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Unformatted text preview: BIOC*2580
Lecture
5.
Analysis
of
amino
acids
 
 1 Synopsis:
 Amino
 acid
 analysis
 usually
 involves
 two
 distinct
 processes,
 first
 separation
 of
 the
 individual
amino
acids
from
each
other
and
from
other
contaminants,
and
then
detection
of
the
 separated
components.
 Separation
 is
 based
 on
 the
 different
 properties
 of
 the
 side
 chains,
 such
 as
 polarity
 or
 charge.
 Separation
is
generally
achieved
by
some
form
of
chromatography.
 Detection
 is
 based
 on
 chemical
 reactions
 that
 generate
 coloured
 or
 fluorescent
 amino
 acid
 derivatives
that
can
be
seen
and
measured.
 Reading:
Lehninger,
p.
85‐92
(4th
ed
p.89‐95)
 
 Amino
acid
analysis
 Amino
acid
analysis
is
a
necessary
aspect
of
experiments
to
determine
protein
structure

 
 Analysis
involves
two
processes:
 
 1. The
 mixture
 must
 be
 separated
 into
 individual
 components
 2. The
components
of
interest
must
be
detected
 • Detection
can
be
qualitative
and
determine
what
 is
present

 • Detection
can
be
 quantitative
and
measure
 how
 much
is
present.
 
 Chromatography
 is
 an
 important
 method
 for
 separating
 components
 of
 a
 mixture

 Particles
of
solid
are
chosen
with
a
given
property.
 
 For
 example,
 silica
 gel
 contains
 HO‐Si‐OH
 groups
 that
 are
 effective
 in
 forming
 hydrogen
 bonds
 with
 polar
 amino
 acids.

This
makes
up
the
stationary
phase
 
 Liquid
 solvent
 or
 buffer
 flows
 past
 the
 particles
 and
 is
 nonpolar.

This
makes
up
the
mobile
phase.
 
 Amino
acids
rapidly
exchange
between
phases.
 
 Polar
 amino
 acids
 (P)
 spend
 more
 of
 their
 time
 hydrogen
 bonded
 to
 the
 stationary
 silica
 ‐
 they
 move
 more
 slowly
 Nonpolar
 amino
 acids
 (N)
 spend
 more
 time
 in
 the
 moving
 solvent,
and
move
almost
as
fast
as
solvent.
 Page
1
of
7
 BIOC*2580
Lecture
5.
Analysis
of
amino
acids
 
 2 Thin
layer
chromatography
 
 The
silica
gel
is
spread
as
a
thin
layer
on
a
 plastic
or
glass
sheet.
 Samples
 are
 applied
 to
 the
 silica
 gel
 in
 a
 small
drop
of
solvent.

Each
sample
forms
a
 spot,
 and
 different
 sample
 spots
 are
 arranged
in
a
row
near
the
bottom
edge
of
 the
sheet.
 The
 lower
 edge
 of
 the
 sheet
 is
 dipped
 in
 solvent.

As
solvent
soaks
up
the
sheet,
the
 sample
 spots
 shift
 as
 the
 solvent
 moves
 past.
 
 Different
 substances
 move
 at
 different
 rates,
 so
 the
 components
 of
 an
 initial
 mixture
 are
 separated.

Pure
samples
of
substances
suspected
to
be
in
the
mixture
are
also
applied.
Spots
in
 the
mixture
can
be
identified
if
they
move
the
same
distance
as
one
of
the
pure
samples.
 Polarity
is
the
basis
for
separation
of
substances
by
thin
layer
chromatography.
 The
 rate
 at
 which
 a
 given
 sample,
 e.g.
 an
 amino
 acid,
 moves
 up
 the
 sheet
 depends
 on
 its
 relative
 preference
 for
 stationary
 phase
 
 (silica
 gel)
 or
 mobile
 phase
 (nonpolar
 solvent).
 
 A
 very
 polar
 amino
 acid
 such
 as
 aspartate
will
spend
most
of
its
time
stuck
to
the
silica
gel
 and
will
barely
move.

A
very
non‐polar
amino
acid
such
 as
leucine
will
spend
most
of
its
time
in
the
solvent,
and
 will
 move
 up
 the
 sheet
 almost
 as
 fast
 as
 the
 solvent.

 Amino
 acids
 with
 intermediate
 polarity
 will
 be
 in
 equilibrium
between
the
two
phases,
and
will
 move
part
 way
up
the
sheet.
 Relative
mobility,
RF
 The
highest
point
the
solvent
reaches
is
called
the
solvent
 front.
 
