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CHM 373 2015

Experiment 1: Determination of amino terminal groups in


peptides and proteins
This experiment will be supplemented with a presentation based on chapter sections 5.3 and 5.4
of your textbook, Garrett and Grisham, any edition.
References
1. W. Gray and B. Hartley. The structure of chymotryptic peptide from pseudomonas cytochrome C551. Biochem J. 89, p379 (1963)
2. W.R. Gray. End -group analysis using dansyl chloride. Methods in Enzymology, Vol. 25, Part B (Ed:
Hirs and Timasheff), Ch 8, p121-138 (1972)
For methods on modern peptide sequencing, see the following papers:
i) K. F. Medzihradszky. In-solution digestion of proteins for mass spectrometry. Methods in
Enzymology, 405, p50-65 (2005)
ii) K. F. Medzihradszk. Peptide sequence analysis Methods in Enzymology, 402, p209-224 (2005)
iii) J. W. Morgan, J. M. Hettick, D. H. Russell. Peptide sequencing by MALDI 193 nm photodissociation
TOF MS. Methods in Enzymology, 402, p186-209 (2005)
STRUCTURE OF PROTEINS: SEQUENCE DETERMINATION
It has been clearly established that the amino acid sequence or primary structure of a protein
determines the overall three-dimensional structure of the molecule and hence its biological activity.
Sequence analysis of a protein has provided valuable information regarding many structure-function
relationships of proteins, as well as insight into gene structure and evolution. In addition, the chemical
synthesis of many important hormones is now possible because the amino acid sequence of these
molecules is now known. Many pharmacological and clinical applications involve the use of fluorinated
or unnatural amino acids, for which peptide synthesis is straightforward.
One important step in sequence determination is the identification of the N-terminal amino acid. A
number of methods are currently available, and in our experiment we will use the reaction of free amino
groups with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) as shown in Figure 1.
This reagent reacts with any free amino groups that include both the -amino group of the N-terminal
amino acid and the -amino group of lysine. Other groups may also react to a small extent. After the
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reaction, the peptide is hydrolysed and the dansyl amino acid is separated from the free, unreacted
amino acids by thin layer chromatography (TLC) (Figure 2). In cases where an entire peptide may need
to be sequenced, the carboxyl terminus of the peptide may first be covalently attached to a bead,
whereupon the N-terminal amino acid is reacted with dansyl chloride. The dansylated amino acid is
then selectively detached by the addition of anhydrous acid. The fluorescent derivative can be washed
off and identified by chromatography, and the cycle can be repeated. The efficiency of each step is
about 98%, which allows about 50 amino acids to be reliably determined.
Identification of the dansyl amino acid is made by comparison of TLC profiles with known
standards. The dansyl derivative can be readily detected by viewing the thin layer plate under a UV
lamp, as the dansyl derivatives are fluorescent.
This image cannot currently be display ed.

Figure 1. Reaction of dansyl chloride with terminal amino groups and identification by TLC.
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In the following experiment, you will determine the sequence of an unknown dipeptide. In the first
laboratory session you will prepare the standard dansylated amino acids, and dansylate the dipeptide.
Your sample will be hydrolysed overnight and analyzed in the following laboratory session.
For the analysis, thin layer chromatography (TLC) silica plates will be used to determine the dansyl
amino acids. To determine the unreacted amino acid (or the C-terminal amino acid) cellulose TLC
plates will be used. The principals involved in the separation procedures will be outlined in the lab.

Week 1: Dansylation of dipeptides and amino acids


Safety

Consult Materials Safety Data Sheet (MSDS) for all reagents used in this procedure for detailed
safety information
Solutions are stabilized with acids or bases, which are potentially corrosive. Wear appropriate eye
protection, gloves and a lab coat at all times.
Perform all procedures in the fume hood.

