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Amino acids Amino acids Glycine

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                                 Amino acids

Background

Amino acids are the building blocks of proteins. Although there are several hundred amino
acids, only twenty are common in most living organisms. All amino acids have several
structural features in common. They possess at least one carboxyl group and at least one
amino group.

                                         H
                                   +              -
                                 H3N      C    COO


                                         R

Distinctive physical, chemical, and biological characteristics of amino acids are dependent
upon the R group that is attached to the alpha carbon. They can be divided into various
classes that are dependent upon functional groups or properties associated with composition
of the R group. For example, four broad classes of amino acids have been defined: acidic
amino acids containing extra carboxyl groups, basic amino acids containing positively
charged nitrogen groups, polar but uncharged amino acids, and nonpolar or hydrophobic
amino acids. See the table below for the distribution of the most common twenty amino
acids.

Hydrophobic        Polar Uncharged            Acidic         Basic

 ala                  gly                     asp             lys
 val                  ser                     glu             arg
 leu                  thr                                     his
 ile                  cys
 pro                  tyr
 met                  asn
 phe                  gln
 trp


In general, amino acids are white crystalline solids with relatively high melting points.
Their solubility in water varies greatly depending upon what class they belong to. The most
common amino acids are found in the L form although the D form of amino acids are also
present in some living organisms.
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Acid base characteristics

In solution amino acids exist in a charged or ionized form. Because they have at least two
different ionizable groups, they are amphoteric and can act as Bronsted acids (proton
donors) or Bronsted bases (proton acceptors). For example, two protons can be dissociated
from glycine as shown below.

H3N+CH2COOH              H3N+CH2COO- +          H+    pKa 2.4

H3N+CH2COO-              H2NCH2COO-        +   H+     pKa 9.6

There can be three forms of glycine present depending upon the pH of the solution. Each
reaction can be described by an ionization constant, Ka, and a pKa. Most of the alpha
carboxyl groups have pKa's between pH 2 and 3 while the alpha amino groups have pKa's
between 9 and 10. The pKa's associated with other ionizable functional groups attached to
the alpha carbon vary depending upon the nature of the functional group. Histidine and
cysteine have groups with pKa's around pH 6 and 8 respectively. Because of the ionizable
groups in amino acids they can also act as buffers. Therefore, a titration curve for glycine
will result in two plateau regions, one associated with each pKa.
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Each plateau will also represent a buffering region where that particular ionizable group can
act as a buffer. Within the titration curve, there should exist some pH value that corresponds
to the point at which the amino acid contains a net zero charge. This pH point is called the
isolectric point (pI). For diprotic amino acids, the pI is halfway between the two pKa's.
Glycine has a pI of approximately 6.0. For acidic amino acids, the pI is 1/2(pK1 + pK2).
For basic amino acids, the pI is 1/2(pK2 + pK3).


Amino acid               pK1           pK2            pK3              pI
                        carboxyl       amino          R group

ala                    2.3             9.7                            6.0
arg                    2.2             9.0            12.5           10.8
asn                    2.0             8.8                            5.4
asp                    2.1             9.8              3.9            3.0
cys                    1.7            10.8              8.3            5.0
glu                    2.2             9.7              4.3            3.2
gln                    2.2             9.1                             5.7
gly                    2.3             9.6                             6.0
his                    1.8             9.2              6.0            7.6
ile                    2.4             9.7                             6.1
leu                    2.4             9.6                             6.0
lys                    2.2             9.0            10.5             9.8
met                    2.3             9.2                             5.8
phe                    1.8             9.1                             5.5
pro                    2.0            10.6                             6.3
ser                    2.2             9.2                            5.7
thr                    2.6            10.4                             5.9
trp                    2.4             9.4                             5.9
tyr                    2.2             9.1             10.1            5.7
val                    2.3             9.6                             6.0



Separation of amino acids

Several techniques can be used to separate and identify amino acids. These techniques
include ion exchange chromatography (IEC), electrophoresis, high performance liquid
chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), and
two-dimensional electrophoresis. In many of these techniques separation occurs because of
differences in charge and polarity among the twenty amino acids. Automated machines are
capable of analyzing amino acids at the picomole level but these are not generally available
to typical biochemistry laboratories.