 The
 ratio
 of
 distance
 moved
 by
 a
 sample
 and
 by
 the
solvent
front
is
called
relative
mobility,
RF
 Very
 polar
 solutes
 will
 have
 RF
 close
 to
 zero;
 Very
 non
 polar
 solutes
 will
 have
 RF
 close
 to
 1.0.

 Most
substances
will
be
spread
out
in
between
these
two
extremes.
 The
value
of
RF
will
depend
on
the
solvent
used
and
solvent
must
be
carefully
chosen
for
a
given
 mixture
of
compounds.
 Page
2
of
7
 BIOC*2580
Lecture
5.
Analysis
of
amino
acids
 
 3 Other
formats
for
chromatography
 Column
chromatography
–
Protein
Biochemistry
Work‐horse
 A
granular
solid
such
as
silica
gel
is
packed
 into
a
glass
tube
or
 column;
silica
is
usually
held
in
 place
by
a
porous
disk
at
the
bottom.
A
sample
mixture
is
applied
at
the
top,
and
then
solvent
or
 buffer
solution
is
allowed
to
flow
through.
Sample
solutes
travel
with
the
flow
of
buffer
solution
 to
the
bottom
of
the
column.
Substances
that
bind
more
strongly
to
the
solid
phase
require
more
 buffer
to
pass
through
or
be
 eluted
from
the
column.
The
main
advantage
is
that
the
separated
 components
of
the
mixture
can
be
collected
allowing
additional
experiments
to
be
performed
 on
the
individual
components.


 Collection
 tubes
 are
 changed
 after
 a
 fixed
 volume
 of
 buffer
 or
 solvent
 has
 passed
 through,
 whether
 sample
 has
 come
 through
 or
 not.
 
 When
 enough
 tubes
 have
 been
 collected,
 their
 contents
 are
 detected,
 and
 the
 quantity
 of
 sample
 in
 each
 tube
 is
 graphed
 as
 a
 function
 of
 volume
 buffer
 that
 has
 passed
 since
 the
 start.
 
 The
 volume
 of
 buffer
 needed
 to
 move
 a
 given
 sample
through
the
column
is
called
its
elution
volume.
 
 High
performance
liquid
chromatography
(HPLC)
 Column
chromatography
using
specially
designed
columns
and
with
solvent
pumped
through
for
 greater
efficiency.
This
is
the
usual
method
in
research
labs.
 
 
 Page
3
of
7
 BIOC*2580
Lecture
5.
Analysis
of
amino
acids
 
 4 Detection
of
separated
amino
acids
 All
20
amino
acids
are
colorless
substances,
and
quantities
in
an
analysis
may
be
anything
from
 10‐6
to
10‐10
moles,
not
enough
to
see,
let
alone
weigh
out.
 The
 separated
 amino
 acids
 must
 be
 detected
 by
 special
 means,
 which
 involve
 reaction
 with
 a
 color
or
fluorescence‐generating
reagent
 Ninhydrin:
reacts
with
‐amino
N
to
give
purple
color
(10‐8
mol
detectable) Fluorescamine:
 reacts
 with
 ‐amino
 N
 to
 give
 yellow
 fluorescence
 (10‐10
 mol
 detectable).
 When
 illuminated
with
UV
lamp,
sample
emits
a
yellow
glow.
 Since
 fingerprint
 sweat
 contains
 significant
 traces
 of
 amino
 acids,
 ninhydrin
 and
 fluorescamine
 are
both
used
by
police
investigators
to
detect
otherwise
invisible
fingerprints.
 The
reagents
are
either
sprayed
onto
the
chromatography
paper,
or
added
to
the
solvent
as
it
 emerges
 from
 the
 column.
 The
 intensity
 of
 color
 or
 fluorescence
 is
 recorded
 and
 plotted
 as
 a
 graph.
Color
intensity
is
proportional
to
the
number
of
moles
of
each
amino
acid.
 An
alternative
method
often
used
in
conjunction
with
HPLC
is
to
prelabel
the
sample
compound
 with
 a
 coloured
 or
 fluorescent
 dye
 before
 separation,
 and
 record
 the
 color
 intensity
 as
 each
 amino
acid
emerges
from
the
column.
This
method
is
often
preferred
for
quantitative
analysis,
 since
the
dye
reaction
can
be
allowed
to
go
to
completion
ahead
of
time.
 Dyes
 used
 for
 prelabelling
 include
 fluorodinitrobenzene,
 dansyl
 chloride,
 dabsyl
 chloride,
 phenylisothiocyanate,
Lehninger
Fig.
3‐25
p.94
(4th
ed
p.
98)
 
