Chapter 3: Amino Acids

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Chapter 3 Amino Acids Polypeptides

• Amino acid H2N-CHR-CO2H
• There are 20 different R groups.? thus, 20
  different amino acids

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Zwitterion

• In aqueous solution, the amino and carboxylic
  acid groups will ionize to give the zwitterionic
  formH3N-CHR-CO2-

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Stereochemistry

• Note that the R group means that the ?-carbon is
  a chiral center.
• All natural amino acids are L-amino acids.
• This means that almost all have the S
  configuration. (Exceptions glycine and cysteine
  can you tell why?)

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You should know

• the structures of the side chains of all 20
  natural amino acids
• both the 1-letter and 3-letter codes (see Table
  3-1).
• the pKa's of the 7 ionizable R groups (only to 1
  decimal place)
• Although you don't have to memorize all of the
  pKa's for every carboxylate and amine, you should
  know that they are all 2 and 9-10, respectively
  (with a few exceptions). You should should be
  able to calculate pI if given the pKa's.

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Can group into several categories

• 1) alkanes A V I L (P)
• 2) aromatics F W Y (H)
• 3) carboxylates D E
• 4) corresponding amides N Q
• 5) positively charged K R (H)
• 6) Sulfur-containing C M
• 7) hydroxyls S T Y

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Can group into several categories

• 8) ?-branched V T I
• 9) small G A S C (V T)
• 10) large W R Y F (M)
• 11) H-bond donors S T Y N Q K R H W (D E, if
  protonated)
• 12) H-bond acceptors S T Y N Q D E R H W

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How to calculate pI

• The isoelectric point (pI) of an amino acid or
  peptide is the pH at which the charge of the
  molecule 0.
• It can be calculated simply as the arithmetic
  mean of the 2 pKa's corresponding to the
  transitions generating the 1 and -1 forms.

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How to calculate pI

• Heres how to do it
• Identify all ionizable groups
• Assign pKas to each ionizable group
• Start with each ionizable group in protonated
  form (very low pH maybe 0 or 1) and calculate
  its net charge
• Slowly move up in pH to the first ionizable
  groups pKa and deprotonate it (reduce charge by
  1)
• Do this until each group is deprotonated. Now
  you have identified all charged forms and at
  which pH each transition occurs.
• Identify the form with net charge 0
• Take the pKa on either side of the electrically
  neutral form and take their average. This is the
  pI.

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How to calculate pI

• Take Glycine as an example it has only 2
  ionizable groups. The transition (from low to
  high pH) would beGly1 ? Gly0 ? Gly -1
• pKa (-CO2H) 2.34 pKa (-NH3) 9.60
• pI (2.34 9.60)/2 11.94/2 5.97

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How to calculate pI

• Glutamate has an ionizable group (-CO2H pKa
  4.25) that generates a negative charge when
  deprotonated.Its transitions would be
• Glu1 ? Glu0 ? Glu-1 ? Glu-2
• The relevant pKa 's are
• pKa(-CO2H) 2.19 pKa(R) 4.25
• pI (2.19 4.25)/2 6.44/2 3.22

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How to calculate pI

• Histidine has an ionizable group (imidazole pKa
  6.00) that is positively charged when
  protonated. Its transitions would be
• His2 ? His1 ? His0 ? His-1
• The relevant pKa 's are
• pKa(R) 6.00 pKa(-NH3) 9.17
• pI (6.00 9.17)/2 15.17/2 7.59

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Making dipeptides

• H3N-CHR-CO2- H3N-CHR-CO2- ?
• H3N-CHR-CONH-CHR-CO2- H2O
• This process can be repeated to make a tripeptide
  and so on
• H3N-CHR-CONH-CHR-CO2- H3N-CHR-CO2- ?
  H3N-CHR-CONH-CHR-CONH-CHR-CO2-

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Making dipeptides

• The C-N bond has partial double bond character,
  making the -CONH- moiety planar. This limits the
  orientations available to the polypeptide
• (e.g. the barrier of rotation about the C-N bond
  in formamide is 18 kcal/mol)

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Hydrolysis of polypeptides amino acid analysis

• Polypeptides can be hydrolyzed to constituent
  amino acids.
• This is typically done by boiling the polypeptide
  in 6 M HCl for 24 hours.
• H3N-CHR-CONH-CHR-CONH-CHR-CO2- 2 H2O ? 3
  H3N-CHR-CO2-

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Hydrolysis of polypeptides amino acid analysis

• The R groups remain intact, except for
• Trp indole ring damaged
• Asn, Gln converted to Asp, Glu

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Hydrolysis of polypeptides amino acid analysis

• The amino acids can be derivatized with
  o-phthalaldehyde to make fluorescent derivatives
  that are easy to detect.
• These are chromatographed by reverse-phase HPLC
  (high-pressure liquid chromatography).
• The characteristic retention times are used to
  identify the amino acids.
• The fluorescence level can be quantified to
  determine the amount of that amino acid.

