BL483 Biochemistry Techniques

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BL 483 Biochemical Techniques -- Lecture 2 - Characterizing an Acid Phosphatase

PDF File for Printing: Lecture 2

Text Reading:

• General Introduction to Amino Acids, Proteins and Enzymology, pages 67-77
• Enzymology and Enzyme Kinetics, pages 77 to 85
• Most relevant this week, pages 82 to 85
• Expt #9 Kinetics of Acid Phosphatase

1. General Principles Underlying Enzyme Assays

2. Finding a Valid Amount of Enzyme for Assay Conditions

3. Finding Optimum pH and Temperature

4. Acid Phosphatase - Assay System

5. Optimizing the Phosphatase Assay

Expt #2 - Characterization of Wheat Germ Acid Phosphatase

This Week you will:

1. Establish Assay Method
2. Determine valid [Phosphatase], ie find best enzyme concentration for the assay conditions being used.
3. Determine the pH optimum

Next Week you will:

• Analyze the Kinetics of Phosphatase using Initial Velocity Measurements
• Find Km and Vmax in absence of Inhibitors
• Find Km' and Vmax' in presence of 2 Inhibitors
• Use Competitive Inhibitor (inorganic phosphate, a reaction product)
• Use a Non-Competitive Inhibitor (fluoride ion)

Copyright ©1996, 1997, 1998, 1999 Wilbur H. Campbell, All Rights Reserved; wcampbel@mtu.edu

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