BIOCHEM LAB 2.0

• Factors Affecting Enzyme Activity

  • pH
  • Temperature
  • Substrate Concentration
  • Enzyme Concentration
  • Activators
• pH


  pH value of enzyme = works at maximum efficiency ; OPTIMAL pH (pH 7 / 7.4 )

  pH > / < optimal pH = enzyme activity DECREASES ↓ > stop
• Temperature


  Narrow range of temp = enzyme activity

  Certain temp for enzyme activity to occur = more active

  OPTIMAL TEMP = 37 – 40 °C

  Enzyme activity ↓ = temperature ↑ > optimal temp ; until it stops - STOP = change of natural composition

  Enzyme activity ↓ = temperature ↓ < optimal temp ; minimum temp / least enzyme activity - STOP = 0°C

  If temp rise again = enzyme activity reactivated
• Substrate Concentration


  Enzyme activity ↑ = substrate concentration ↑ - until LIMIT RATE is reached ; will become saturated

  HIGH subs. Concentration = no significant change in enzyme reax ; all enzyme active site will have substrate bound to them. – SATURATED

  Excess substrate mol CANNOT REAX until substrate bound to enzyme – reacted / been released

  
• Enzyme Concentration


  Enzyme concentration ↑ = reaction velocity ↑

  Determine activities of serum enzymes during diagnosis of disease

  
• Activators


  Require INORGANIC METALLIC IONS = optimum activity - Mg2+ ; Mn2+ ; Zn2+ ; Ca2+ ; Co2+ ; Cu2+ ; Na+ ; K+

  • Zinc, Iron Copper

  Rare ; anion = Chloride ion for AMYLASE
• Correlate the biological significance of carbohydrates, proteins, and cholesterol in the blood.
• Carbohydrates


  • Form of glucose
  • Energy source for cells
  • For proper brain function & metabolic process
  • Blood glucose regulation
  • Essential to avoid hypoglycemia or hyperglycemia
• Proteins


  • Contains enzyme, antibodies, transport proteins
  • Hemoglobin
  • For immune response, nutrient transport, metabolic regulation
  • Identify & neutralize pathogens
• Cholesterol


  • Cell membrane integrity
  • Hormone production & regulation
  • Helps in digestion & absorption of fatty acids
  • Overall, to maintain homeostasis, support & development, protect against chronic diseases
• TRIPEPTIDE


  A peptide consisting of three amino acids
• Amino acids in a tripeptide

  • Alanine
  • Glycine
  • Phenylalanine
• LIPIDS


  Fatty acids
• Lipid bilayer


  • Polar head = hydrophilic
  • Non-polar tails = hydrophobic
• Lipids are insoluble in water but soluble in organic solvents (chloroform, benzene, acetone)
• Triglycerides


  Lipids that store energy, consisting of 1 glycerol and 3 fatty acids
• Hydrolyzable lipids


  • Waxes
  • Glycolipids
  • Phospholipids
  • Neutral wax
• Non-hydrolyzable lipids


  • Steroids
  • Fat-soluble vitamins (A, D, E, K)
• Test for unsaturation of lipids

  1. Add bromine

  2. Brown to clear = unsaturated

  3. Brown precipitate remained = saturated
• Solubility test of fats

  • Mustard oil in water = -
  • Mustard oil in ether = +
  • Mustard oil in chloroform = +
  • Mustard oil in benzene = +
  • Mustard oil in ethyl alcohol = +
• Reactions of fats and oils

  1. Hydrolysis: Fats, oils + H2O > H+ ; lipase > glycerol + 3 fatty acids

  2. Saponification: Fats, oils + NaOH > glycerol + 3 salts of fatty acid soaps

  3. Hydrogenation: Oils + H2 > Ni ; catalyst > more saturated; double bond disappears = margarines; less unsaturated
• Protein


  Molecules made up of amino acids
• Proteins contain carbon, hydrogen, oxygen, nitrogen and sometimes sulfur
• Amino acids


  The 20 different molecules that make up proteins
• Polypeptide


  One or more chains of amino acids
• Proteins are biologically active due to the sequence of the amino acid chain, which causes the polypeptide to fold
• Proteins are encoded in genes
• All amino acids in proteins are L-isomers with an alpha-carbon
• Formation of peptide bond

  1. Remove O2 from preceding amino acid + 2 hydrogens from subsequent amino acid

  2. Connect the two groups with an amide bond

  3. Always recover H2O
• Physical properties of proteins

  • Colorless
  • Tasteless
  • Homogenous
  • Crystalline
  • Globular or fibrous
  • Large molecular weights; giant size
  • Have colloidal properties
  • Amphoteric; act as acids and bases
  • Have a fixed isoelectric point; move in an electric field
  • Solubility influenced by pH
• Albumin


  Water-soluble, heat-coagulable protein found in egg white and milk
• Casein


  Chief protein in milk, a phosphoprotein that is insoluble in water but soluble in dilute acid/alkali, and is non-heat coagulable
• Gelatin


  Insoluble in cold water but soluble in hot water, a non-heat coagulable protein derived from collagen and rich in proline, hydroxyproline, glycine and hydroxylysine
• Gelatin does not contain tryptophan, giving it a low biological value
• Protein denaturation


  Loss of secondary, tertiary and quaternary structures of a protein, with disruption of the primary structure (hydrolysis of peptide bonds to produce free amino acids)
• Factors that cause protein denaturation

  • Strong acids and bases
  • Organic solvents
  • Reducing agents
  • High salt concentration
  • Heavy metals
  • Temperature changes
  • Mechanical stress
• Enzyme


  A biological catalyst that increases the rate of a chemical reaction without being consumed or permanently altered
• Fundamental properties of enzymes

  • Able to increase the rate of a reaction without altering the chemical equilibrium between reactants and products

  
• Enzyme-substrate interaction


  Lock and key model: substrate precisely fits the active site

  Induced fit model: configuration of enzyme and substrate is modified, distorting the substrate to lower activation energy
• Nucleic acids


  DNA and RNA, consisting of nucleotides with a sugar, phosphate and nitrogenous base