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Amino acid analysis.
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Disulfide bonds

• 2 cysteine ? cystine
• 2 R-SH ? R-S-S-R (Note This is an oxidation)
• Intracellular conditions are maintained
  sufficiently reducing to inhibit formation of
  most disulfide bonds.
• Extracellular conditions (as well as those found
  in some organelles) are more oxidizing, favoring
  disulfide formation.
• Thus, extracellular proteins containing cysteines
  often have disulfides, while intracellular
  (cytosolic) proteins rarely have disulfides.

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Reactions with amino acids

• I. Amino group
• Acylation ? R-(CO)-NH-R
• Ninhydrin reactionCauses oxidative
  decarboxylation of ?-amino acids, and release of
  ammonia, which reacts with a second molecule of
  ninhydrin to form a purple product.(You dont
  need to know details just know that it reacts
  with any free amino group and the final product
  is purple.)

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Reactions with amino acids

• I. Amino group
• Ninhydrin reaction

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Reactions with amino acids

• Fluorodinitrobenzene reaction
• Dansyl chloride reaction

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Reactions with amino acids

• Fluorescamine reactionSimilar to dansyl chloride
  forms fluorescent adduct.(Dont need to know
  details.)

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Reactions with amino acids

• o-phthalaldehyde reaction
• Schiff's base formation
• R-HCO NH2-R' ? R-HCN-R' H2O
• 8. Edman degradation (more later)

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Reactions with amino acids

• Carboxyl group
• Amide formation
• Ester formation
• Acyl halide formation
• Reduction to alcohol (via aldehyde)

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Reactions with amino acids

• Side chains
• R-SH (cysteine) R-S-S-R (cystine)
• Reduction of disulfide with ?-mercaptoethanolR-S-
  S-R' HS-CH2CH2OH ?? R-S-S-CH2CH2OH
  R'-SHR-S-S-CH2CH2OH HS-CH2CH2OH ?
  HOCH2CH2-S-S-CH2CH2OH R-SH(driven by mass
  action)
• Can also accomplish with dithiothreitol (DTT)
  (only requires 1 molecule to reduce 1 disulfide)

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Reactions with amino acids

• Coupling to N-ethylmaleimide(blocks disulfide
  formation after reduction)
• Carboxymethylation with iodoacetate (introduces
  carboxylate) R-SH I-CH2-COOH ?
  R-S-CH2-COOH HI

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Reactions with amino acids

• Reaction with ethyleneimine (introduces amino
  group)
• 5. Performic acid oxidation to cysteic acid
• 3 HCOOOH R-SH ? R-SO3H 3 HCOOH 5 HCOOOH
  R-S-S-R H2O ? 2 R-SO3H 5 CHOOH
• 6. Reaction with mercurials R-SH R'-Hg-Cl ?
  R-S-Hg-R' HCl

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Reactions with amino acids

• B. Imidazole (histidine)
• 1. Acetylation (with IAA)
• C. Phenol (tyrosine)
• Nitration

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Reactions with amino acids

• Acetylation
• iodination (important use isotopic labeling
  with 125I)

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Reactions with amino acids

• D. ?-NH2 (lysine)
• Acylation R-NH-R
• Fluorodinitrobenzene reaction
• Dansyl chloride reaction
• o-phthalaldehyde reaction
• Schiff's base formation
• cyanylationR-NH2 HNCO ? R-NH-CONH2
• amidination

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Reactions with amino acids

• R-COOH (glutamate, aspartate)
• Amidation
• Esterification
• Acyl halide formation
• Reduction to alcohol
• R-CONH2 (glutamine, asparagine)
• DeamidationR-CONH2 H2O ? R-COOH NH3

