Protein Purification and Enzymes March 11 2003

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Protein Purification and EnzymesMarch 11 2003
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Strategy of Purification

• Fractionation procedures or steps to isolate
  protein based on physical characteristics.
• Characteristic
  Procedure
• Charge 1. Ion exchange
• 2. Electrophoresis
• 3. Isoelectric focusing
• Polarity 1. Adsorption
  chromatography
• 2. Paper chromatography
• 3. Reverse phase chromatography
• 4. Hydrophobic interaction

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• Characteristic
  Procedure
• Size 1. Dialysis and ultrafiltration
• 2. Gel electrophoresis
• 3. Gel filtration
• 4. Ultracentrifugation
• Specificity 1. Affinity chromatography
• 2. Immunopurification
• Solubility 1. Salt precipitation
• 2. Detergent solubilization

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Salting out
Use (NH4)2 SO4 it is a Very Soluble salt that
does not harm proteins. Refer to the Hofmeister
series
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Chromatography

• Materials can be visualized by
• Radioactivity
• Fluorescence
• UV absorbency
• Stained with one of several dyes
• Ninhydrin
• Iodine
• Sulfuric acid

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Two dimensional separation of Amino acids
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Chromatography

• Analytical methods used to separate molecules.
  Involves a mobile and a stationary phase.
• Mobile phase is what the material to be separated
  is dissolved in.
• Stationary phase is a porous solid matrix which
  the mobile phase surrounds.
• Separation occurs because of the differing
  chemistries each molecule has with both the
  mobile and stationary phase.
• Chemistries are different depending on the
  specific method.

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Types of chromatography

• Gas - liquid Mobile phase is gaseous, stationary
  phase is liquid usually bound to a solid
  matrix.
• Liquid - Liquid Mobile phase is gaseous,
  stationary phase is liquid usually bound to a
  solid matrix.
• If separation is based on ionic interaction the
  method is called Ion Exchange chromatography.
• If separation is based on solubility differences
  between the phases the method is called
  adsorption chromatography.
• If the separation is base on size of molecule the
  method is called gel filtration or size
  exclusion.
• If the separation is base on ligand affinity the
  method is called Affinity chromatography.

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Ion Exchange Chromatography

• A solid matrix with a positive charge i.e. R can
  bind different anions with different affinities.
• We can swap one counter ion for another
• (RA-) B- ? (RB-) A-
• R Resin and exchanges Anions (-)
• This is an anion exchange resin.
• There are also cation exchange resins. The type
  of an R group can determine the strength of
  interaction between the matrix, R and the counter
  ion.
• If R is R-
• (R-A) B ? (R-B) A-

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Proteins have a net charge.
The charge is positive below pI, while the charge
is negative above pI The choice of exchange
resin depends on the charge of the protein and
the pH at which you want to do the
purification. Once the protein binds, all unbound
proteins are washed off the column. Bound
proteins are eluted by increasing the ionic
strength, changing the counter ion or changing
the pH altering the charge on the protein or the
column.
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Gel filtration can be used to determine the
molecular mass of proteins
Ve elution volume Vo exclusion volume Common
matrix dextran, agarose, or polyacrylamide also
desalts proteins
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Affinity Chromatography
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Affinity Chromatography
Based on molecular complementary between an
enzyme and substrate. The substrate (R) is linked
to a matrix with a spacer arm
Only protein that binds R will stick to column.
put citrate on column citrate dehydrogenase will
specifically bind. Add excess citrate and the
enzyme will be released.
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Protein Assays

• An assay is a method of detection
• Specific
• Sensitive
• Convenient to use

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Activity Measurements
In order to follow the purity of an enzyme, you
need a method to measure its activity. Spectrapho
tometric analysis- is one common method to
measure activity. Substrate S
Product P a change of S with time if S is
colored absorbs light we can use Beers Law. A
eb c c - concentration e - millimolar
extinction coefficient A - absorbance b - path
length T - percent transmittance
A - log T
if ? A then ? c at ? max
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• Enzyme-linked Immunosorbent Assay
• Usable in a complex mixture
• High sensitivity

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Electrophoresis
The migration of ions in an electric field Fe
qE where q is the charge E is the electric
Field strength
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Electrophoresis
a) Fe qE Opposing this is is the frictional
force b) Ff µv where v velocity of
migration µ is the coefficient of
friction. Therefore substituting equation a) into
b) qE µv
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Electrophoresis
qE µv Therefore when Fe Ff vqE/µ
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Separates on charge and size
pH matters as well as the pI of the protein. Can
be run at several pH values depending on
proteins. DNA can also be separated on agarose
gels. Genomic sized DNA can also be separated but
requires more sophisticated equipment.
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Paper electrophoresis
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Acrylamide gel electrophoresis
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Disc gel using a glass tube
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Proteins can be visualized by several methods
Stained with a Dye Coomassie blue Fluorescamin
e stain for fluorescence Silver staining
very sensitive proteins can be labeled with
radioactivity and visualized by exposure
to X- ray film
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sodium dodecyl sulfate (SDS)-PAGE
Add SDS, a 12 carbon detergent to denature and
give a negative charge to the protein. The
proteins are separated by molecular mass
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Zonal Ultracentrifugation
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Dialysis is a process that separates molecules
according to size through the use of
semipermeable membranes containing pores of less
than macromolecular dimensions