 Ninhydrin
and
fluorescamine
can't
be
used
to
label
amino
acids
before
separation
since
the
color‐ forming
reaction
destroys
the
amino
acid.
 Different
mechanisms
of
chromatography
 Reversed
phase
chromatography:
 Instead
 of
 polar
 silica
 gel,
 a
 non‐polar
 hydrocarbon
 silicon
 derivative
 is
 used
 as
 the
 solid
 stationary
phase;
instead
of
 non‐polar
solvent,
 polar
solvent
is
used
as
mobile
phase.
The
order
 of
 passage
 is
 reversed,
 since
 now
 polar
 solutes
 don't
 bind
 and
 have
 high
 RF,
 while
 non‐polar
 solutes
do
bind
and
have
low
RF.
(Used
because
it's
better
at
distinguishing
subtle
differences
in
 hydrocarbon
side
chains
of
amino
acids.)
 
 Page
4
of
7
 BIOC*2580
Lecture
5.
Analysis
of
amino
acids
 
 5 Ion
exchange
chromatography
 The
silica
gel
or
cellulose
stationary
phase
is
replaced
by
ionic
resins:
 Cation
exchange
resins
are
based
on
polymers
with
 negative
carboxylate
groups,
and
will
bind
 positive
ions
or
cations.
 
 Anion
 exchange
 resins
 are
 made
 from
 polymers
 with
 positive
 amino
 groups,
 and
 will
 bind
 negative
ions
or
anions.
 
 Solutes
 will
 now
 bind
 according
 to
 their
 charge
rather
than
polarity,
e.g.
 positive
 amino
 acids
 NH3+‐CHR‐CO2H
 bind
to
negative
charged
resin.
 
 All
amino
acids
can
be
made
positive
 to
 some
 degree
 by
 lowering
 the
 pH,
 e.g
to
pH
3.5.

The
exact
charge
on
a
 given
 amino
 acid
 will
 depend
 on
 its
 exact
pKa
value,
and
there
is
enough
 difference
that
all
20
amino
acids
are
 easily
distinguished.
 
 The
amino
acids
can
be
eluted
either
 by
adding
NaCl
to
the
buffer,
so
that
 Na+
binds
in
exchange
for
the
+ve
amino
acid
NH3+‐CHR‐CO2H.
More
weakly
bound
amino
acids
 are
 displaced
 by
 a
 low
 NaCl
 concentration,
 while
 tightly
 bound
 amino
 acids
 require
 a
 higher
 concentration
of
NaCl
to
be
eluted.
 
 Alternatively,
 pH
 may
 be
 increased.
 As
 pH
 rises,
 amino
 acids
 become
 deprotonated
 giving
 a
 net
 +‐CHR‐CO ‐,
 and
 neutral
 charge,
 NH3 their
 2 binding
 to
 the
 negative
 resin
 is
 weakened.
 
 Amino
 acids
 are
 detected
 and
 concentration
 measured
 as
 they
 come
out
of
the
column.
 
 The
volume
of
buffer
needed
to
move
a
given
amino
acid
from
top
to
bottom
of
the
column
is
 also
 measured:
 this
 is
 the
 elution
 volume
 for
 that
 amino
 acid.
 Elution
 volumes
 may
 be
 normalized
by
comparing
with
elution
volumes
of
a
common
standard
such
as
Ala
or
Leu.


 Elution
 volumes
 are
 characteristic
 for
 a
 given
 amino
 acid
 and
 this
 allows
 amino
 acids
 to
 be
 identified.
 Page
5
of
7
 BIOC*2580
Lecture
5.
Analysis
of
amino
acids
 
