Protein Isolation: Casein and Glutenin

Here are the study notes:

• Effect of Various Substances on Proteins*

• Isopropyl alcohol:

  • Denatures casein and gliadin
  • Coagulates egg white

• ZnSO4:

  • Causes precipitation of casein
  • Coagulates gliadin and egg white

• CuSO4:

  • No effect on casein
  • Coagulates gliadin and egg white

• HgCl2:

  • Causes precipitation of casein
  • Coagulates gliadin and egg white

• Na4Fe(CN)6:

  • No effect on casein and gliadin
  • No effect on egg white

• Denaturation of Proteins*

• Denaturation: loss of native structure and biological activity

• Causes of denaturation:

  • Heat: disrupts non-covalent bonds, leading to unfolding of protein structure
  • pH: extreme pH values disrupt electrostatic interactions
  • Chemicals: detergents, urea, guanidine hydrochloride, and beta mercaptoethanol disrupt hydrophobic and electrostatic interactions

• Reversibility of denaturation: some denaturations can be reversed, while others are permanent

• Properties of Amino Acids*

• Zwitter ions: amino acids exist as zwitter ions in the body, with both acidic and basic properties

• Acid-base properties: amino acids have pK1, pI, and pK2 values, which describe the pH ranges for different ionic forms

• Tertiary structure: 3-dimensional shape of amino acids, influenced by interactions between side chains and prosthetic groups

• Color Reactions of Amino Acids*

• Ninhydrin test:

  • Positive result: purple or violet color, indicates presence of amino acids or proteins
  • Used to detect amino acids, peptides, and proteins

• Biuret test:

  • Positive result: yellow color, indicates presence of phenols or imines
  • Used to detect tyrosine, tryptophan, and other aromatic amino acids

• Pauli's test:

  • Positive result: violet color, indicates presence of guanidine or amino groups
  • Used to detect arginine, histidine, and other amino acids with guanidine or amino groups

• Sakaguchi's test:

  • Positive result: red color, indicates presence of guanidine or amino groups
  • Used to detect arginine, histidine, and other amino acids with guanidine or amino groups

• Ehrlich's test:

  • Positive result: pink color, indicates presence of aromatic amino acids
  • Used to detect tryptophan, tyrosine, and other aromatic amino acids

• Xanthoproteic test:

  • Positive result: yellow or orange color, indicates presence of aromatic amino acids
  • Used to detect tryptophan, tyrosine, and other aromatic amino acids

• Lipids*

• Solubility:

  • Lipids are insoluble in polar solvents, but soluble in non-polar organic solvents
  • Examples of non-polar solvents: petroleum ether, hexane, chloroform

• Extraction methods:

  • Hexane extraction: used for non-polar lipids
  • Chloroform-methanol extraction: used for polar lipids

• Types of triglycerides:

  • Saturated: solid at room temperature, examples include lard and butter
  • Unsaturated: liquid at room temperature, examples include olive oil and cod-liver oil

• Protein Isolation and Analysis*

• Isolation of casein and glutenin:

  • Casein: can be isolated from milk using acidification, rennet, or heat
  • Glutenin: can be isolated from wheat flour using organic solvents

• Color reactions:

  • Ninhydrin test: used to detect amino acids, peptides, and proteins
  • Biuret test: used to detect phenols or imines
  • Pauli's test: used to detect guanidine or amino groups
  • Sakaguchi's test: used to detect guanidine or amino groups
  • Ehrlich's test: used to detect aromatic amino acids
  • Xanthoproteic test: used to detect aromatic amino acids### Carbohydrates and OsaZone

• OsaZone is a product formed from sugars, characterized by a crystalline structure when viewed under a low-power microscope.

• The time required for OsaZone formation is a specific property of a sugar.

Sugar Classification and Tests

• Reducing sugars: Arabinose, Fructose, Galactose, Glucose, Xylose
• Non-reducing sugars: Cellulose, Inulin, Lactose, Maltose, Starch, Sucrose
• IAA (Indole-3-Acetic Acid) test: detects Fructose and Sucrose
• Iodine test: detects Starch

Polysaccharides

• Long chains of monosaccharides
• Classification:
  • Homopolysaccharides: Starch, Glycogen, Cellulose (contain same sugar units)
  • Heteropolysaccharides: Pectin, Chitin, Agar (contain different sugar units)
  • Mucopolysaccharides: Glycosaminoglycans (repeating sugar units)

Starch and Glycogen

• Starch: mixture of Amylose and Amylopectin
• Amylose: 20% of Starch, 300 units of D-glucose linked by alpha 1-4 glucosidic bonds, forms a helix
• Amylopectin: major constituent of Starch, branched, majority of units linked by alpha 1-4 glucosidic bonds
• Glycogen: analogous to Amylopectin, larger and more branching

Color Reactions of Carbohydrates

• Molisch test: detects Carbohydrates, purple/red-violet color with alpha-napthol and concentrated sulfuric acid
• Anthrone test: detects Carbohydrates, blue-green color with anthrone and concentrated sulfuric acid
• Selivanoff test: differentiates aldoses and ketoses, cherry red color for ketoses, yellow to faint pink color for aldoses
• Bial's test: differentiates pentoses and hexoses, blue-green color for pentoses, purple-red color for hexoses