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Protein Sequencing Strategy

• 1) Purify protein (methods discussed later)
• Cleave disulfides react with
• reducing agent followed by alkylating agent
• DTT or ?-ME
• NEM or IAA
• performic acid

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Protein Sequencing Strategy

• Determine ends
• N-terminus react with FDNB or dansyl chloride
  followed by acid hydrolysis and HPLC(Can also
  perform Edman degradation with intact protein to
  get N-terminal sequence, if the N-terminus is not
  blocked.)
• C-terminus digest with carboxypeptidase(not
  often done)

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Protein Sequencing Strategy

• Cleave polypeptide into smaller peptides
• A) Endopeptidases
• trypsin cleaves after Lys and Arg
• chymotrypsin cleaves after Phe, Tyr, or Trp
• endoproteinase Glu-C cleaves after Glu

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Protein Sequencing Strategy

• Cleave polypeptide into smaller peptides
• B) Chemicals
• CNBr cleaves after Met(Lactone will be
  hydrolyzed during acid hydrolysis.)

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Protein Sequencing Strategy

• Purify peptides
• Characterize peptides(i.e. spectroscopy, amino
  acid analysis, chromatography, etc.)
• Most important determine amino acid sequence
  by Edman degradation

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Protein Sequencing Strategy

• 7) Reassemble sequence through overlaps of
  peptides created by different means
• 8) Map disulfides by cleaving protein into
  peptides before disulfide bond cleavage. After
  purification of disulfide-linked peptides and
  cleavage of their disulfide bonds, sequencing of
  the peptides should reveal which cysteines are
  linked in disulfide bonds.

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Edman degradation

• Q Why can you not use it to sequence long
  polypeptides?
• A Each step is completed with lt100 yield.
  Eventually, less than half of the peptides have
  the "current" amino terminal amino acid residue.

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Example (inefficiency exagerated)

• H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-H2N-V-D-R-G-
  T-H-K-L-S-F-E-W-Q-C-V-N-H2N-V-D-R-G-T-H-K-L-S-F-
  E-W-Q-C-V-N-H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-
  H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-H2N-V-D-R-
  G-T-H-K-L-S-F-E-W-Q-C-V-N-
• After 1st step
• H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-H2N-D-R-G-T-H-
  K-L-S-F-E-W-Q-C-V-N-H2N-D-R-G-T-H-K-L-S-F-E-W-Q-
  C-V-N-H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-H2N-D-
  R-G-T-H-K-L-S-F-E-W-Q-C-V-N-H2N-V-D-R-G-T-H-K-L-
  S-F-E-W-Q-C-V-N-
• After 2nd step
• H2N-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-H2N-R-G-T-H-K-L-
  S-F-E-W-Q-C-V-N-H2N-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-
  H2N-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-H2N-D-R-G-T-H-
  K-L-S-F-E-W-Q-C-V-N-H2N-D-R-G-T-H-K-L-S-F-E-W-Q-
  C-V-N- And so on

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Synthesis of polypeptide

• Artificial (Merrifield process)
• Utilizes amino acid coupled to a solid phase
  support through the carboxylate.
• Amino acids with protected amino groups and
  activated carboxylates react with the amino acid
  to make N-terminally protected dipeptide.
• After removal of the protecting group, another
  protected and activated amino acid can be added
  to the N-terminus.
• The cycle is repeated until the desired
  polypeptide has been synthesized.
• It can then be removed from the solid support by
  treatment with strong acid. (This also removes
  the protecting groups on the amino acid
  sidechains that prevent unwanted reactions.)

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Natural polypeptide synthesis

• Performed on the ribosome, a large aggregate of
  RNA and protein in effect, "solid phase".
• Protecting groups are not necessary, as the
  sequence is determined by genetic programming.
• The C-terminus is activated by coupling to a
  nucleic acid (tRNA).
• This tRNA serves both as the activating group and
  the connection to the ribosome. The polypeptide
  is synthesized starting from the N-terminal amino
  acid.

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Protein Purification

• In an organism, any one protein is present as a
  very small percentage of the total biomolecules.
  
• In order to characterize a protein fully, it is
  necessary to purify it.
• There are many possible strategies that one could
  use to purify a protein.
• In general, the more that you know about the
  protein's properties, the better strategy you
  could design to purify it.
• A typical protein purification strategy will be
  comprised of several stages, each one taking
  advantage of different characteristics of the
  protein.