A. Preparation of dansylated amino acid standards and unknown dipeptide.


1. You will be given 0.5 mM solutions of the following amino acids: glutamic acid, glycine, alanine and
phenylalanine, and an unknown dipeptide dissolved in pH 9 0.2 M NaHCO3. Pipette 0.2 mL of each
solution into separate microfuge tubes, add 0.2 mL of 5 mg/mL dansyl chloride solution in acetone
to each tube and gently mix. Why is this reaction performed under basic conditions?
2. Also prepare a control reaction; add 0.2 mL of the dansyl chloride solution to 0.2 mL of just the
0.2 M pH 9 NaHCO3 buffer.
3. Close the tubes, mix well by flicking, and incubate at 37oC for 1 hr.
4.

After incubation precipitate the excess bicarbonate by adding 1.6 mL of acetone and mix. Make
sure you condition your micropipette for using acetone.

5.

Isolate the precipitate by centrifugation for 5 minutes at 5000 rpm.

6.

Except for the unknown dipeptide, aspirate the liquid layer with a Pasteur pipette to a new set of
microfuge tubes.

7.

Label your tubes with tape and leave them open in the fume hood to allow the solvent to dry for the
next lab session.

8.

For the unknown dipeptide, aspirate the liquid layer into a vial from your locker and dry it down
using a gentle stream of air while placing the vial in a beaker of hot tap water.
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9.

Once the unknown dipeptide is dry add 0.2 mL of 6 M HCl (careful, strong acid) to the residue
and mix using gentle vortexing until dissolved.

10. Transfer this solution to an ampule labelled with tape, Parafilm and submit to the TA. The ampule
will be sealed by a glass blow torch for you, and then be placed in an oven at 110C for 18 hours.
The samples will be returned to you in the following lab period for analysis. How does acid
hydrolysis occur?
Would the above method be adequate for longer polypeptides, such as proteins?
Disposal
Rinse the microfuge tubes with water to suspend the precipitate and dispose the solution into the
organic waste container. Once clean, the tubes can go in the garbage.
Discard used plastic pipette tips in the garbage.
Discard used Pasteur pipettes in the broken glass container.

Week 2: Thin layer chromatography


Chromatography (General)
References
1. E. Lederer and M. Lederer. Chromatography; a review of principles and applications.
2. E. Heftmann. Chromatography (Second Edition).
3. J.M. Bobbitt. Thin Layer Chromatography.
4. A.I. Vogel, A.R. Tatchell. B.S. Furnis, A.J. Hannaford, and P.W.G. Smith Vogels Textbook of
Practical Organic Chemistry (5th Edition) ISBN 0582462363
5. J.M. Stoddard, L. Nguyen, H. Mata-Chavez, K. Nguyen. TLC plates as a convenient platform for
solvent-free reactions. Chemical Communications 12, 1240-1241 (2007). This is an interesting
application of TLC. A description is given for the use of TLC plates for solvent free chemistry.
The separation of pure compounds from complex mixtures is a major occupation in biochemistry as
in other branches of chemistry. The most powerful and commonly used separation techniques are
those based on the principle of chromatography.
The name chromatography is something of a historical accident, and as mentioned above refers
to a principle rather than a specific technique. A Russian botanist, Tswett, reported in 1900 that a
petroleum ether solution of leaf pigments, when filtered through a bed of starch in a glass column, is
separated into several coloured zones which move at different velocities through the column. He called
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the pattern of coloured zones produced from a specific mixture of pigments the chromatogram of the
mixture, and thus chromatography is the method used to produce a chromatogram. Tswett's method is
actually a simple example of adsorption chromatography. The technique has nothing whatever to do
with colour however, as many non-coloured substance can be separated. In addition, there are many
chromatographic techniques which do not depend on adsorption. Fortunately all of these have the
same general principles.
Theory of Chromatography
Relative mobility
The simplest chromatographic set-up is a cylindrical column containing an insoluble, granular,
porous material (the packing), permeated with a solvent which flows through the column with an
overall velocity vsol. The volume of packing material plus solvent trapped between the particles and in
the pores is called the bed volume, or total volume, and is given the symbol Vt . In addition to the inert
material, i.e. resin, there are two chromatographically important phases: the mobile phase and the
stationary phase, which is normally bound to an inert resin. The mobile phase is the flowing solvent
whose volume in the bed is denoted by Vo (also called the void volume). The volume of the stationary
phase in the bed is called the inner volume and is denoted by the symbol Vi. The volume occupied by
the stationary phase itself, which is inaccessible to solvent, is denoted as Vinert. The total volume of the
bed is defined as:

Vt = V0 + Vi + Vinert

The substances being chromatographed (or separated) are called the solutes.

