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Experiment #2: Isolation and characterization of proteins

Casein from skimmed milk


Isoelectric precipitation
Casein (calcium caseinate), lactalbumins, lactoglobulins
pH to 4.6 (10% CH3COOH)
Filter by: vacuum, gravity filtration
Albumin from skimmed milk
Heat denaturation
Soluble in H2O and dilute salt solutions
Denatured by heat
Gluten from wheat flour
Difference in solubility (selective dissolution)
Washing the dough with H2O; removes starch
Test with I2 until negative result
Insoluble material (crude gluten)
Myoglobin from beef heart
Salt-induced precipitation
Oxygen carrier
In muscles; red color
Isolated by ammonium sulfate ppt.
Mix 70% (NH4)2SO4 to release myoglobin; dark-red extract
Proteins
- Most abundant organic molecules
- Polymers of amino acids (20)
- Catalyst
Considered in the isolation of protein from its source:
- 3D structure
- Interactions
- Acid-base property
- Solubility
Solubility altered by changing pH
Isoelectric pH insoluble; net charge = 0
Denaturation
- Disrupt the native conformation
- Extremes of heat and pH; denaturing solvents
- Alters the protein function
Qualitative
- Colorimetric reactions
- Paper chromatography

Quantitative
- Spectrophotometric
Acid hydrolysis of intact Protein
- 6M HCl to isolated protein
- 1M NaOH to neutralize
- Characterization test; chromatography
Alkaline hydrolysis of intact Protein
- 4M NaOH to isolated protein
- 1M HCl to neutralize
- Characterization test; chromatography
Enzymatic hydrolysis of intact protein
- Proteases (peptidases or proteolytic enzymes)
- Cut peptide bonds
- 6 classes:
1. Serine
2. Threonine
3. Cysteine
4. Aspartate
5. Glutamate
6. Metallopeptidases
- 0.1M phosphate buffer, pH 7.5
- Thin layer chromatography
Biuret test
- Detect the presence of peptide bonds
- 2.5M NaOH
- 0.1 CuSO4
Ninhydrin test
- Typical test for an alpha amino acid
- 0.1% ninhydrin solution
- blue-violet
Xanthoproteic test
- Detects side chains of aromatic amino acids
- conc. HNO3
- conc. NaOH
Millons test/ Hopkins-Cole test
- Determine tyrosine and tryptophan residues
- Millons reagent/ Hopkins-Cole reagent
- ? / conc. H2SO4
Sakaguchi test
- 10% NaOH
- 0.02% naphthol solution
- 2% NaOBr

Nitroprusside test
- If sulfur-containing amino acids are present
- 3M NaOH
- 2% nitroprusside solution
- Red solution
Fohls
-

test
30% NaOH
5% (CH3COO)2
Dark brown or black sediment

Test for Amides


- Detect R-groups for asparagine and glutamine
- 20% NaOH
Pauly
-

test
Diazo reagent, 1% sulfanilic acid
5% NaNO2
10% Na2CO3
Red coloration

Separation and identification of Amino Acids


- For qualitative analysis
- Thin-layer chromatography
- Amino acid standards:
1. 2% w/v Tryptophan
2. Arginine
3. Proline
4. Cysteine
5. Serine
6. Aspartic acid
7. Tyrosine
8. Histidine
9. Glycine
10.Alanine
Bradford assay
- Determine the total protein concentration (protein assay)
- Based on the binding of Coomassie dye in acidic soln.
- Leads to increased absorbance at 595 nm
- Sensitive: 20 to 200 mcg protein