ESSCOSMOS/2009:Enzymes: Difference between revisions
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===Reagents=== | ===Reagents=== | ||
====Buffer==== | ====Buffer==== | ||
50 mM sodium acetate, pH 5.0 | 50 mM sodium acetate, pH 5.0 | ||
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25 mM L-dihydroxyphenylalanine (L-DOPA) | 25 mM L-dihydroxyphenylalanine (L-DOPA) | ||
WARNING: L-DOPA is hazardous; wear gloves. | '''WARNING''': L-DOPA is hazardous; wear gloves. | ||
====Homogenate==== | ====Homogenate==== | ||
For each mushroom species: | For each mushroom species: | ||
*Combine 2 g sample and 125 mL [[ESSCOSMOS/2009:Enzymes#Buffer|buffer]] in a blender and blend for one minute; pour homogenate into a flask labeled with the mushroom species name. | *Combine 2 g sample and 125 mL [[ESSCOSMOS/2009:Enzymes#Buffer|buffer]] in a blender and blend for one minute; pour homogenate into a flask labeled with the mushroom species name. | ||
====Hydrogen Peroxide==== | ====Hydrogen Peroxide==== | ||
0.3% H<sub>2</sub>O<sub>2</sub> | 0.3% H<sub>2</sub>O<sub>2</sub> | ||
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#For assay cuvettes (3,4), record the at which homogenate and L-DOPA are combined. | #For assay cuvettes (3,4), record the at which homogenate and L-DOPA are combined. | ||
#Cover cuvettes and invert to mix every fifteen minutes | #Cover cuvettes and invert to mix every fifteen minutes | ||
While you are waiting, research the different species of mushroom, and predict which mushroom species will have the highest enzyme activities based on what you find out about their habitat and physiology. | |||
#After 1-2 hours, samples will be ready to read | #After 1-2 hours, samples will be ready to read | ||
#Record absorbance at 450 nm using spectrometer | #Record absorbance at 450 nm using spectrometer |
Revision as of 23:49, 20 July 2009
Based on Steve Allisons Colormetric Enzyme Assay Protocol
Overview
Objectives
Background
Materials
- Cuvettes: three per sample
- Spectrophotometer
- Pipettes
- Mushroom substrate from three mushrooms: Pleurotus eryngii, Hypsizygus marmoreus, and Grifola frondosa that was collected on 7/18/2009 from Golden Gourmet Mushrooms / Hokto Kinoko Company in San Marcos, CA.
Reagents
Buffer
50 mM sodium acetate, pH 5.0
Enzyme Substrate
25 mM L-dihydroxyphenylalanine (L-DOPA)
WARNING: L-DOPA is hazardous; wear gloves.
Homogenate
For each mushroom species:
- Combine 2 g sample and 125 mL buffer in a blender and blend for one minute; pour homogenate into a flask labeled with the mushroom species name.
Hydrogen Peroxide
0.3% H2O2
Methods
- For each species, make four cuvettes:
- homogenate control for homogenate absorbance
- substrate control for L-DOPA absorbance
- assay for polyphenol oxidase
- assay for peroxidase
- For assay cuvettes (3,4), record the at which homogenate and L-DOPA are combined.
- Cover cuvettes and invert to mix every fifteen minutes
While you are waiting, research the different species of mushroom, and predict which mushroom species will have the highest enzyme activities based on what you find out about their habitat and physiology.
- After 1-2 hours, samples will be ready to read
- Record absorbance at 450 nm using spectrometer
Homogenate control
Label first cuvette "Ho", add:
- 0.5 mL homogenate
- 1.5 mL buffer
Enzyme substrate control
Label second cuvette "Su" (enzyme substrate), add:
Polyphenol Oxidase Assay
Label third cuvette "PPO", add:
- 0.5 mL homogenate
- 1.5 mL L-DOPA
Peroxidase Assay
Label third cuvette "Per", add:
- 0.5 mL homogenate
- 1.5 mL L-DOPA
- 0.1 mL Hydrogen Peroxide, 0.3%
Results
Compute activity as μmol L-DOPA converted per hour per g sample as follows:
Activity (μmol h-1 g-1) =
OD/[(EC/μmol/ml)/(2.0 ml/assay)(incubation, hr)(g Sample/ ml sample homogenate)(0.5mL homogenate/assay)]
where:
- OD=Sample Absorbance - (Substrate Absorbance + Homogenate Absorbance)
- Extinction Coefficient, EC = 0.403
Peroxidase activity is the difference in activity between the PPO and peroxidase assay.
Analysis
- Which species has the highest PPO activity?
- Which species has the highest Peroxidase activity?