(1)

In column

chromatography the mixture of solutes is initially applied to the top of the column in a small volume of
solvent.

Elution is achieved by continually applying pure solvent. The various solutes then move
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through the column as zones or bands. Due to differential retention of solutes in the stationary phase,
each zone moves along at a characteristic fraction, R, of the mobile phase velocity. The quantity R, the
relative mobility, is the basic parameter of chromatographic theory. On a particular column, each
individual solute would have a characteristic R value.
Although the zone appears to migrate smoothly, at the molecular level migration occurs in a
stepwise fashion, with each solute molecule progressing in a stop-and-go sequence. Each time a
molecule affixes itself to the stationary phase, its motion is interrupted. Thus the velocity of migration of
a single molecule depends on the average fraction of time, R, it spends in the mobile phase. If the
molecules spend a fraction R of their time in the mobile phase, the molecules, and hence the zone as a
whole, move with a velocity (v) that is slower than the velocity of solvent front (vsol). This accounts for
the relative mobility in molecular terms.
The essential basis for chromatography is partitioning.

We can define the term partition

coefficient, K, as the equilibrium ratio of the concentration of the solute in the two phases (K=Cs/Cm,
where Cs and Cm are the concentrations of the solute in the stationary and mobile phases,
respectively). It should be intuitively obvious that if a mixture contains 2 solutes with different Ks, then
theoretically one of these solutes will move faster in the mobile phase than the other. The solute with
the higher K value will spend more time in the stationary phase and will be eluted later.
The relative mobility of the solute can be defined as:

R=

V0
V0 + KVi

(2)

Because the column parameters Vo and Vi are constant for a given column, the relative mobility will
increase as K decreases.
The ability of a solute to partition between a mobile and stationary phase does need not be based
only on preferential solubility. Any physical method of retaining one solute preferentially in one phase
will also work. In ion exchange chromatography, the solute is held preferentially in the stationary phase
(or resin) because of electrostatic interaction with the resin.

In adsorption chromatography, the

stationary resin itself also serves to bind the solute, but not through electrostatic interactions. But in all
cases, the same principle applies i.e. differential partitioning or distribution between mobile and
stationary phases, the latter being the liquid, a liquid-solid matrix or a solid support.
Calculating the relative polarity of the mobile phase is made possible by the Snyder Polarity
Scale/Index (P'), which is a measure of the ability of the solvent to interact with the following polar test
solutes: ethanol, dioxane, and nitromethane. Solubility measurements of each of the test solutes in a
given solvent are used to determine adjusted partition coefficients, kg', where kg' is the solute
distribution coefficient corrected for the molecular weight of solvent and solute. The polarity index (P') is
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given by the following equation:
P' = log (kg') ethanol + log (kg') dioxane + log (kg') nitromethane
The equation for a mixture of solvents is given as:
=

% + % + + %
100

Thin Layer Chromatography


In thin layer chromatography, a glass plate is uniformly coated with a thin layer of adsorbent (silica
gel, alumina, or cellulose). The adsorbent also contains a binder, such as CaSO4, which helps it adhere
to the glass plate. The layer is dried and activated by heating. An aliquot of the sample to be
analysed is applied at one end and the plate is developed in a closed tank by immersing the lower end
of the plate in a suitable solvent. The solvent rises up the plate by capillary action. The solvent
normally contains several components, one of which will adhere more tightly to the silica gel. This is
usually the more polar phase, such as water. In this case, the water is the stationary phase and the
non-polar component of the solvent is the mobile phase. However, in some applications of thin layer
chromatography interaction of a solute may occur directly with the silica gel (which is polar) so that if a
highly non-polar solvent is used, the stationary phase is the silica gel itself.
As the solvent rises up the plate it carries with it, the solutes in solution to be separated. These
solutes will have differing solubilities in the two phases and will migrate up the plate at rates depending
on their relative partition coefficient. In thin layer and paper chromatography, the term Rf is commonly
used. Rf is a measure of the relative mobility of a solute under specified conditions and is defined as:

