Molecular Physiology: Plant Peroxidase

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Molecular Physiology Plant Peroxidase
Peroxidase Family of enzymes
What is an enzyme?
What is meant by term family when referring to
enzymes?
How are all plant peroxidases related?
ALL use H2O2
ALL include heme prosthetic group in addition to
polypeptide (protein) component
ALL defined by the following reaction
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Classes of peroxidases
Class I Sequence similar to those found in
bacteria In plants this class is comprised of
the ascorbate peroxidases
Class II Sequences similar to those found in
fungi Currently no plant peroxidase fall into
this class
Class III Secretory plant peroxidases Largest
class of plant peroxidases In Arabidopsis
thaliana 73 genes known
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Why do plants need so many peroxidases?
We do not know the complete answer to this
question!!!!!
Some postulated roles of plant peroxidases
Wound response
Pathogen response
Cell wall stiffening lignin synthesis
Cell wall loosening
Reactive oxygen scavenger
Stress response
Breakdown of auxin
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Many peroxidases are expressed in a tissue
specific manner Brassica rapa example
Many studies Correlation between peroxidase
activity and location and/or treatment
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Analyzing Peroxidase Activity
Total soluble peroxidase activity solution
studies
Activity of specific peroxidases
electrophoretic assay
How can specific peroxidases be detected using
electrophoresis?
Different peroxidases have different amino acid
sequences
Some amino acid residues have charges
Basic amino acids, lysine and arginine,
positively charged (gained proton)
Acidic amino acids, glutamic and aspartic acid,
negatively charged (lost proton)
Histidine is a tweener
Different peroxidases can differ by the number of
acidic and basic amino acids they contain
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Going from plant to peroxidase analysis
Determine plant, tissue type, condition for
analysis
Remove appropriate tissue(s) from plant
Extract soluble peroxidases using a buffer
solution and mechanical disruption
Centrifuge the sample to separate the insoluble
portion (pellet) from the soluble (supernatant)
Remove a portion of the supernatant and mix with
gel loading buffer
Load an agarose gel (buffered at pH 8.5) with
sample wells in center with the appropriate
samples and markers
Place gel in buffer and expose to electrical
current to separate the proteins by charge
Stain gel with a solution containing H2O2 and
chloronaphthol peroxidase specific stain
Make a standard curve using distance migrated and
isoelectric points of standards, determine
isoelectric points and amount of specific
peroxidases in each sample lane
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