Biochem Proteins Lab

Qualitative Color Reactions Biuret Test for Presence of a peptide bond Reagents CuSO4 + NaOH Principle Formation of coordination
complex Cu2+ and 4 Nitrogen atoms Oxidative decarboxylation (+) result Purple color of solution (faint purple for not isolated) Blue-violet color Yellow Proline (pyrrolidine ring) Intense=hydroxylate Alpha-Amino Acid Yellow color of solution with HNO3, Orange color of solution with NaOH (Tyrosine = outside polar +) Red color
Ninhydrin
Alpha-Amino acids (groups)
Ninhydrin (Triketohydrinden e hydrate)
Xanthoproteic
Aromatic amino acids
HNO3, NaOH
Nitration of aromatic rings via SeAr
Millons
Tyrosine (Phenyl ring)
Hopkins-Cole
Tryptophan
Sakaguchi
Arginine
Nitroprusside Fohls Test for Amide Paulys
Sulfur-containing Amino Acids Sulfur-containing Amino Acids Asn, Gln
Complexation (Mercuration and nitration/nitrosation/ Complexation of nitrohydroxyphenyl derivative Hg+ Glacial acetic acid, Reduction of oxalic acid glyoxylic acid, to glyoxylic acid and acid conc. H2SO4 catalyzed condensation of 2 Alpha-naphthol, Complexation and NaOBr, NaOH, condensation of alphaurea naphthol with groups of arg (guanido) NaOH, Complexation reaction nitroprusside 6M NaOH, lead Fusion Ionic reaction acetate crystals NaOH Basic Hydrolysis
Salt of Hg dissolved in HNO3
Purple color at interphase (Humin Appears as black ppt) Red color
Red color Black-brown precipitate Evolution of gas, presence tested using litmus paper Red Color
Histidine, Tyrosine
Sulfanilic acid in NaNO2 solution, Na2CO3
Formation of Azodyes
Results Color Reactions Biuret Ninhydrin Xanthoproteic Millons Hopkins-Cole Sakaguchi Nitroprusside Fohls Test for Amide Paulys Intact +++ +/+ +++ +++ +/+++ Acid +++ ++ +++ +++ +/+++ Base +++ +++ +++ ++ +++ Enzyme ++ ++ +++ +++ ++ ++ +/+++
Experimentation Protein Casein Description - Phosphoprotein (Phosphate groups that attach to OH groups of ser/thr) that exists as calcium caseinate - Prevent micelles in milk - Storage protein in milk - Isolated from milk via Isoelectric Precipitation (Principle) - Isoelectric pH 4.6 (Beyond/less, proteins become soluble) - Solubility of proteins decreases as it reaches isoelectric point - Curd-like precipitate - 2nd major protein in bovine milk - Metalloprotein that can bind to several metal ions like calcium and zinc - Can serve as regulatory protein lactose biosynthesis - Isolated from whey by Heat Denaturation (Principle) [in acidic solution] - Small, bright red protein common in muscle cells - Stores oxygen (used when muscles are hard at work) - Hemoprotein contains heme group at ip center - Isolated by Salt Precipitation (Principle) Salting in (filtrate) Salting out (add salt crystals) - Isolated by Difference in Solubility in Water (Principle) - Storage proteins responsible for elasticity and extensibility of dough - Consists of gliadin and glutenin - Isolated gluten free of starch when (-) to iodine test [presence of starch: Blue-black]; [no starch = yellow/brown]
Alpha-Lactabulin (Albumin)
Myoglobin
Gluten
Protein Hydrolysis Types: Complete Incomplete/Partial Hydrolysis Complete Use of strong acids/bases + high temperature (products = Amino Acids) Enzymes called protease (products = mixture of Amino Acids + Oligopeptide) Advantage Commonly used: 6N HCl Disadvantages Complete destruction of trp Partial destruction of cys and tyr Incomplete liberation of val and ile Racemization and Destruction of ser and thr Asn & gln converted to asp & glu Trp not destroyed Arg, asn, gln, ser are destroyed
Alkaline Incomplete
Use NaOH / KOH Specialized peptide bonds hydrolyzed by proteases like: trypsin, chymotrypsin, repsin, bromelain, papain
Classification of Proteins Globular - Polypeptide chains fold into spherical/globular shape - Soluble in aqueous system - Example: enzymes Further divided to: Simple Fibrous - Polypeptide chains arranged in long strands or sheets - Water insoluble - Examples: Keratin, Collagen
Composition
Biological Function
Yield only Amino Acids upon hydrolysis breaking of peptide bonds Conjugated/Complex Combination of simple proteins + Non-Protein substances 1. Nucleoprotein (nucleic acid + histones) 2. Phosphoprotein (casein calcium phosphates) 3. Hemeproteins (hemoglobin) Catalyst/Catalytic Activity = Invertase / Enzymes Protein (Hb) Nutrient and Storage Proteins = Gluten Structural Proteins = Collagen (Provides general structure) Contractile and Motile = myosin and actin Defense Proteins = Immunoglobulins Regulatory Proteins = Hormones

Biochem Proteins Lab

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Biochem Proteins Lab

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Qualitative Color Reactions Biuret Test for Presence of a peptide bond Reagents CuSO4 + NaOH Principle Formation of coordination

Alpha-Amino acids (groups)

Ninhydrin (Triketohydrinden e hydrate)

Aromatic amino acids

Nitration of aromatic rings via SeAr

Tyrosine (Phenyl ring)

Nitroprusside Fohls Test for Amide Paulys

Sulfur-containing Amino Acids Sulfur-containing Amino Acids Asn, Gln

Salt of Hg dissolved in HNO3

Purple color at interphase (Humin Appears as black ppt) Red color

Sulfanilic acid in NaNO2 solution, Na2CO3

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