POSTLAB Report on

Exercise No. 5
Dipeptide Sequence Determination

Bolandos, Kim Leonard C.

CHEM 161.1 – 2L
1st Semester AY 2018-2019

Groupmate:
Joel Amado
Giorgia Escalante
CJ Carlos
Pamela Macalagay

Date performed: October 16, 2018

Date submitted: October 23, 2018

Sir Bong Remillion

Results and Discussion

The primary structure of protein is the order in which amino acids are linked together in a
protein. Each protein has its own unique amino acid sequence. Aside from the numbers and kind of
amino acid present, the primary protein structure also involves the order of attachment of the amino
acids to each other through peptide bonds. The proteins’ primary structure is the same regardless of
where the protein is found within an organism (Stoker, 2010).

Conventionally, the protein primary structure begins at the amino-terminal (N) and continues
until the carboxyl-terminal (C) end. The amino acid sequence of a protein or peptide is a prerequisite for
the determination of the three dimensional structures of proteins thereby understanding its function,
identify it in a sample, and categorize its post translational modification. Proteins with novel properties
can be generated by varying the sequence of known proteins. Protein sequencing is the process of
determining the amino acid sequence (Smith, 2000).

Chemical method, such as acid and base hydrolysis, may be used to initially determine the
amino acid sequence as it break the bonds between amino acid residues. Unfortunately, the hydrolysis
procedure chemically modifies or destroys asparagine, glutamine, and tryptophan residues. Asparagine
and glutamine are converted into their corresponding acids (Asp and Glu). Tryptophan is completely
destroyed and is best determined spectrophotometrically using unhydrolysed protein. It is followed by a
separation method for the identification of the amino acid residues found in the protein. Separation and
analysis of amino acid which can be done by different methods such as Ion-exchange chromatography,
high performance liquid chromatography, gas chromatography, and paper chromatography. After
identification, several methods are then used to determine the sequence of the identified amino acid
residues. Sequencing which can be done by end group analysis (amino termini & carboxyl termini) and
by chopping off residue one at a time via Edman Degradation and carboxypeptidase (Boyer, 2012).

One of the most commonly used method in determination of the N-terminal amino acid is by
employing Sanger’s method. The reagent 1-fluoro-2, 4-dinitrobenzene (FDNB) was developed by Sanger.
After treatment of protein with FDNB, the amino terminal residue is labeled with FDNB and the
polypeptide is hydrolyzed to its constituent amino acids. The marker, dinitrophenyl residue, remains
intact during hydrolysis of peptide. The hydrolysate contains one dinitrophenyl amino acid that is
colored yellow and easily identified (Stretton, 2002).

A substituted amide linkage, called a peptide bond, can covalently join two amino acid molecule
to form a dipeptide. Such linkage is formed by the removal of the elements of water from the α-carboxyl
group of one amino acid and the α-amino group of another. The peptide bond formation is an example
of condensation reaction happening inside a living cell. The carboxyl group must be chemically modified
or activated so that the hydroxyl group can be more readily eliminated and to make the reaction more
thermodynamically favored (Nelson & Cox, 2008).

The amide bonds of proteins and peptides can be hydrolyzed using strong acid or base.
Treatment of either of these conditions yields a mixture of the constituent amino acids. Thus, complete
hydrolysis of the dipeptide sequence in the exercise is represented in the Reaction 5.1. In this
experiment, the amino acid composition was determined by total acid hydrolysis of the sample and
subsequent paper chromatography. The total hydrolysis of the dipeptide was done by heating a sample
of unknown dipeptide with 6 N HCl using an oven at 110 °C overnight. The disadvantage of this method
if tryptophan is present, it is completely destroyed. Serine and threonine are dehydrated to give
corresponding alkenes. Also, sulfur-containing amino acids are oxidized if oxygen is not excluded (Wilson
& Walker, 2010).

Amino acid 1- Amino acid 2( Acid or base)→ Amino acid 1 + Amino acid 2 (Reaction 5.1)

Other reactions may occur in the released amino acids when proteins are subjected to
hydrolysis in acidic medium. This includes:

a.) Dehydration of serine and threonine

Threonine

b.) Oxidation of methionine, cysteine, and cysteine

c.) Hydrolysis of asparagine and glutamine

Consequently, base hydrolysis is done by boiling sample with strong NaOH solutions and is used
only for separate estimation of Tryptophan. The disadvantage of this method is that it converts L-amino
acids into racemic mixtures and completely destroys serine, arginine, cysteine, and threonine (Wilson &
Walker, 2010).