We will use a relatively simple technique, TLC, to demonstrate how amino acids can be
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separated and identified. This same technique can also be used to show how different chiral
forms of amino acids can be separated using chiral TLC. Thin layer chromatography
consists of a stationary phase and a mobile phase. The stationary phase can be silica,
cellulose, polyamides, or other material bonded to some solid support such as glass plates or
plastic backings. The mobile phase consists of a organic or organic/water solvent.
Typically, a small amount of sample is spotted on the stationary phase/solid support. The
support is then placed in a container so that the mobile phase can move up the solid support
by capillary action. As the liquid phase is moving up the solid support, the sample becomes
partitioned between the mobile phase and the solid phase. The amount of time spent in the
mobile phase vs the stationary phase is dependent on the attraction of the amino acid to the
mobile phase vs its attraction to the solid phase. Because each amino acid is partitioned to
different extents or spends a different amount of time in the mobile phase, they will each
migrate to a different position on the solid support within a given time frame. Thus, each
amino acid will display a characteristic Rm, or relative mobility, for a given solvent system
and solid support. This Rm is a measure of the distance traveled by the amino acid divided
by the distance that the solvent moves.

Chiral TLC is a another type of TLC separation technique. A CHIRALPLATE is a TLC
plate coated with reversed phase silica gel impregnated with a chiral selector (proline) and
copper II ions. Optically active isomer separation is based upon ligand exchange and
partitioning between the solid and mobile phases.

There are many methods to detect amino acids once they have been separated. One of the
easiest is to treat the sample or chromatogram with ninhydrin. Ninhydrin reacts with
primary amines, such as amino acids, to yield a blue-purple colored product when heated at
110 oC. All amino acids react to give this blue colored product except proline that gives a
yellow product because it contains a secondary amine (imine). Qualitative and quantitative
procedures can be used with ninhydrin.
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Experimental

Your goal is to examine the acid-base characteristics of an amino acid by titrating this amino
acid and determining its pKa values and pI. You will also examine characteristics of amino
acid separation using TLC.


Amino acid titration

You will be given an amino acid - histidine, histidine HCl, cysteine, or cysteine HCl.
Prepare 100 mL of a 25 mM solution of this amino acid. Note whether it is soluble or
insoluble. If it is insoluble, what must you do to make it soluble?

1. Titrate 40 mL of your amino acid solution with 50 mM HCL. Add the acid in 500 uL
increments and record the pH. Try to get to pH 2 if possible.

2. Titrate 40 mL of your amino acid solution with 50 mM KOH. Add the base in 500 uL
increments and record the pH. Add enough KOH to reach pH 12.

3. Plot mL of added acid or base vs pH. Identify buffering regions and pK values for your
amino acid. You may also want use other plotting methods to locate pKs (see buffer
experiment). Locate and determine the pI of your amino acid. Draw the chemical species
of your amino acid and ratio of species at the end points, each pKs, pI, and one pH unit on
either side of the pK.


Thin Layer Chromatography

1. Spot 0.5 uL of the amino acids given to you and your unknown on a silica gel, cellulose,
or a HPTLC plate. Put the plate into the chromatography chamber and let the solvent move
to within 1 cm of the top. Choose either butanol:acetic acid; water (4:1:1) or propanol:water
(7:3) as a solvent. Both solvents should be prepared fresh. Amino acids standards and
unknowns are at 10-20 mg/ml.

2. Remove the plate, mark the solvent front, and blow dry with hot air (hair dryer). Then
dip in a 0.1% ninhydrin solution and dry it in the oven at 110 C for 10 min. Plastic backed
supports can only be heated for a few minutes with a hair dryer.
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Description: Amino acids Amino acids Glycine