mL of conc. H2SO4Color of interphase notedHopkins Cole Test2 drops Hydrolyzate2 drops protein suspensioResultsIntact Casein Grp #Biuret TestSakaguchi testNinhydrin testXanthoproteic testHopkins-Cole Test 1Purple clear solutionRed orange turbid solutionIndigo with violet precipitateLight yellow ppt. colorless solnPurple interphaseDarker yellow ppt, yellow soln2Light purple clear solutionRed orange clear solution; jellylikeLight violet solution with light violet pptclearWhiteOrange soln3Violet solnRed-orange solnMurky soln w/ purple pptColorless w/ yellow pptPurple interphaseYellow-orange soln w/ whitish yellow ppt

4Purple solnOrange- red orange solnPurple soln w/ purple pptSmall yellow ppt w/ cloudy white solnPurple interphaseClear orange soln yellow orange ppt5Light Purple solnOrange solnLight purple solnClear, colorless w/ suspended particlesPurple interphaseOrange soln w/ suspended particlesIntact Casein Grp #Biuret TestSakaguchi testNinhydrin testXanthoproteic testHopkins-Cole Test 6Purple solnOrange solnClear w/ light violet ppt.Clear soln w/ white pptWhite interphaseRed orange ppt7Clear Purple solnRed orange solnPale blue-violet soln w/ purple pptTurbid soln w/ white particles

Light purple interphase

Orange soln w/ big red& fine white ppt

8Light blue violet solnIntesnse red-orange, slightly turbid solnColorless turbid soln w/ white precipitateturbid colorless solnLight purple interphaseturbid yellow soln w/ yellow precipitate

9Purple solnRed-orange solnColorless soln w/ white pptcolorless soln, white pptFaint purple interphaseRed orange ppt w/ yellow translucent soln 10Clear light violet solnOrange solnLight violet turbid pptTurbid colorless solnPurple interphaseTurbid orange pptAcid HydrolyzateGroup #Biuret TestSakaguchi testNinhydrin TestXanthoproteic test

Hopkins-Cole Test1Blue clear solutionLight yellow clear solutionmaroon clear solutionBright yellow clear solnColorless interphaseRed orang soln3Blue solnOrange solnDark violet/Rusty solnBright yellow solnClear InterphaseOrange soln

5Dark gray w/ pptYellow orange solnMaroon/ dark red solnYellow solnColorless turbid interphaseRed orange soln w/ susp. particles7Green-gray soln with particlesOrange soln Clear brown liquidClear, light yellow solnClear solnRed-orange soln w/ suspended particles9light blue solnYellow- orange solnDeep translucentNo change, translucent solnNo interphaseOrange soln w/ red orange ppt.

22Base HydrolyzateGrp #Biuret TestSakaguchi testNinhydrin TestXanthoproteic test

Hopkins-Cole Test2Clear gray solnClear light yellow solnDark reddish brown non turbid solnYellow soln Pinkish interphaseDark orange soln4Light purple solnLight yellow solnBrown clear soln

Small yellow ppt, cloudy white solnLight purple interphaseYellow-orange ppt and clear orange soln6Light yellow solnLight yellow-orange solnDark violet solnYellowish white solnPurple interphaseRed orange & clear soln8Light brownish yellow soln Turbid pale yellow soln Clear dark violet-dark red soln Darker yellow clear soln Light purple interphaseOrange yellow clear soln10Light brown/yellow solutionYellow orange solutionDark violet solnClear brownish orangeColorless interphaseTurbid, yellow pptDISCUSSION

ResultsSakaguchi Test

presence of argininePositive result: red-orange to red-colored solutionGuanido group in arginine reacts with naphthol in the presence of an oxidizing agent to give off the red color.Condensation reaction

oxidizing agent:NaOB

Crondensationis a chemical process by which 2 molecules arejoinedtogether to make a larger, more complex, molecule, with theloss of water.oxidizing agent:NaOBr

----- Meeting Notes (7/11/13 08:25) -----hyperlink27Sakaguchi TestAddition of 10% NaOH: catalyzes the reactionAddition of 0.02% naphthol solution: condensation reaction with arginineAddition of 2% NaOBr after 3 minutes: NaOBr and NH in guanido group produces a red complexSakaguchi TestPositive result for Intact casein and acid hydrolyzate. Arginine is present.Base hydrolyzate will give off a light yellow or yellow-orange solution. Alkaline hydrolysis decomposes arginine.