The determination of amino acid composition was done using paper chromatography. The
hydrolysates were spotted on a chromatographic paper alongside the standard amino acids. The
chromatography paper was developed in a chromatographic chamber previously equilibrated with
solvent (4:1:1 butanol-acetic acid-water) to allow its vapor to saturate the paper. The chromatogram
was removed from the chamber when the solvent is approximately 2/3 on the chromatography paper.
The solvent front was then marked and the chromatogram was blow-dried. After drying, Ninhydrin
solution was applied by spraying as the visualizing agent. Upon heating using a blow-dryer, the α-amino
acid reacts with 2 molecules of ninhydrin to yield a purple colored product wherein the intensity of the
colored product is proportional to amino acid concentration.
 Figure 5.1. Reaction of Ninhydrin to the Amino acid.

The retardation factor (Rf) of the hydrolysates and standards were obtained to deduce the
amino acids present in the sample. The fate of each amino acid in the mixture depends on the affinity of
each substance to the mobile and stationary phases. If an amino acid has a higher affinity for the mobile
phase than the stationary phase, it will tend to travel with the solvent front and be relatively unhindered
by the chromatographic paper. On the contrary, if the amino acid has a higher affinity for the paper than
the solvent, it will tend to “stick” to the paper and travel slower than the solvent front. It is these
differences in the amino acid affinities that lead to their separation on the paper. Hence, amino acids
have unique Rf values. The difference in Rf values can be accredited to the R-group side chain of the
amino acid (Clark, 2007).

Table 5.1. Data on the Rf value of hydrolysates and standard amino acids using paper chromatography.

Sample Distance Travelled, cm Rf Possible identity

Solvent 7.8
Amino Acid standards
A1-Gly 1.6 0.205 ---
A2-Val 4.0 0.513 ---
A3-Ala 2.5 0.321 ---
A4-Phe 4.5 0.577 ---
A5-Leu 5.4 0.692 ---
Sample
1a 2.5 0.321 Alanine
1b 5.3 0.679 Leucine
2a 1.5 0.192 Glycine
2b 4.6 0.590 Phenylalanine
 Figure 5.2. The chromatogram for the paper chromatography.

As shown on Table 5.1, each amino acid standard was also analyzed using paper
chromatography to determine their respective Rf value which will be used to determine the component
of the dipeptide sample. As shown on Figure 5.2, the first five where the amino acid standards followed
by the 2 hydrolysates which contains two amino acids. Knowing the values of the Rf value of the samples
H1 and H2, their corresponding amino acid component was determined by comparing their Rf value to
the Rf value of the amino acid standards. We found out that H1 has an amino acid component of Alanine
and Leucine due to their Rf value of 0.321 and 0.679 respectively while H2 has an amino acid component
of Glycine and Phenylalanine due to the Rf value of 0.192 and 0.590.

After the composition of the dipeptide was identified, the amino acid sequence must be
determined to know the exact identity of the dipeptide. Sequential analysis of amino acids is best
initiated by analysis of terminal amino acids. In this exercise, N-terminal identification of the dipeptide
was done using Sanger’s method. The sanger’s method uses 1-fluoro-2,4-dinitrobenzene reagent to
form 2,4-dinitrophenyl derivatives. The α-amino group of the n-terminal amino acid must be
deprotonated for the reaction to take place. The reagent reacts similarly with ε-NH2 group of lysine,
imidazole group of histidine, and phenolic group of tyrosine (Berg et al., 2012). If either lysine, histidine,
or tyrosine is part of a peptide sequence as the N-terminal, the reaction of these amino acid with
Sanger’s reagent are illustrated below respectively.

Lysine

Histidine

Tyrosine

On the other hand if lysine, histidine and tyrosine is part of a peptide chain as carboxyl and
internal residue, the following reactions are obtained with Sanger’s reagent.

Lysine

Histidine
 Tyrosine

Table 5.2. Data on the Rf value of hydrolysates and standard amino acids using Thin Layer
Chromatography (TLC).

Sample Distance Travelled, cm Rf Possible identity

Solvent 7.9
Amino Acid standards
A1-Gly 4.5 0.570 ---
A2-Val 5.9 0.747 ---
A3-Ala 5.8 0.734 ---
A4-Phe 5.9 0.747 ---
A5-Leu 5.9 0.747 ---
Sample
Dipeptide 1 6.0 (Nearest at 2) 0.759 Valine or Leucine
Dipeptide 2 4.7 0.594 Glycine

Prior to the preparation of the DNP derivative, the pH was ensured for the reaction to proceed.
The pH was adjusted to around 8 using NaHCO3. NaHCO3 was used to keep the alpha-amino group
deprotonated which makes it suitable for DNP-dipeptide formation. NaOH was not used as the base to
adjust the pH since this experiments works on a dipeptide which can hydrolyze in a present of a strong
base like NaOH. NaHCO3 results to produce water and CO2 when reacted to an acid.