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Modulating Solubility

• 1) Precipitation at the pI
• A protein's average net charge at its isoelectric
  point is 0. Above or below this pH, the protein
  molecules are negatively or positively charged,
  respectively, causing them to repel each other.

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Modulating Solubility

• 2) Salting in
• Many proteins are poorly soluble in pure water,
  but are much more soluble in salt-containing
  solutions. Thus, lowering ionic strength can be
  used to precipitate certain proteins.

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Solubility of lactoglobin as a function of pH at
several NaCl concentrations.
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Modulating Solubility

• 3) Salting out
• At very high concentrations (gt1 M) of certain
  salts, proteins solubility is reduced due to a
  competition with the protein for interaction with
  water molecules.

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Solubility of hemoglobin at its pI as a function
of ionic strength and ion type
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Ammonium sulfate precipitation

• Precipitation with ammonium sulfate is a common
  first step in protein purification.
• Because proteins precipitate at different
  concentrations of salt, one can perform a first
  "cut" with a concentration that will leave the
  desired protein soluble, remove the precipitate,
  and then add sufficient salt to precipitate the
  desired protein.

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Solubilities of several proteins in ammonium
sulfate solutions.
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Ammonium sulfate precipitation

• Example
• Your protein will remain soluble at 30 (w/v)
  ammonium sulfate, but precipitates at 40
  ammonium sulfate.
• You slowly add the salt to your protein extract
  until it reaches a concentration of 30, allow
  precipitation to occur, and then centrifuge the
  solution to remove the precipitate.
• You take the supernatant, and add ammonium
  sulfate until it reaches a concentration of 40,
  allow precipitation to occur, and then centrifuge
  the solution to remove the precipitate.
• You dissolve the precipitate in a buffer and
  dialyze it to remove the salt.

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Dialysis

• The protein is put in a bag of cellulose
  membranes having small pores of controlled size.
  
• Proteins bigger than the pores are retained,
  while smaller molecules may diffuse out.
• As the volume of the buffer surrounding the bag
  is many times (100-1000x) the volume within the
  bag, the smaller molecules can be effectively
  removed after several changes of the outer buffer.

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Dialysis
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Gel filtration chromatography(a.k.a. "molecular
sieve", "size exclusion")

• The protein is applied to the top of a column
  consisting of porous beads made of a hydrated
  material, such as agarose, dextran, or
  polyacrylamide.
• The pores of the beads are of a controlled size
  and this regulates which proteins can enter the
  beads.
• The larger proteins will be excluded from the
  beads and will flow through the column faster
  than the smaller molecules, which experience a
  much larger volume.
• In practice, there exists a range of pore sizes,
  and proteins are separated by their sizes (and
  shapes) the largest are eluted first, and the
  smallest elute last.

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Gel filtration chromatography
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Gel filtration chromatography

• Vbed Vbeads Vvoid
• The void volume is the volume surrounding the
  beads.
• The bed volume is the total volume of the column.
• Vvoid is typically about Vbed /3
• A protein can be characterized by its elution
  volume (Velution), which is the volume of solvent
  required to elute it from the column. The
  relative elution volume ( Velution / Vvoid) is a
  quantity independent of the particular column
  used.
• By standardizing the column with proteins of
  known size, one can use this technique to
  estimate molecular weight (assuming that the
  shape is close to that of the standards more or
  less spherical).

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Commonly Used Gel Filtration Materials
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Ion exchange chromatography

• This technique uses the charge on a protein to
  separate it from other proteins. In the process
  of ion exchange, ions in solution replace ions
  that are electrostatically bound to an inert
  support carrying groups with the opposite charge.
• Cation exchangers bear negatively charged groups.
• Anion exchangers bear positively charged groups.
• Polyelectrolytes, such as proteins, can bind to
  either cation or anion exchangers, depending on
  their net charge (i.e. depending on the pH).

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Ion exchange chromatography

• In most cases, a column is prepared with the ion
  exchanger, which is then equilibrated with the
  same buffer used to dissolve the protein. The
  exchanger and pH are chosen such that the protein
  will bind relatively tightly to the ion
  exchanger.
• The protein solution is loaded to the top and the
  column is washed with the buffer, removing all
  proteins with the opposite charge or low charge.
• The protein can be eluted from the column either
  by changing the pH or by increasing the salt
  concentration, which shields the charges and thus
  decreases their attraction. Elution is most often
  carried out by applying a salt gradient to the
  column.

