BIOCHEM LAB LE #1

• Proteins are the most abundant macromolecules found within living cells
• Organic compounds of high molecular weight
• Made of alpha amino acids joined by means of peptide linkage
• Proteins are the fundamental constituents of the protoplasm of the cells
• Proteins are one of the building blocks of life
• Proteins act as catalysts, structural elements, lubricants, and cellular communication agents
• Proteins contain elements carbon, hydrogen, oxygen, nitrogen, sulfur, and phosphorus, together with traces of iron, copper, iodine, manganese, and zinc
• Experiment 1: Elementary Composition of Proteins
• Heating Casein in a low flame:

  • Charring indicates the presence of Carbon
  • Moisture indicates the presence of Hydrogen
• Mixing Soda Lime and Casein, heating slowly, and exposing red litmus paper to vapors:

  • Red litmus paper turns blue, indicating the presence of Nitrogen
  • Ammonia Gas NH3 turns the litmus paper blue, confirming the presence of Nitrogen
• Fusion Mixture:

  • Sodium Carbonate Na2CO3
  • Potassium Nitrate KNO3
  • Casein (C81H125N22O39P)

  Purpose: To oxidize sulfur to sulfate and phosphorus to phosphate
• Mixing fusion filtrate and diluted Hydrochloric Acid HCl, resulting in Barium Sulfate BaSO4 precipitate
• Mixing fusion filtrate and conc. Nitric Acid HNO3, adding Ammonium Molybdate (NH4)2 MoO4, and forming Ammonium phosphomolybdate (NH4)3PO4*12(MoO3) precipitate
• Experiment 2: Color Reaction of Proteins
• Biuret Reaction:

  • General test for proteins
  • Positive reaction for native and derived proteins with 2 carbonyl groups connected by single Nitrogen or Carbon atom
  • Violet color indicates long chain, pink color indicates short chain
  • Simple Amino acids do not give a positive test
  • Negative test results in a blue colored solution
• Xanthoproteic Reaction:

  • Presence of Phenyl Group leads to nitration of phenyl rings in aromatic amino acids
  • Yellow nitro-substitution products turn orange when alkali is added
• Millon’s Test:

  • Mixing 2% Albumin and Millon’s reagent, heating to form a red flocculent precipitate due to the presence of Tyrosine
• Glyoxylic Acid Reaction (Hopkins-Cole):

  • Mixing 2% albumin, Hopkins-Cole reagent, and conc. Sulfuric Acid H2SO4 to form a violet ring due to the presence of Tryptophan
• Heller’s Ring Test:

  • Detects the presence of Albumin in Urine by forming a white precipitate due to denaturation of proteins
• Reduced Sulfur Test:

  • Positive test shows the presence of reduced sulfur groups in Amino Acids by forming Lead (II) Sulfide (PbS)
• Adamkiewicz Reaction:

  • Mixing Albumin, Glacial Acetic Acid CH3COOH, and conc. Sulfuric Acid H2SO4 to form a yellow ring indicating the presence of indole derivatives
• Precipitation and heat coagulation occur best near the isoelectric point of protein
• Albumin coagulates when heated
• Aqueous solution of proteoses, peptones, gelatin, and casein do not heat coagulate
• Heat Coagulation denatures proteins forming lumps via disulfide chains
• Isoelectric point is the pH where the molecule has no net charge
• Denaturation is the process where proteins or nucleic acid lose quaternary, tertiary and secondary structure because of the presence of stress: acid, base, salt or heat
• Casein needs a dehydrating agent for coagulation
• Acidic medium brings the pH closer to isoelectric pH where proteins have minimum solubility and precipitate out
• Ethyl Alcohol and Albumin solution mixed with water results in white precipitate that does not dissolve in water
• Organic solvents precipitate proteins
• Alcohol disrupts intra hydrogen bonds in proteins, making tissues shrink and harden
• Alcohol is used as an antiseptic because it can precipitate proteins, preventing microorganisms from multiplying
• Solid Ammonium Sulfate and 2% Albumin mixed, resulting in a precipitate tested through Millon’s reaction and Biuret Test
• Positive Millon's Test indicates the presence of Tyrosine containing protein
• Negative Biuret Test means all proteins were salted out in Precipitation by Salting Out
• Adding salts and electrolytes to a protein solution causes the protein to precipitate
• Salting out is a purification method that makes molecules less soluble in a high ionic strength solution
• Precipitation by Heavy Metal Ions: 2% Albumin solution in three test tubes with Lead (II) Acetate, Silver Nitrate, and Cupric Sulfate results in different precipitates
• Egg albumin is used as an antidote for mercury or lead poisoning