Experimental protocol:

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Enzyme assay for alkaline phosphatase to
determine Km Ki and Vmax and hence determine mode
of inhibition by Pi

• Experimental protocol
• Initial rate of reaction of alkaline phosphatase
  determined by measurement of chromogenic
  substrate photospectrometrically at A405.
• Reaction conducted in absence and presence of
  phosphate inhibitor.
• Construct Lineweaver-Burke plot and graphically
  determine Km Ki and Vmax.
• Use these data to characterise the mode of
  inhibition of the enzyme by phosphate.
• 
  alkaline phosphatase
• p-nitrophenol phosphate ? p-nitrophenol

Conclusions It can be seen from the above plot
that there is a reduction of Vmax when phosphate
is added to the reaction mixture. This suggests
phosphate is an irreversible inhibitor.
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Alkaline phosphatase assay to determine Km KI
and Vmax of alkaline phosphatase.

• Experimental protocol
• Determine initial rate of enzyme catalysis over
  10 min period by monitoring A405 of chromogenic
  substrate.
• A405 measured for different substrate.
• Initial rate measured both in presence and
  presence of a phosphate inhibitor.
• Graphically determine Km Ki and Vmax using
  Lineweaver-Burke plot.
• Use these data to characterise the mode of
  inhibition of the enzyme by phosphate.