Rf =

distance migrated by solute


distance migrated by solvent front

(3)

Rf will be constant for a particular solute in a defined solvent and will vary between 0 (no migration from
origin) to 1 (migration along with solvent front).
Cellulose Chromatography
The principle of TLC using cellulose is basically the same as that of TLC using silica. In this case,
the inert support is the cellulose matrix, which is very polar. As the solvent permeates the cellulose, it
separates into a water-rich phase, which binds to the cellulose, and the organic solvent or non-polar
rich phase that is mobile. Solutes that are more soluble in the aqueous layer have lower Rf values.
Amino acids can be separated by cellulose chromatography generally because of their differences
in polarity. For example, glutamic acid will be more soluble in the water-rich phase than isoleucine.
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Isoleucine will migrate up the chromatogram carried by the non-polar phase of the solvent and will be
separated from the glutamic acid zone. Remember that all the amino acids except proline contain an
NH2 group and all amino acids contain an COOH group. Therefore, it is the properties of the side
chains which essentially determine differences in mobility.
Detection of compounds
After chromatography, the solutes can be detected by a number of methods that depend on their
properties. These detection methods include:
(1) fluorescence; i.e. dansyl group coupled to an amino acid (dansyl-AA)
(2) colorimetric; i.e. amino acids react with ninhydrin to produce a purple intermediate
(3) other; e.g. charring with sulphuric acid to detect organic compounds in thin layer
chromatography.
Identification of solutes using these methods is based on comparison of the chromatogram of the test
solute with that of known standards.
In the following experiment, the amino acids are separated by thin layer chromatography.
Chromatography on cellulose coated plastic sheets will be used to separate the dansylated free amino
acids.

Week 2: Chromatography of amino acids and dansyl amino acids on


silica and cellulose TLC
Safety

Consult Materials Safety Data Sheet (MSDS) for all reagents used in this procedure for detailed
safety information
Solutions are stabilized with acids or bases, which are potentially corrosive. Wear appropriate eye
protection, gloves and a lab coat at all times.
Do not inhale loose silica powder from the TLC plates.
TLC tanks will contain solvent that emit strong fumes; do not move the tanks from the fume hoods.
Allow wet TLC plates to dry in the fume hood.
Perform all procedures in the fume hood.

A. Silica TLC for N-terminal determination


1. Retrieve your amino acid standards and hydrolysed dipeptide samples.
2. Dry down the hydrolysed dipeptide samples as you did the previous week using a stream of air and
a beaker of hot tap water.
3. Two groups will share a 20 cm x 10 cm pre-cut TLC plate on aluminium backing, pre-spread with a
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layer of silica gel. The layer is fairly stable but should not be touched by hand. Handle plates by
their sides with gloves.
4. Plan and organize such that each group will spot their half of the plate, which is in a landscape
orientation, as follows with the peptides spotted in duplicate:

DNS-Glu

DNS-

DNS-

DNS-

control

Phe

Peptide

DNS-Gly

DNS-

DNS-

Peptide

Ala

DNS

5. When you are ready to spot the samples, reconstitute them by adding 100 microL of acetone and
mix completely by vortexing.
6. Dip a capillary tube into one of your dansylated amino acid samples and apply ONE spot of no more
than 2-3 mm in diameter to one of the pencil dots. Multiple dabs will give you big spots that will
smear and you will NOT get good results. Repeat with the rest of the dansylated standards. To save
glassware you should use the other end of the capillary tube to spot another sample. If you want to
practice, try spotting acetone only at the top of the TLC plate above the solvent front.
7. Place the plate in a pre-equilibrated tank containing the solvent (chloroform/methanol/acetic acid,
95:10:1). Let it run until the solvent front has reached 1 cm from the top of the plate. Calculate the P
of this solvent mixture.
8. Remove the plate and immediately mark the solvent front. Allow the plates to completely dry in the
fume hood (1-2 minutes) before viewing under ultraviolet light.
9. Record the Rf values in the table provided at the end of this procedure. Also note the colours of all
spots.
B. Cellulose TLC for C-terminal determination
1. Three groups will share a 20 cm x 10 cm pre-coated cellulose TLC plastic plates. Handle the plates
as little as possible and only at the very edges with gloved hands.
2. Plan and organize such that each group will spot their third of the plate, which is in a landscape
orientation, as follows with the peptides spotted in duplicate:
Glu

