Post-Lab 1: Qualitative Analysis of Proteins

Amino acids

Building blocks of proteins

Amino and carboxylic acid

_____ and _____ groups comprise the functional groups in a class of biologically important compounds, amino acids

Proteins

Polymers composed of amino acids that are linked together by a peptide bond (amide) bond

Peptide bonds (amide bonds)

Proteins are linked together by _____

Complete hydrolysis

_____ of proteins yields the component amino acids

R groups, peptide backbone

Different types of intermolecular forces of attraction between ____ and the _____ maintain the structures of proteins

Secondary and tertiary

The biological activity of a protein depends on its _____ and _____ structures

Protein denaturation

Associated with alteration of protein structures, causing changes in physical properties (precipitation or coagulation) and loss of biological activity

Heat, light, chemical reagents (such as alcohols, strong acids and bases, detergents, heavy metal ions like Hg(II) and Pb(II))

Denaturation may be caused by action of (?)

Gelatin

Derived from protein collagen, connective tissue protein by boiling with water

Collagen

Insoluble in water and resistant to animal digestive enzyme

True

(True/False) Gelatin is easily digestible and soluble

Ninhydrin Test

• Test for amino acids and proteins with free -NH2 (amino) groups

• Forms a complex with free amino acids and proteins and shows deep purple color for proteins; gives orange color for proline because proline does not have a free NH2 group

• Commonly used in the detection of fingerprints

Deep purple

Ninhydrin test gives a _____ color for proteins

Orange

Ninhydrin test gives a _____ color for proline

Ninhydrin Test

Commonly used in the detection of fingerprints

Ninhydrin in ethanol solution

Reagent for Ninhydrin Test

Blue or violet-colored product

Proline = Orange

Positive result for Ninhydrin Test

Biuret Test

• General test for proteins

• Determines the presence of protein peptide bonds

Biuret Test

Copper ions found in _____ forms a complex with the protein’s peptide bond which gives a characteristic violet/blue violet color

Blue-violet solution

Positive result for Biuret Test

Copper sulfate (CuSO4) in alkane medium

Reagents for Biuret Test

Sakaguchi Test

• Chemical test for detecting the presence of arginine in proteins

• Given by guanidinium compounds

• Arginine reacts with a-naphthol in presence of an oxidizing agent such as bromine water or sodium hypochlorite to give a red colored product

Oxidizing

Bromine water and sodium hypochlorite are examples of _____ agents

a-naphthol in alkaline medium; bromine-water

Reagents for Sakaguchi Test

Red-colored solution

Positive result for Sakaguchi Test

Hopkin’s Test

• Chemical test for detecting the presence of tryptophan in proteins

• aka Glyoxylic acid reaction

• indole group of tryptophan reacts with glyoxylic acid in the presence of cone H2SO4 to give a purple color

indole, glyoxylic acid

In Hopkin’s Test, the _____ group of tryptophan reacts with _____ in the presence of cone H2SO4 to give a purple color

Purple ring at the junction of 2 layers

Positive result for Hopkin’s Test

Hopkins-cole reagent (glyoxylic acid) + H2SO4

Reagents for Hopkin’s Test

Lead Sulfide Test

• Chemical test for sulfur containing amino acids (Cysteine, Cystine, Methionine)

• aka Lead acetate test

• Sulfur-containing amino acid (sulfhydryl/thiol group) are degraded in strongly alkaline media to release sulfide ion (S2-) in the form of H2S (hydrogen sulfide)

• The sulfide ions can react with lead (II) acetate to form a brownish-black precipitate

Brownish-black precipitate

Positive result for Lead Sulfide Test

Lead acetate (Pb(CH3COOH) + NaOH

Reagents for Lead Sulfide Test

Keratin, cysteine

In Lead Sulfide Test, hair strand will test positive due to the presence of _____ protein which contain _____ residues

Heat Coagulation Test

• Chemical test to detect coagulable proteins: albumin & globulin found in proteins

• Positive outcome of the heat coagulation test can be manifested by the creation of an extremely dense coagulum on top of the mixture

  • The lower portion of solution functions as a buffer

isoelectrical

Coagulation process involves heating proteins at their _____ pH

irreversible

Protein coagulation is an _____ process which occurs at the highest pH of proteins

• Denaturation of agglutination

• Dissociation of the protein denatured in the form it is in

2 phases of the process of coagulation by heat of proteins

methyl red indicator, 1% acetic acid

Reagent/pH indicator for Heat Coagulation Test

Dense coagulum (cloudiness)

Positive result for Heat Coagulation Test

orange

In Heat Coagulation Test, a solution giving an _____ color indicates the pH of around 5.1

Gelatin

Deficient in tryptophan and cysteine; non-coagulable protein

acetylated

NAC is negative in Ninhydrin test since the amino group is _____ (not free)