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Ion exchange chromatographyusing stepwise
elution.
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Device for generating a linear concentration
gradient.
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Molecular formulas of cellulose-based ion
exchangers.
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Some common ion exchangers.
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Affinity chromatography

• This technique takes advantage of the fact that
  many proteins specifically bind other molecules
  as part of their function. One can use this
  information to construct a column containing the
  ligand covalently attached to a matrix.
• Upon passing the protein solution through such a
  column, only the proteins that can bind the
  ligand will be retained on the column.
• Then the conditions can be adjusted to effect
  release from the ligand. Often this can be done
  simply by eluting with the soluble version of the
  ligand.

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Covalent linking of ligand to agarose
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Derivatization of epoxy-activated agarose.
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Example purification of staphylococcal nuclease
by affinity chromatography on bisphosphothymidine-
linked agarose
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Preparative vs. Analytical

• Preparative methods used to purify proteins
• Able to handle large amounts of protein at once
• The chromatographic techniques just discussed are
  all preparative.
• Analytical methods used to analyze proteins
• Usually deal with small amounts of protein
• Some preparative techniques can also have
  analytical formats.

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Analytical techniques

• Separation based on
• Mass
• Charge
• Shape
• Different combinations of the above

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Density Gradients

• An equilibrium sedimentation experiment can be
  set up with linear gradients of sucrose or
  glycerol.
• The protein is loaded on top, and centrifugation
  is started.
• The advantage of this technique is that the
  gradients tend to be rather stable and allow one
  to remove the protein of interest from them. The
  disadvantage is that they are not so accurate for
  determination of S.
• Often such gradients are run with molecular
  weight standards of known molecular weights to
  allow estimation of molecular weight. The
  sedimentation coefficient of a protein is
  approximately proportional to the 2/3 power of
  its molecular weight. Note that this only holds
  true for proteins with a globular shape (i.e.
  quasi-spherical).Sunknown/Sstandard
  (Munknown/Mstandard)2/3

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Zonal ultracentrifugation
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Zonal ultracentrifugation
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Gel electrophoresis

• General technique to analyze mixtures of
  proteins, and for limited purification of
  proteins.
• The protein is driven through a viscous solvent
  by an applied electric field (E), due to the
  charge of the protein (z) v Ez/f (f the
  protein's frictional coefficient)
• A proteins electrophoretic mobility (µ) is
  defined µ v/E
• Typically this is performed in the presence of a
  gel support, such as polyacrylamide
• prevents convection currents
• enhances separation by serving as a molecular
  sieve

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Polymerization of acrylamide and
N,N-methylenebisacrylamide
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Native gel electrophoresis

• The native protein migrates through the gel
  according to the charge of the protein at the pH
  of the buffer system.
• Useful for getting relatively pure protein, but
  in small amounts.
• Not used very often.

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SDS-polyacrylamide gel electrophoresis(SDS-PAGE)

• Most common form of PAGE, but not useful for
  purification of proteins in their native
  conformation.
• Proteins are solubilized with the detergent SDS
  (sodium dodecyl sulfate)
• binds polypeptide at a ratio of 1 SDS per 2
  amino acid residues
• denatures protein ? converts to roughly rod-like
  shape
• protein has a negative charge roughly
  proportional to its mass
• The additional negative charge is much greater
  than the protein's intrinsic charge, which can
  usually be ignored.

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SDS-polyacrylamide gel electrophoresis(SDS-PAGE)

• As charge/mass ratio is almost constant and the
  molecular shapes are all similar, separation is
  on the basis of size.
• Smaller polypeptides migrate faster and larger
  ones migrate slower, due to the gel filtration
  effect.
• There is an empirical relationship between
  mobility and molecular weight µ ? 1/log Mr
• Average pore size of the gel can be controlled by
  varying the concentration of acrylamide before
  initiating polymerization. Higher percentage
  polyacrylamide gels (smaller pore sizes) will
  result in better resolution of smaller
  polypeptides.

