ASI, Ranelle Janine L.

Chemistry 160.1 Section 3L

2015

Date performed: July 3, 2015

Date submitted: July 8,

EXERCISE 6. Dipeptide Sequence Determination

Post-Laboratory Report
The structural and chemical properties of a protein can be understood by
determining the monomers that comprise it in order. The differences in sequence
primarily determine protein function as amino acids vary in the identity of the R
group. All amino acids consist of an amino group and a carboxyl group and the
dipeptide bond is formed between these two groups forming a direction to which all
the amino acids face, hence a peptide chain has a designated N-terminal and Cterminal end.
When two amino acids make a dipeptide molecule, the amino group and
carboxyl group form an amide bond (the peptide bond) with the release of a water
molecule in a dehydrogenation reaction as seen in Figure 6.1.

R1 O R2 O
H3+
NCC H3+
NCC
O
H
H O

H2O
RO
1 R2 O
H3+
NCCNCC
O
H
H
H
N-terminus
C-terminus
Peptide bond

Fig 6.1. Reaction scheme of peptide bond formation between two amino acids and
designation of the terminal ends of the dipeptide.
Dipeptide proteins can be deconstructed as the peptide bond can be
hydrolyzed by strong acids and bases. Acid hydrolysis is preferred as it is less
destructive on the amino acids. A protein is completely hydrolyzed by an acid into
constituent amino acids upon exposure to high heat. Once in their individual forms,
the amino acids are characterized using separation methods like paper

chromatography (PC) and thin-layer chromatography (TLC), both of which are used
in this experiment.
Total Hydrolysis of the Dipeptide and Paper Chromatography
As previously discussed, strong acids can hydrolyze peptide bonds. In this
experiment, hydrochloric acid (HCl) was added to an unknown peptide sample at a
concentration of 6N to ensure high reaction occurrence. Hydronium ions (H 3O+)
dissociated from HCl attack the double bond of the carbon at the peptide link and
water molecules (H2O) bind to the same carbon, eventually forming the free
carboxyl group of the first amino acid. H 3O+ then donates its hydrogen atoms to the
nitrogen at the amino terminal of the other amino acid, forming the free amino
group of the second amino acid. Samples are then heated at 110C overnight as
quantitative fission of most amino acids requires high energy reactions. The
resulting samples are then characterized using PC.
In chromatography, the paper acts as a stationary phase while the
chromatography solvent is the mobile phase. Different amino acids are
characterized by their affinity for the solvent phase and are carried at different rates
along the paper via capillary action, resulting to different distances travelled by
their corresponding spots. The stationary phase used in this experiment is Whatman
Paper No. 1, which is thick and smooth and made from cellulose polymers which
consistently let solvents pass at an ideally linear rate. The mobile phase used is
butanol-acetic acid-water in a 4:1:1 volume ratio which is a common compound
used in chromatography of proteins which readily dissolve in this particular solvent.
After the run in PC, ninhydrin is used to visualize the spots as pink-purple
blots upon exposure to high heat, illustrated in Figure 6.2. Ninhydrin only reacts with
free amino acids, so any compounds that do not contain such will not be detected
by this method.

Fig 6.2. The reaction of ninhydrin with the free amino group of an amino acid.

During the ninhydrin reaction, two H2O molecules are lost in the formation of
a bond with the ninhydrin molecule, and a loss of a proton on the free amino group
of the amino acid. Resonance stabilization and loss of CO 2 then result, followed by
loss of another H2O molecule and an aldehyde group. Finally, another molecule of
ninhydrin reacts with the originally modified ninhydrin molecule to give the purple
colored product.
In both types of chromatography, Rf value is computed as a quantifiable basis
of comparison among blots, computed as follows:
Rf = distance travelled by the sample
distance travelled by the solvent
During PC, standards, or amino acids with known R f values and estimates of
blot color are run along unknown samples. Although some amino acids are easily
characterized using qualitative comparison of color and distance, R f comparison is
more accurate as not all solvent phases during chromatographic runs rise in the
same height.
In this experiment, the amino acid constituents of two unknown samples of
dipeptides were determined, the results of which are shown in Table 6.1 and 6.2.
Table 6.1. Rf values of amino acid standards from paper chromatography.
Amino acid
Distance travelled
Distance travelled
Rf
standards
by the sample (cm) by the solvent (cm)
Met
2.9
5.9
0.49
Phe
3.0
5.5
0.55
Ala
2.9
5.6
0.52
Leu
2.6
6.3
0.41
Gly
0.8
6.8
0.12
Glu
0.3
6.7
0.04
Table 6.2. Amino acid composition of the acid hydrolyzed dipeptide sample.
Amino acid
Distance travelled
Distance travelled
Rf
Identity of
sample
by the sample
by the solvent (cm)
the amino
(cm)
acid
D1 spot 1
2.0
6.1
0.33
Leu
D1 spot 2
2.7
6.1
0.44
Met
D2 spot 1
0.8
6.5
0.12
Gly
D2 spot 2
4.0
6.5
0.62
Ala
Table 6.1 shows the Rf values of the standards used in the experiment:
methionine, phenylalanine, alanine, leucine, glycine and glutamate, the Rf values of
which were compared with the four amino acids released from the hydrolysis of the
two unknown dipeptides. Each of the two hydrolysate columns had two spots; D1
spots had Rf values of 0.33 and 0.44, which are closest to the values of leucine and
alanine, respectively, which indicates the presence of the two amino acids. Although
there is a notable difference in R f values, qualitative comparison of the spots size
and color aided in determining their identity. D2 hydrolysates had Rf values of 0.12

