Protein Biochemistry Flashcards
What are the primary reasons for assaying enzyme activity?
To detect the enzyme
To establish basic properties of enzyme
To determine amount of enzyme
What are the four most important characteristics of enzyme assays?
Specificity
Sensitivity
Accuracy/ Reliability
Convenience
Name six environmental conditions to consider when assaying and enzyme:
pH Temperature Degree of oxidation Heavy metal contamination Ionic strength Protease contamination
What are the alternative methods for a spectrophotometric assay if no natural substrate gives an absorbance change?
Convert product to derivative
Use a synthetic substrate
Use a coupled reaction
Give continuous assay alternatives to spectrophotometry:
Electrochemical methods
ATP hydrolysis
What is the yield of an enzyme?
The ratio of total activity of the purified enzyme to the total activity of the starting material
What is the specific activity of an enzyme measured in?
1 unit/mg
What is the purification factor of an enzyme?
The ratio of a preparation to the specific activity of the starting material
What are the general stages of enzyme purification?
Crude broken cell preparation
Centrifugation
Crude methods to reduce to a smaller quantity
Ion exchange or affinity chromatography to concentrate
Subsequent column steps for final purification
Assay at each stage to monitor
What are the three column chromatography methods?
Gel filtration
Ion exchange
Affinity methods
Describe ion exchange chromatography:
The gel beads are charged, meaning that oppositely charged molecules filter out first
Describe gel filtration chromatography:
The gel beads are porous, meaning that smaller molecules filter out last
Describe solvent flow chromatography:
The gel beads have attachment sites for some molecules, meaning that the complimentary molecules filter out last