HCO3- + H+ → H2CO3 → H2O + CO2 Reaction 5.2

Based on the Rf values obtained, dipeptide 1 was said to be close to the amino acid standard
no. 2 which was valine or it could be a Leucine and dipeptide 2 has the Rf value close to the amino acid
standard glycine. However, for dipeptide 1, for paper chromatography, has no valine as an amino acid
component, refer at Table 5.1. Dipeptide 1 has leucine and alanine as the amino acid component
according to the paper chromatography. Therefore there is a source of error in the analysis especially on
the TLC since the amino acid standards has almost the same Rf values which will give us a hard time to
determine the amino acid on the sample. Therefore, error was observed in the results. But since the Rf
value for valine is the same with Leucine, a component of the dipeptide 1, we can assume that it is
leucine rather than valine. Therefore, with the assumption, the structure would suggest that it is
leucine-alanine.

For dipeptide 2, the identified amino acid was glycine, suggesting that the structure of the
dipeptide is glycine-phenylalanine. This was confirmed by the TLC plate since this would result to the N-
terminal amino acid of the dipeptide.
Table 5.3. Enzymes used in sequence analysis of peptides.

Enzyme Specificity
Aminopeptidase Cleaves peptide bond involving the carboxyl
side of N-terminal amino acids
Carboxypeptidase Cleaves a peptide bond involving the amino
side of C-terminal amino acids
Thermolysin Cleaves a peptide bond involving amino side
of aromatic amino acids (phe, trp, tyr) and
amino acids with bulky non-polar side chains
(leu, ile, val)
Trypsin Cleaves a peptide bond involving the
carboxyl side of aromatic amino acid

If an unknown pentapeptide is treated with Sanger’s reagent similar in the experiment, but no
DNP acid was obtained, it can be concluded that the sample does not contain lysine, histidine, nor
tyrosine; and/or that the pentapeptide is cyclic.
Sample Calculations

1.) Retardation factor (Rf) in Paper Chromatography

Solvent front: 7.8 cm

Valine: 1.6 cm
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 1.6
𝑅𝑓= 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
= 7.8
= 0.205

2.) Retardation factor (Rf) in TLC

Solvent front: 7.9 cm

Glycine: 4.5cm
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 4.5
𝑅𝑓= 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
= 7.9
= 0.570
Summary and Conclusion

Proteins are made up of amino acids connected by peptide bonds. Primary structure of
proteins is the sequence of amino acids which dictates the form of the preceding structural levels and
the function of the protein itself. The amino acid sequence of a protein or peptide is a prerequisite for
the determination of the three dimensional structures of proteins thereby understanding its function,
identify it in a sample, and categorize its post translational modification.

In this exercise, the amino acid composition and sequence of two dipeptides were determined.
The hydrolyzed samples were used in paper chromatography to determine the identity of the amino
acid components in which ninhydrin was used as the visualizing agent. Based on the Rf values calculated,
dipeptide 1 contains alanine and leucine while dipeptide 2 contains glycine and phenylalanine.

After the composition of the dipeptide were identified, the amino acid sequence must be
determined to know the exact identity of the dipeptide. Sequential analysis of amino acids is best
initiated by analysis of terminal amino acids. In this exercise, N-terminal identification of the dipeptide
was done using Sanger’s method. The sanger’s method uses 1-fluoro-2,4-dinitrobenzene reagent to
form 2,4-dinitrophenyl derivatives. The obtained DNP derivatives were subjected to thin layer
chromatography alongside the amino acid standards. No visualizing agent needed for TLC. Based on the
Rf values, it was determined that the N-terminal for dipeptide 1 was leucine and glycine for dipeptide 2.

Based on the results obtained from the paper chromatography and thin layer chromatography,
it can be concluded that the structure the dipeptides were Leu-Ala for dipeptide 1 and Gly-Phe for
dipeptide 2.
References

 Berg J.M., J. L. Tymoczko, and L. Styler. 2012. Biochemistry. 7th ed. W.H. Freeman and

Company.

 Boyer, R. 2012. Biochemistry Laboratory: Modern theory and techniques. 2nd ed. New

Jersey: Pearson Education, Inc.

 Clark, J. 2007. Paper chromatography. Retrieved from

http://www.chemguide.co.uk/analysis/chromatography/paper.html.

 Nelson, D. L. and M. M. Cox. 2008. Lehninger Principles of Biochemistry. 5th ed.

New York: W.H. Freeman and Company.

 Smith, Y. 2000. Amino acids and protein sequence. Retrieved from

https://www.news-medical.net/life-sciences/Amino-Acids-and-Protein-Sequences.aspx.

 Stoker, H.S. 2010. General, Organic, and Biological Chemistry. 5th ed. USA: Brooks/Cole

Cengage Learning.

 Wilson K. and Walker, J. 2010. Principles and Techniques of Biochemistry and Molecular

Biology. 7th ed. Cambridge: Cambridge University Press.