Laboratory

1
Laboratory 8

• Enzymes

2
Chemical Reactions

• Chemical Reactions
• substrates (reactants) ? products
• Activation energy energy required to cause
  reaction to proceed

http//www.stark.kent.edu/cearley/ChemWrld/thermo
/thermo.htm
3
Heat and Chemical Reactions

• Heat adds energy to reactants
• In uncatalyzed reactions or reactions with
  inorganic catalysts, increased temperature
  increases reaction rates.

4
Catalyst Characteristics

• increase reaction rates (by decreasing activation
  energy)
• are effective in small amounts
• do not add energy to reaction
• are not consumed (permanently altered) by
  reaction
• are chemicals (inorganic or organic heat is not
  a catalyst)

5
Enzymes As Catalysts

• Enzymes consist of proteins often with a
  non-protein component
• Protein this other component holoenzyme
• Non-protein part may be inorganic (called a
  cofactor) or organic (called a coenzyme)
• Some vitamins act as coenzymes

http//www.people.virginia.edu/rjh9u/actenz1.html
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Enzymes As Catalysts

• Advantages of enzymes over inorganic catalysts
• Enzymes are SPECIFIC for particular reaction or
  type of reaction
• Enzymes allow CONTROL of rate of reaction
• Control comes because enzymes depend on their
  tertiary configuration which depends on the
  amino acid side chains!

7
Enzymes As Catalysts

• Enzyme activity depends on its tertiary
  configuration (shape in space).
• Shape can be changed by environmental changes
• Enzymes that are altered are said to be
  denatured and do not function normally
• Denaturating can be reversible or irreversible

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Denaturing Enzymes

• Things that denature enzymes
• Increased temperature
• Altered pH
• Altered ion concentrations (salts)

9
Optima

• For each environmental factor, there is an
  optimum
• Enzyme activity is lower on each side of optimum
• Each enzyme has its own optimum

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How Enzymes Work

• Enzyme forms an enzyme-substrate complex.
• Activation energy is decreased
• Products are released and enzyme can be reused.

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Reaction Studied

• H2O2 catalase ? H2O2 catalase complex ? H2O
  O2 catalase

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Measuring Reaction

• Spectrophotometer used to measure change in
  absorbance due to production of oxygen and
  reaction of oxygen with indicator
• Indicator guiacol
• Turns brown when oxidized
• darker color means more oxygen was produced
• Activity determined as slope of best fit line

13
Experiments Enzyme Availability

• 0.5 ml 1.0 ml 2.0 ml
• 7 cuvettes table, p. 106
• Record on pg. 109
• Determine enzyme activity
• ? absorbance / ? time
• (absorbance120 absorbance20)
• (120 seconds 20 seconds)
• Record activity on p. 110

14
Experiments Temperature

• 0-4 oC (ice bath) 24-28 oC (room temp) 37 oC
  48 oC 100 oC
• 11 cuvettes table p. 107
• Record on pg. 111
• Determine enzyme activity
• ? absorbance / ? time
• (absorbance120 absorbance20)
• (120 seconds 20 seconds)
• Record activity on p. 112

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Experiments pH

• Effect of pH 3 pH 5 pH 7 pH 9
• 9 cuvettes table p. 108
• Record on pg. 112
• Determine enzyme activity
• ? absorbance / ? time
• (absorbance120 absorbance20)
• (120 seconds 20 seconds)
• Record activity on p. 113

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Enzyme Short Report

• Raw Data Tables pp. 109, 111, 112
• Graphs (enzyme availability temperature pH
  based on data tables)
• Calculated Activities Tables (based on graphs)
  pp. 111, 112, 113
• Conclusions p. 115 1, 3, 7, 9, 11

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Conversion and Dosage Bonus

• Convert 50 µl to ml
• A drug comes as a suspension of 30 mg/ml. A
  person needs 45 mg of the drug. How ml should she
  receive?