and 0.62, which were near to values of glycine and alanine, respectively. Glycine
had an identical value with the hydrolysate and alanine was determined to be closer
to the second spot in terms of blot appearance.
There were several errors during PC, most notable of which were the varying
solvent fronts and the slight incongruity of some standard Rf values with the
hydrolysate values. The solvent front should have ideally been a straight line but
during the placing of the chromatographic paper in the chromatogram, the solvent
may have been slightly shaken and rose up at different heights, hence affecting the
rates of some spots during the run, resulting to inconsistent R f values.
Preparation of the DNP-derivatives and Thin-Layer Chromatography
The first reagents added were saturated sodium bicarbonate (NaHCO 3) and
ethanolic dinitrofluoro-benzene (DNFB) or Sangers reagent. Sodium bicarbonate
neutralized the solution by acting as a buffer and bringing the pH to around 8.0 and
DNFB reacts with the free amino group of the dipeptide to produce dinitrophenylamino acids in a reaction shown in Fig 6.3 and was subjected to high heat to
facilitate and speed up the reaction.

Fig 6.3. Reaction scheme of dinitrofluorobenzene (DNFB) with the free

amino group of a peptide chain.
After production of the DNP-derivative, ether was used as an extracting
solvent and HCl was used to bring the pH to 1.0. Ether dissolved all non-polar
residues, acting as the organic phase of the layered solution, and left the charged
residues in the aqueous phase. Since most DNP-derivatives are uncharged at low pH
(in this case, pH 1.0), they will be extracted into the ether phase; hence the ether
solution was used for Thin Layer Chromatography (TLC) to determine the amino acid

at the N terminus of the dipeptide. Ether extraction was performed three times to
ensure maximum dissolution.
Like in PC, several amino acid standards were prepared also by
dinitrophenylation and extraction using ether. All the samples and standards were
then blotted on Whatman Paper No.1 and submerged in a chromatogram with the
solvent methylenechloride-methanol-acetic acid at 95:4:1 volume ratio. Ninhydrin
was not used as the reaction between the DNP-derivatives and the solvent
components can produce already colored spots. Results of TLC of the standards and
the unknown dipeptide samples are shown in Table 6.3 and 6.4.
Table 6.3. Rf values of DNP-amino acid standards from TLC.
Amino acid
Distance travelled
Distance travelled
standards
by the sample (cm) by the solvent (cm)
Met
4.0
Phe
4.1
Ala
0.4, 2.9, 4.7
6.7
Leu
0.4, 3.7, 4.4
Gly
0.4, 1.9, 4.2
Glu
4.3

Rf
0.6
0.61
0.06, 0.43, 0.70
0.06, 0.55, 0.66
0.06, 0.28, 0.63
0.64

Table 6.4. Identity of N-terminal amino acid of the unknown dipeptide.

Amino acid
Distance
Distance
Identity of
Sample
travelled by
travelled by
Rf
the amino
the sample
the solvent
acid
(cm)
(cm)
D1
4.3
6.7
0.64
Met
D2
0.3, 2.9, 4.8
0.04, 0.43, 0.72
Ala
The amino standards used were the same with the ones used in PC. Some
columns contained more than one blot as a result of the corresponding dipeptides
having formed intermediate derivatives during the run as in the case of alanine,
leucine, glycine and the D2 hydrolysate. From comparison of R f values, the amino
acid at the N-terminus of D1 was determined to be methionine and alanine for D2.
The values in TLC are more consistent than in PC, and identities of the amino acids
were simply obtained quantitatively.
Identities and sequence of the unknown dipeptides
In conclusion, the two unknown dipeptides D1 and D2 have the following
chemical structures:

Met-Leu
(D1)

Ala-Gly
(D2)

Other Methods for Sequence Determination of Dipeptides

Sangers method is only one of the more popular methods used to determine
peptide sequence. An alternative to Sangers reagent is dansyl chloride which
produces acid-stable sulfomides. Edmans reagent can also be used to sequentially
cleave the N-terminal residue of a peptide without the need for total hydrolysis.
If a peptide ester is reduced with lithium borohydride (LiBH 4) then hydrolyzed
with 6M acid, the C-terminus residue can be identified as an amino acid alcohol. If a
peptide is reacted with hydrazine, all amino acids except the C-terminus residues
will be isolated as the acid hydrazide.

REFERENCES
Cazes, J. 2005. Encyclopedia of Chromatography, vol 1. CRC Press.
Stahl, E. (ed). 2013. Thin-Layer Chromatography: A Laboratory Handbook. Springer
Science & Business. USA.
Campbell, M. K. and Farrell, S. O. 2009. Biochemistry. 6 th ed. Thompson Brooks/Cole.
Canada.
Wilson, M. C. (n.d.). Determination of the Amino Acid Sequence of an Unknown
Dipeptide. University of North Carolina, Asheville, North Carolina. USA.
Retrieved
from
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%20444%20Lab%20Report%202.pdf
Inglis, A. S., et al. 1971. Hydrolysis of the Peptide Bond and Amino Acid Modification
with
Hydriodic
Acid.
J.
bio.
Sci.
Australia.
Retrieved
from
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