What are enzymes again

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What are enzymes again?
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What are enzymes again?

• Enzymes are
• proteins ? long chains of amino acids
• biological catalysts ? speed up reactions
• Enzyme Activity is
• specific for particular substrates reactions
• pH-dependent
• temperature-dependent
• salt-dependent

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Peroxidases
Enzymes that catalyze the oxidation of phenolic
substrates at the expense of hydrogen peroxide
Peroxidase activity can be visualized by using
the artificial substrate chloronaphthol
clear, soluble
blue, insoluble
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Peroxidase Functions

• cell wall synthesis
• cross-linking of phenolic residues of cell wall
  polysaccharides and glycoproteins
• synthesis of lignin
• protection from microorganisms and toxins
• detoxification of phenols
• protection from stress
• H2O2 is a side-product of electron transport
  gone awry ? any stress destabilizing membrane
  electron transport results in H2O2 release

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Isoenzymes

• Enzymes that catalyze the same chemical reaction
  but are not the same protein.
• can be encoded by different genes
• ? have different amino acid sequence
• OR
• can be encoded by the same gene, but be
  modified in different ways after translation
• ? have same amino acid sequence but different
  sugars or phosphates attached

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Plants make up to 200 or more different
peroxidase isoenzymes, the exact number depends
on the plant species.
Why do they need so many?

• different functions
• different compartments
• adapted to different stress conditions (temp,
  salt, pH,......)

How can these different peroxidases be told
apart from each other?
Most isoenzymes have different isoelectric points.
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Isoelectric Point
The pH at which a protein does not migrate in an
electric field.

• at this pH, the positive and negative charges
  of the amino acid side chains balance each other.

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How is the charge of the side groups dependent on
the pH again?
acid solution
basic solution
Carboxy groups
- COOH
- COO -
Amino groups
- NH3
- NH2
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How come different peroxidase isoenzymes have
different isoelectric points?
If different peroxidase isoenzymes have different
amino acid sequences, then their numbers of
positively and negatively charged amino acid side
chains differ.
If different peroxidase isoenzymes have different
phosphates or charged sugars attached, then their
net charge differs also.
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How can peroxidase isoenzymes with different
isoelectric points be separated from each other?

• By gel electrophoresis
• separates proteins according to net charge by
  allowing them to migrate in an electric field.
• Peroxidase isoenzymes with a net positive
  charge (more amino than carboxy groups in side
  chains) will migrate towards the negative
  electrode.

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Gel Electrophoresis Setup
-

gel made from agar buffer slows proteins
down and keeps them where they are once the
current is turned off
wells indentations in the gel allow loading
of protein extracts
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Gel Electrophoresis in Action Loading
-

gel pH 8
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Gel Electrophoresis in Action Running
-

gel pH 8
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How can peroxidase isoenzymes be made visible
after the gel has stopped running?
Soak gel in the artificial substrate
chloronaphthol
peroxidase
chloro-naphthol
chloro-naphtholox
H2O2
H2O
O2



clear, soluble
blue, insoluble
? get blue bands wherever a peroxidase is present.
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Effects of Wounding on peroxidase expression in
tobacco plants Heather Tardio Brandon
Grove Erika Paustenbach Kathleen Sperdute
horse radish peroxidases
anionic - strong negative net charge
anionic - slightly negative net charge
cationic
Sample loaded in lane 1 - horse radish peroxidase
standards 2 - cytochrome c standard 3 - serum
albumin / hemoglobin standard 4 - bottom leaf of
wounded plant 5 - leaf below wound 6 - leaf of
control plant (not wounded) 7 - leaf above
wound 8 - top leaf of wounded plant
serum albumin
hemoglobin
cytochrome c
bromephenol blue -disregard this for your
analysis- its the loading dye you added to help
you load and track your sample
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use the formula for the trendline to calculate
the isoelectric points of the peroxidase
isoenzymes in your sample extracts
protein
isoelectric
point
cytochrome c
10.2
hemoglobin
7.2
serum albumin
4.8
horse radish
9.0
peroxidase 1
horse radish
7.1
peroxidase 2
horse radish
6.4
peroxidase 3
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What kinds of experiments can be done using this
system?

• Done by students in the past
• wounding
• light
• touch (wind)
• hormones
• nutrients, salts, osmotic stress
• heat or cold stress
• smoke, toxins, heavy metals, human saliva
• For inspiration
• Browse titles of primary research papers, read
  bits of review papers. Remember to bring at
  least one paper to class by Wednesday!

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What kinds of tissues are available for this
experiment?

• Two sources for plant tissues
• grow plants from seed
• sunflower (grows fast and works great!)
• Fast Plants (have mutants available)
• corn, wheat (fainter bands than sunflower but
  work fine)
• other suggestions?
• use plants that happen to be around
• (greenhouse, outside, office plants)

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Review of Seedling Development Monocots
true leaves
coleoptile
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Review of Seedling Development Dicots
true leaves
cotyledons
hypocotyl hook
hypocotyl
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