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slab gel electrophoresis
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Isoelectric focusing

• A pH gradient is set up by electrophoresing
  polyampholytes (300-600 Da oligomers bearing
  amino and carboxylate groups in varying rations)
  in a gel tube.
• The more basic ones (cationic) will accumulate
  near the cathode and the more acidic (anionic)
  will accumulate near the anode, thereby
  establishing a continuous pH gradient.
• When a protein is applied to the gel, it will
  migrate toward the anode if it is negatively
  charged or toward the cathode if positively
  charged, until it reaches the region
  corresponding to its pI, where it will stop.
• Proteins are often denatured in 6M urea, which
  does not change the protein's charge (unlike SDS).

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General formula of the ampholytes used in
isoelectric focusing.
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Example

• A protein with a pI of 5.2 is introduced near the
  cathode end, which has pH of 9.5 (in this gel).
• It is then negatively charged and will migrate
  toward the anode.
• As it migrates in this direction, the pH will
  steadily decrease, and the amount of negative
  charge carried by the protein will also decrease,
  as more and more ionizable groups become
  protonated.
• Thus it will slow down, until it hits the region
  where pH 5.2, where it will experience no
  further force. If it diffuses in either
  direction, it will pick up charge and the
  electric field will force it back.
• This results in the protein being concentrated
  (focused) in a very narrow region of the gel.

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2-dimensional gels

• This is a powerful technique that combines
  isoelectric focusing with SDS-PAGE.
• Proteins are separated in the first dimension by
  isoelectric focusing (IEF).
• Then this tube is attached to the side of an
  SDS-polyacrylamide gel. SDS-PAGE provides the
  second dimension.
• Each protein migrates to a semi-unique spot
  according to its pI and molecular weight (MW).

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Two-dimensional (2D) gel electrophoresis
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Protein Purification Practical Aspects

• All cells contain proteases enzymes that
  catalyze hydrolysis of peptide bonds.
• Upon breaking cells, these are released into the
  extract, where they can degrade the protein you
  want to purify.
• In order to inhibit proteolysis and denaturation,
  protein purification is usually carried out in
  the cold (on ice or in a cold room) in the
  presence of protease inhibitors (small molecules
  that inhibit specific proteases).

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Protein Purification Practical Aspects

• You often have to choose your source of material
  carefully
• unicellular organism
• organ of a metazoan (or plant)?
• Where is the enzyme located?
• In cytosol?
• Within membrane-bound organelle?
• Secreted?
• You can use differential centrifugation to do a
  (crude) separation of various cellular
  compartments to get a starting point.
• If the protein is in a specific organelle,
  density gradient centrifugation is usually used
  to purify the organelle first.

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Purification table
Activity definition of 1 unit (U) will vary,
depending upon enzyme Specific activity measure
of enzymes purity activity/protein Yield of a
step Units retained after that step/Units
input(Measure of how well enzyme activity is
preserved by the step.) Purification factor
specific activity after the step/before(Measure
of how effective the step is.)
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Purification table

• Yield and purification factor can be expressed
• for the individual step OR
• as cumulative yield or purification factor,
  taking into account all steps up to to that
  point
• You need to be careful to distinguish between
  them.

141
Example Purification of Rat Liver Glucokinase
142
Modern tricks

• Genetic engineering assists protein purification
• In the past, one often had to purify a protein in
  order to obtain enough sequence information to
  search for the gene.
• Nowadays, one often has a gene encoding a protein
  long before the protein has been purified.
• In either case, "engineering" the gene can ease
  protein purification.

143
Modern tricks

• One can use strong promoters (DNA elements that
  direct RNA polymerase to transcribe the gene) to
  overproduce the protein, either in a homologous
  system, or in a very different system. This
  increases the specific activity of your starting
  material.
• One can attach sequence elements to the gene,
  such that the protein will have additional
  polypeptide segments that can be used as "tags"
  for affinity purification.
• Example The sequence H-H-H-H-H-H (hexahistidine)
  is capable of binding Ni2 ions. Attachment of
  the "His6-tag" to a protein allows it to bind to
  a nickel-nitrilotriacetic acid (NTA-Ni2) column
  and be eluted with imidazole.

144
Example Engineering removable affinity tag

• Clone gene into plasmid to express in bacteria
• Fuse protein to intein and chitin-binding domain
  (CBD)
• Makes chimeric protein
• Pass through chitin column and wash off all other
  proteins.
• DTT allows intein to cleave link between itself
  and protein.
• Protein elutes, leaving intein-CBD on the column.