Protein Biochemistry Flashcards

1
Q

What are the primary reasons for assaying enzyme activity?

A

To detect the enzyme
To establish basic properties of enzyme
To determine amount of enzyme

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2
Q

What are the four most important characteristics of enzyme assays?

A

Specificity
Sensitivity
Accuracy/ Reliability
Convenience

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3
Q

Name six environmental conditions to consider when assaying and enzyme:

A
pH
Temperature
Degree of oxidation
Heavy metal contamination
Ionic strength
Protease contamination
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4
Q

What are the alternative methods for a spectrophotometric assay if no natural substrate gives an absorbance change?

A

Convert product to derivative
Use a synthetic substrate
Use a coupled reaction

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5
Q

Give continuous assay alternatives to spectrophotometry:

A

Electrochemical methods

ATP hydrolysis

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6
Q

What is the yield of an enzyme?

A

The ratio of total activity of the purified enzyme to the total activity of the starting material

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7
Q

What is the specific activity of an enzyme measured in?

A

1 unit/mg

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8
Q

What is the purification factor of an enzyme?

A

The ratio of a preparation to the specific activity of the starting material

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9
Q

What are the general stages of enzyme purification?

A

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

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10
Q

What are the three column chromatography methods?

A

Gel filtration
Ion exchange
Affinity methods

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11
Q

Describe ion exchange chromatography:

A

The gel beads are charged, meaning that oppositely charged molecules filter out first

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12
Q

Describe gel filtration chromatography:

A

The gel beads are porous, meaning that smaller molecules filter out last

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13
Q

Describe solvent flow chromatography:

A

The gel beads have attachment sites for some molecules, meaning that the complimentary molecules filter out last

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