ResultsSakaguchi TestPossible errors:Solution will lose its red coloration if left for an extended period of timeErrors for Acid hydrolysis: hydrolyzate will require neutralization by addition of a base. If excess base is added, arginine may be destroyed.Results----- Meeting Notes (7/11/13 08:25) -----delete causing31detect ammonia or primary and secondary aminesfree -NH2 group and proteins W/ free amino group reacts with ninhydrin purple-blue complex.Ruhemann's purple = reacts to free aminesMost of the amino acids, except Proline, are hydrolyzed and react with ninhydrin*PROLINE GIVES A COLOR YELLOW

Ninhydrin degrades amino acids into aldehydes, ammonia, and CO2 the net result is ninhydrin in a partially reduced form hydrindantin:

Ninhydrin then condenses with ammonia and hydrindantin to produces an intensely blue or purple pigment, sometimes called Ruhemann's purple:

PROTEIN SUSPENSIONACID HYDROLIZATEBASE HYDROLIZATENINHYDRIN TESTLight violet solution with purple precipitateViolet/Dark violetViolet/Dark violetNINHYDRIN TESTgroupIntact CaseinAcid hydrolyzateBase hydrolyzate1PositivePositive2PositivePositive3PositivePositive4PositivePositive5PositivePositive6PositivePositive7PositivePositive8PositivePositive9PositiveNegative10PositivePositiveoxidative deamination and decarboxylation; reduction of ninhydrinPositive result:Intact casein: pale violet soln with purple ppt.Base/ Acid Hydrolyzate: violet/ dark violet

Results

ResultsYellow PrecipitateSaltGrp #INTACT CASEIN1Light yellow ppt. colorless solnDarker yellow ppt, yellow soln2clearOrange soln3Yellow solnYellow-orange soln w/ whitish yellow ppt

4Small yellow ppt w/ cloudy white solnClear orange soln yellow orange ppt5Clear, colorless w/ suspended particlesOrange soln w/ suspended particlesGrp #6Clear soln w/ white pptRed orange ppt7Turbid soln w/ white particles

Orange soln w/ big red & fine white ppt

8turbid colorless solnturbid yellow soln w/ yellow precipitate

9colorless soln, white pptRed orange ppt w/ yellow translucent soln 10Turbid colorless solnTurbid orange pptAcid HydrolyzateGroup #Xanthoproteic test

1Bright yellow clear solnRed orang soln3Bright yellow solnOrange soln

5Yellow solnRed orange soln w/ susp. particles7Clear, light yellow solnRed-orange soln w/ suspended particles9Translucent soln, no changeOrange soln w/ red orange ppt.

Base HydrolyzateGroup #Xanthoproteic test

2Yellow soln Dark orange soln4Small yellow ppt, cloudy white solnYellow-orange ppt and clear orange soln6Yellowish white solnRed orange & clear soln8Darker yellow clear soln Orange yellow clear soln10Clear brownish orangeTurbid, yellow pptprotein solution is hydrolyzed by the conc. H2SO4 at the solution interphaseThe indole group of free Tryptophan reacts with glyoxylic acid to produce purple product

Add result later44(+) Positive results = violet ring formed in the junction / purple interphase

Protein SuspensionAcid HydrolyzateBase HydrolyzateHopkins-Cole TestPurple Interphase(+)Colorless Interphase(-)Purple Interphase(+)ResultsAdditional ReactionsPaulys Diazol testdetection of Tryptophan or Histidine reagent: sulphanilic acid dissolved in hydrochloric acidForms a diazonium saltThe diazonium salt couples with tyrosine or histidine in alkaline medium result: red coloured chromogen (azo dye)Millons Testtyrosine reacts with Millons reagent

Millons rgt: Mercurous nitrate in HNO3

Positive: flesh to red complex upon heating. The complex is probably the mercury salt of the phenolic compound.

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Conclusion Through various color reactions, intact casein, and base and acid hydrolyzate were known to contain different amino acids as part of their composition.Milio, Frank R., & Loffredo, William E. (1995). Qualitative Testing for Amino Acids and Proteins.Modular Laboratory Program in ChemistryBenedict, Stanley R. (1999). A Note on the Preparation of Glyoxylic Acid as a Reagent.The Journal of Biological ChemistryBettelheim, F., Vandesberg, J., (2013). Laboratory Experiments for Introduction tp General, Organic, and Biochemistry (8th Ed). Brooks/Cole, Cengage Learning: USA.http://amrita.vlab.co.in/?sub=3&brch=63&sim=1094&cnt=1 Retrieved July 5 2013http://prezi.com/knbu2alx8prs/copy-of-results/ Retrieved July 5Clark, J.M. Jr., Experimental Biochemistry. 1964. New York: W.H. Freeman and Company. pp. 69-96.Espino-Cabatit, B., Biochemistry, 12e.1988. Philippines: UST Publishing House. pp. 135-138, 141, 142-144

Reference