Determining the Amino Acid Composition of a Protein

Determining the Amino Acid Composition of a Protein

In this web cast and
the three that follow we’re going to take a look at the chemical
analysis and synthesis of polypeptide chains. Today, we take for
granted the idea that a naturally occurring
polypeptide like insulin has a unique
chemical composition and a specific arrangement
of amino acids along the chain. That’s its primary sequence. But there was a day when
people believed that polypeptides may be
complex mixtures of closely related substances that might have similar
but not exactly identical amino acid compositions
and sequence. It was Fred Sanger who in
1952 published the paper which really put
those ideas to rest, the ideas that polypeptides
were mixtures. He showed that the
molecule insulin had a very specific arrangement of amino acid sequences and its sequences are shown
along the chain there. The ideas that ah, Fred
Sanger put into practice are essentially the same
ideas that are used today to determine amino
acid composition and although the methods have
gotten more sophisticated basically they involve
the three steps that sho- are shown here. So starting with a
purified protein, the first step is
to hydrolyze those, hydrolyze that protein
into- i- individual constituent amino acids. The amino acids are tagged and then in some
chromatographic way those ah tagged amino
acids are separated and quantified. The hydrolysis step
is basically just breaking down the polypeptides. So here it’s represented as a repeating chain with
undefined ends. The hydrolysis step
breaks the amide linkage and produces
individual amino acids as a mixture of
amino acids. At this point the tagging
step- step takes place and for the most part today, it- it involves a reaction
that I’ll show you on the next slide that involves the
formation of an isoindole. So with the amino
group nitrogen there you can see that the
amino acid is stuck on this molecule of isoindole which is derived from
an- an ortho dialdehyde. The molecule, isoindole,
is fluorescent and so now we can use
fluorescence intensity in order to quantify how much
of the different amino acids are there if we
can separate them. I’ll have more to say
about fluorescence in a subsequent web cast but
basically this last step, the step of separation
and quantification, is involving a chromatographic
-type separation. So this mixture of amino acids where these R-groups
are variable and in variable proportions are put on a column and
the most strongly retained of the amino acids in-
interact with the column the strongest. And so in this
particular example, the column happens to
be negatively charged and so anything with
a negative charge is very quickly eluded
off the column. The things with positive charge like arginine, lysine
and histadine are most strongly retained, and then the other amino
acids have intermeniate- intermediate ah sticking times
or sticking ah interactions with the column and are
eluded at various points. The y-axis is an intensity and that’s a
fluorescence intensity and so we can quantify the,
ah, intensity of the very a intensity as being, ah,
directly proportional to the concentration of
the different amino acids. You’ll notice that proline
doesn’t hardly show up and that’s because if you
look at the chemistry that’s going to be
on the next slide you’ll realized that
only primary amines will react with the
dialdehyde, OPA, to make this
isoindole structure. So let’s take a look
at that chemistry. Basically, what’s
involved here is that we start with this OPA
and an amino acid in the presence of
this molecule which is mercaptoethanol and the beginning of the
reaction involves the formation of an iminium ion. So that is a nitrogen
that’s a primary amine reacts with an aldehyde; it’s a condensation product
and the iminium ion is formed. We’ve discussed this
already in the course so I won’t go through
that in any detail. The next thing that happens
is an, a nucleophile addition to that polarized π bond,
we do an [AdN] step to make this
tetrahedral intermediate. There’s a second, this sets
up a second [AdN] step but it’s an
intramolecular one now. We have another electrophile
in this aldehyde and we have a nucleophile
in this mean- amine so we can do an [AdN] step followed by some
proton transfer steps to give us this
ah intermediate which is ready to lose by β
elimination the hydroxide. So the nitrogen lone pair serves to kick out
hydroxide anion and we begin to set
up the indole ring. At this point, hydroxide
can serve as a base to remove that proton
and the indole- the isoindole ring is formed. That’s the ah, method that’s
used to tag the amino acids and those are then subjected to
the chromatographic separation that I showed you on
the previous slide. The older ah way that this
was done back in Sanger’s day and- and before involved
the reagent anhydron, and I won’t give you the full
details of the mechanism but I will just say that
another way to quantify rather than using fluorescence is the intensity of the
purple dye in anhydron, which is showed here. It involves the nitrogen that
comes from the amino acid and so if you take an
amino acid and anhydron, two- two equivalents, two
molar equivalents, you can produce the
purple dye here. And I won’t give you the full
details of the mechanism but basically there’s an
equilibrium that’s involved, that initial steps
of the mechanism involve a condensation
of this amine with this tautomeric
form of this triketone, anhydron has this triketone
structure in equilibrium and that begins the
process by which the nitrogen of the
amino acid is extracted from the amino acid to make the intensely colored
purple dye ah anhydron.

2 Replies to “Determining the Amino Acid Composition of a Protein”

  1. Can we not tag the amino acids with Phenylisothiocyanate (PITC)? Very sensitive and the detection is by ultraviolet absorption.

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