 6 Separation
of
proteins
from
complex
mixtures
 Protein
 samples
 can
 be
 extremely
 complex
 since
 proteins
 are
 generally
 extracted
 from
 cellular
 sources.
Bacteria
such
as
 Escherichia
coli
or
yeasts
such
as
 Saccharomyces
cerevisiae
are
often
 grown
 as
 sources
 of
 proteins
 and
 enzymes.
 Other
 sources
 include
 extracts
 of
 tissues
 such
 as
 animal
 liver.
 These
 extracts
 may
 contain
 about
 1000
 to
 3000
 different
 proteins,
 and
 three
 problems
must
be
addressed.
 A
typical
single
protein
may
represent
only
0.03
to
0.1%
by
mass
of
the
protein
mixture;
this
 can
be
increased
by
inducing
over‐expression
of
a
gene
inserted
into
yeast
or
bacteria.
 There
may
be
several
other
proteins
present
in
the
extract
with
similar
properties
to
the
one
 you
are
trying
to
isolate.
 Proteins
 are
 easily
 damaged
 under
 to
 harsh
 conditions
 such
 as
 extreme
 pH,
 non‐aqueous
 solvents
 and
 temperature,
 and
 many
 techniques
 must
 be
 carried
 out
 at
 0o‐4o
 and
 near
 neutral
pH
to
minimize
loss
of
sample.
 Complete
 protein
 purification
 involves
 successive
 application
 of
 several
 chromatographic
 or
 other
separation
techniques.
Since
there
may
be
other
proteins
with
similar
charge,
separations
 based
on
other
properties
such
as
size
are
also
applied.
 
 Proteins
are
readily
separated
by
ion
exchange
chromatography
‐
separation
based
on
 charge
 of
protein
 
 Anion
exchangers
are
 positive
charged
polymers
that
bind
and
retain
 negative
charged
 solutes
 (anions)
including
proteins.


 Cation
exchangers
are
 negative
charged
polymers
that
 bind
 positive
charged
solutes
(cations)
 including
proteins.
 Since
a
protein
is
a
long
chain
of
many
different
amino
acids,
each
protein
species
has
a
unique
 electric
charge
which
is
the
algebraic
sum
of
the
number
of
negative
and
positive
amino
acid
side
 chains;
 for
 histidine
 the
 contribution
 may
 be
 a
 fractional
 positive
 charge
 since
 histidine
 is
 only
 partly
protonated
(approximately
+0.24)
at
neutral
pH.
The
N‐terminal
(pKa
=
8)
and
C‐terminal
 (pKa
=
3)
may
also
contribute
charge.
 
 e.g.
 At

 pH
7:
 +1






‐1







0







0


+0.24




0







0






0






0






‐1





+1





0






‐1
 Ala
–
Asp
–
Leu
–
Gly
–
His
–
Gln
–
Tyr
–
Cys
–
Ile
–
Glu
–
Lys
–
Ser
–
Thr

 Due
to

 N‐terminal
 Due
to

 C‐terminal
 
 Net
charge
=

+
1
–
1
+
0.24
–
1
+
1
–
1
=

–0.76

 
 By
changing
the
pH
used
in
an
ion
exchange
column
it's
also
possible
to
net
change
the
charge
on
 a
given
peptide
chain.
However
many
proteins
may
not
tolerate
much
pH
change.
 Page
6
of
7
 BIOC*2580
Lecture
5.
Analysis
of
amino
acids
 
 7 
 Proteins
 with
 the
 appropriate
 charge
 will
 bind
 to
 ion
 exchanger.
 They
 are released
 from
 the
 resin
 by
gradually
increasing
the
concentration
of
neutral
salt
such
as
NaCl
or
KCl,
a
technique
 known
as
gradient
elution.

 The
upper
part
of
the
graph
on
the
right
shows
a
gradually
increasing
NaCl
concentration
in
the
 buffer.
 The
 lower
 part
 shows
 the
 protein
 concentration
 measured
 in
 the
 buffer
 as
 it
 comes
out
of
the
column.
 Proteins
 that
 lack
 charge
 or
 have
 the
 same
 charge
 as
 the
 resin
 will
 not
 bind
 and
 are
 eluted
quickly.
 Proteins
 that
 have
 the
 opposite
 charge
 to
 the
 resin
 bind
 until
 the
 NaCl
 concentration
 has
 risen
 enough
 to
 release
 them.
 Proteins
 that
 have
 a
 higher
 charge
 will
 bind
 more
 tightly,
 and
 a
 higher
 NaCl
 concentration
 is
 needed
 to
 release
 or
 elute
 them.
 Resin
 and
 pH
 are
 chosen
 so
 that
 the
 desired
 protein
 binds
moderately
tightly.
 
 Page
7
of
7
 ...
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This note was uploaded on 09/21/2011 for the course BIOOC 2580 taught by Professor Douger during the Fall '10 term at University of Guelph.

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