Peptide

Ala

Phe

Peptide

Gly

3. Glutamic acid, alanine, glycine and phenylalanine are supplied at concentrations of 0.1 mM.
4. Dip a capillary tube into one of the samples and apply ONE spot of no more than 2-3 mm in
diameter to one of the pencil dots. Multiple dabs will give you big spots that will smear and you will
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NOT get good results. Repeat with the rest of the samples. To save glassware you should use the
other end of the capillary tube to spot another sample. If you want to practice, try spotting acetone
only at the top of the TLC plate above the solvent front.
5. Before running the plate, first equilibrate the plate by perching it on a test-tube rack placed in the
tank. Why is this done? After equilibration, remove the test tube rack and lower the plate into the
solvent to run. The solvent used is butanol: acetone: acetic acid: H2O (10:10:5:2). Calculate the P of
this solvent mixture.
6. Let the chromatograph run until the solvent front is about 1 cm from the top.
7. Remove chromatogram, mark the solvent front, and once dry spray with a solution of ninhydrin
(0.25%) in acetone. Heat the plate in the oven for approximately 2 min, exercising care not to
overheat the plastic. Note colour, shape and size of the spots. Also note the position of each
component with pencil and determine the Rf values. What is the reaction of ninhydrin with the amino
acids?
Disposal
Discard used plastic pipette tips in the garbage.
Discard spotting tubes in the broken glass container.
Carefully rinse glass ampules and microfuge tubes with acetone and discard the rinses into the
organic waste container. The ampule goes into the broken glass container, and the plastic tubes into
the garbage.
Thoroughly wash used the glass vials and return them to your locker for future use.
Discard the TLC plates into a zip-lock bag labelled Solid waste. Make sure you have all the data
you need before disposal!

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Data Sheets (Write the number of the unknown dipeptide you used and print out the tables double-sided and
hand in with lab report, along with pictures of your original TLC plates. Identify your unknown dipeptide.
Silica TLC
Rf value

DNS-Glu

DNS-Peptide

DNS-Phe

DNS-Peptide

DNS-Ala

DNS

1.00-0.90
0.89-0.80
0.79-0.70
0.69-0.60
0.59-0.50
0.49-0.40
0.39-0.30
0.29-0.20
0.19-0.10
0.09-0.00
Cellulose TLC
Rf value

Glu

Peptide

Ala

Phe

Peptide

Lys

1.00-0.90
0.89-0.80
0.79-0.70
0.69-0.60
0.59-0.50
0.49-0.40
0.39-0.30
0.29-0.20
0.19-0.10
0.09-0.00

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Lab report
In addition to what is written in the syllabus, the following guide provides a few more details for this
particular report.
Introduction
For the last paragraph, what do you expect to see on your silica and TLC plates? Think of where the
standards will appear on the plate in terms of polarity.
Results
Provide labelled pictures of both silica and polyamide TLC plates. Identify ALL spots on the TLCs.
Fill out the following table and include it in your results section. Note which dipeptide you analysed.
N-Terminal amino acid
Dipeptide #:
Rf of dipeptide

Rf of matching
standard

C-Terminal amino acid


Rf of dipeptide

Rf of matching
standard

Dipeptide
structure and
name

Discussion
Recall the purpose of the experiment and what techniques are being used to address it.
Look at your TLCs and Rf tables and start to match up and identify spots. Are the spots appearing
where you expect them to in terms of polarity?
If the experiment did not work, suggest how you would change the experiment.
Conclude with restating the purpose and the techniques used to answer it. Identify your unknowns and
state if the purpose was answered or not. If not, how would you change the experiment?
Appendix

References
Calculations
 Sample calculation of Rf values
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 Print out the Rf values tables and include them in this section
Methods: state that the lab manual was followed, provide its reference, and include any deviations
from the procedure here.
Copies of flow charts
Copies of observations and raw data

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