Proteins --- HOH / H+ OH- Hydrolase --- Amino Acids QUALITATIVE ANALYSIS: Q1.

COLOR REACTIONS
A. BIURET - General test for peptides starting from a dipeptide. - Produce pink color from dipeptide to polypeptide. - CuSO4 Oxidizing Agent ; NaOH rxn medium Slightly + to PhenylAlanine because of Conjugative Stability D. MILLONS-NAZZE TEST - For HydroxyPhenyl ring - HgSO4 in H2SO4 + to Tyrosine = Old Rose Complex E. HOPKINS-COLE TEST - For Indole Ring - Glycoxylic Acid + H2SO4 + to Tryptophan = Violet Ring F. BROMINE WATER TEST - For Indole Ring - Br2 . H2O + to Tryptophan = Pink Solution G. ERLICH-DIAZO TEST - For Imidazole -NaNO2 in HNO2 producing highly colored azo dyes for Histidine (Redorange or Lighter Orange) H. REDUCED SULFUR TEST - For Sulfur Containing AA - Pb(OAc) + to Cysteine producing Black ppt. I. C. XANTHOPROTEIC - Test for Phenyl Ring - conc. HNO3 involves nitration of Phenyl Ring producing a Yellow Color + to Tyrosine + to Tryptophan SAKAGUCHI TEST - For Guanidino Group - 10%NaOH + 0.02% naphtol in Alkaline Hypobromite + to Arginine = Red/Orange Color

B. NINHYDRIN - General test for Amino Acids including Single Amino Acids. - Triketohydrinhydrate + AA = Pink / Lavender

COLOR REACTIONS SUMMARY: COMP. OF RGNTS

Triketo Hydrin Hydrate

TEST
1. Ninhydrin

+ RESULTS
Lavander

GRP. RESPONSIBLE
Free amino and Free COOH

IMPORTANCE
+ to AA Dipeptides in determining free AA + to tripeptide up to Protein + To Tyrosine

2. Biuret

NaOH + CuSO4

Violet

Peptide Linkages (At least 2) Hydroxyphenyl Group Benzene Ring

3. Millons

Hg in HNO3

Red

4. Xanthoproteic

Conc HNO3

Lemon Yellow

5. Hopkins Cole 6.Liebermanns

Glyoxylic Acid + conc. H2SO4 Conc. HCl + Sucrose HCHO Conc. H2SO4 Pb(OAc)2 Sulfanilic Acid in HCl, NH4OH KOH + Pb(OAc)2

Violet Ring Violet

Indole Group Indole Group

+ To Tyrosine + To Tryptophan Slightly + To Phe + To Tryptophan + To Tryptophan

7. Acreetosenheim 8. Erlichs

Violet Ring

Indole Group

+ To Tryptophan

Red-orange Lighter-orange Black ppt.

Imidazole Group

+ To Histidine

9. Reduced Sulfur 10. Bromine Water 11 . Molisch

Sulfur Containing / Thiol Indole Group

Br2 . H2O

Pink

+ To Cystine + To Cystine + To Methionine + To Tryptophan

12. Adanskiewez

naphtol in alcohol + conc. H2SO4 Glacial POAc + conc. H2SO4 10% NaOH, 0.02% naphtol in Alkaline Hyprobromite

Violet Ring

Carbohydrate Group Carbohydrate Group Guanidine

+ To Glycoproteins Glycoprotein

13. Sakaguchi

Reddish Violet Ring at the Junction Intense Red Color

+ To Arginine

Q2. CHROMATOGRAPHY - Principal at Partition/ Distribution Law separation of Amino Acids by virtue of their attraction to the mobile phase and to the stationary phase. TECHNIQUES: 1.) Paper Chromatography - Mobile Phase Solvent - Stationary Phase Medium (Paper) [HOH imbibed in the Cellulose of the paper] Rate of Flow Increases the affinity of the solute to the solvent. Rf = Distance traveled by the Solute Distance traveled by the Sovent - A solute with high Rf value has great affinity with the solvent. - A solute with low Rf value has less/weak affinity w/ the solvent but has high affinity with the medium. (Least soluble) EX: A mixture of Amino Acids (Leucine, PhenylAlanine, Serine) has eluted using paper chromatography, solvent used is Acetone. Leucine traveled 10cm from the starting point while Serine creeped the paper 7cm about the distance traveled by Leucine. Phenyl Alanine Landed in between Leucine and Serine. Calculate the Rf value. Given: Leucine = 10cm Serine = 3cm Phenyl Alanine = 6.5cm [ 10-3 = 7/2 = 3.5 + 3cm (Serine Distance to make it in between Ser and Leu)] Solution:

Q3. ELECTROPHOESIS - Separation of Amino Acids using Electric Current. - Migration of Amino Acids towards Electrolytes.

pI pH in which the net charge of Amino Acid is equal to zero. EX: A solution containing Lysine (pI = 9), Alanine (pI = 6.8), and Glutamic Acid (pI = 2.3) was electrophorized at pH 6.79. Describe the direction of migration towards the electrode. SOLUTION: Anode = Glutamic Acid (since pH 6.79 is above pI which is 2.3) Solution = Alanine (since pH is equal to pI) Cathode = Lysine (since pH 6.79 is below pI which is 9) STRUCTURE CHANGES:

Q4. CHEMICAL REACTION/PRECIPITATION A. USING SALT OF HEAVY METALS - Proteins pH must be at the basic side of pI - Protein ins anionic and attracted w/ positively charged metal and precipitates as proteinate * Used in Pb poisoning B. STRONGe ACIDS AND ALKALINE BASES - Disrupts salt bridge and ionization of acids and bases portion of the Amino Acids C. BY HEAT - Proteins are Hydrophilic Calloids, they take up large quantity of water that aids in their dispersibility when water is removed by heat, protein is precipitated out.

A. BY HEAT - 1drp. HOAc + 5drps. Egg Albumin - - > (heating) Coagulation B. BY HEAVY METALS - 5drps Egg Albumin + 2drps 10%Pb(OAc) - - > White - 5drps Egg Albumin +2drps 5% FeCl3 - - > Yellow Orange C. BY STRONG MINERAL ACIDS - 5 drps Egg Albumin + 1drp. Conc. HNO3 - - > White Ring/Ppt. - 5 drps Egg Albumin + 1drp. H2SO4 - - > White Ring/Ppt. D. ALKALOIDAL REAGENTS - 5 drps Egg Albumin + Tannic Acid - - > Brown - 5 drps Egg Albumin + Picric Acid - - > Yellow E. BY ALCOHOL - 5 dprs Egg Albumin + 2dprs HOAc + 2mL 95% Ethanol - - > Precipitation/Coagulation

F. COLOR REACTIONS (All 5drps Egg Albumin Except for Indicated) 1. BIURET - Negative 2. MILLONS - Negative - Tyrosine Red 3. HOPKIN COLES - Negative - Histidine Pink Ring 4. MOLISCH - Violet 5. REDUCED SULFUR - Negative - Cysteine Black ppt.

G. ACIDITY/BASICITY 1. 2. 3. 4. 5. 6. Glutamic Acid - 5 Aspartic Acid - 3 Arginine 10 Lysine - 8 PhenylAlanine - 7 Leucine - 7