Post Laboratory Report on
Exercise 6 Dipeptide Sequence Determination
Aldrin John N. Alviar Chem 160.1 8L 2nd Semester, A.Y. 2016-2017
Groupmates: Bane, Danica Rabino, Mikaela Bernadette Santos, Maura Mercedes
Prof. Marvin Bilog
Table 6.1. Rf values of amino acid standards from paper chromatography. Amino acid standards Glycine Alanine Valine Leucine Glutamic acid Phenylalanine Methionine
Distance travelled by the sample (cm) 1.2 2.5 4.3 5.2 2.0 6.9 3.2
Distance travelled by the solvent (cm)
7.5
Retardation factor (Rf) 0.16 0.33 0.57 0.69 0.27 0.92 0.43
Table 6.2. Amino acid composition of the acid hydrolysed dipeptide sample. Amino acid sample
H1
A B C D
H2
Distance travelled by the sample (cm) 5.4 4.5 2.6 2.0
Distance travelled by the solvent (cm) 7.5
Retardation factor (Rf)
Identity of the amino acid
0.72 0.60 0.35 0.27
Leucine Valine Alanine Glutamic
Possible identity of the dipeptide sequence LeucineValine AlanineGlutamic acid
Sample Calculation: Rf glycine = =
ℎ () ℎ ()
1.2
= 0.16
7.5
Table 6.1 shows the distance travelled by the solvent and sample (i.e. amino acid standards) and their corresponding retardation factor (Rf). On the other hand, table 6.2 also shows the distance of samples and solvent and their corresponding Rf, also their possible identity of the dipeptide sequence. According to the data, leucine-valine and alanine-glutamic acid are the possible identity of the dipeptide due to the comparison of the Rf value of the amino acid standards. Also, the two peptide in the dipeptide sequence were labelled from A to D to differentiate and get the corresponding amino acid based on the standard. In addition to that, it can also be concluded that there is a direct relationship between the distance travelled by the sample and Rf value. As the distance travelled by the sample increases, the Rf value also increases. This can be proven by glycine and phenylalanine having lowest and highest Rf value, respectively. Glycine has the shortest distance travelled while phenylalanine has the longest distance travelled in the chromatogram.
Figure 6.1. Result of the Paper Chromatography of the labeled Dipeptide sequence (H1, H2) alongside with amino acid standards.
After paper chromatography, the samples were run in thin layer chromatography (TLC) to determine the N-terminal of the dipeptide sequence.
Table 6.3 Rf values of DNP-amino acid standards from TLC. Amino acid standards Methionine Phenylalanine Glycine Valine Leucine Glutamic acid Alanine
Distance travelled by the sample (cm) 6.4 8.1 2.2 1.3 5.5 0.6 4.1
Distance travelled by the solvent (cm)
9.4
Retardation factor (Rf) 0.68 0.86 0.23 0.14 0.59 0.06 0.44
Table 6.4. Rf value of the N-terminal of the dipeptide sequence and its corresponding identity. Amino acid sample
DP1 DP2
Distance travelled by the sample (cm) 5.1 3.9
Distance travelled by the solvent (cm) 9.4
Retardation factor (Rf)
Same Rf in amino acid standard
Identity of the DNP-amino acid
0.54 0.41
Leucine Alanine
DNP-Leucine DNP-Alanine
Table 6.3 shows the Rf values of amino acid standards from TLC. This data also shows the direct relationship of distance travelled by the sample and the Rf. On the other hand, table 6.4 shows the Rf values of the two dipeptide sequence (DP1 and DP2) and its corresponding DNPamino acid identity. The amino acid sample (H1 and H2) were added with 1-fluoro-2,4dinitrobenzene (FDNB) resulting to DP1 and DP2. After TLC, the resulted band only consists the N-terminal of the amino dipeptide sequence. The distance travelled by N-terminal of the two samples were also measured and compared to the amino acid standards to determine the identity of the N-terminal. The Rf value of the N-terminal of DP1 has 0.54 which is the closest to the Rf value of leucine while DP2 has 0.41 which is closest to alanine. Therefore, it can be concluded that the N-terminal of DP1 (H1+FDNB) and DP2 (H2+FDNB) is leucine and alanine, making it to be DNP-Leucine and DNP-Alanine, respectively.
Figure 6.2. Result of TLC of the DP1 (H1+FDNB) and DP2 (H2 +FDNB) alongside amino acid standards.
+
H1 (Leucine-Valine)
FDNB
DNP-Leucine-Valine
Figure 6.3. Reaction of the dipeptide sequence (H1) with FDNB.
Paper chromatography is used as a qualitative analytical chemistry technique for identifying and separating colored mixtures like pigments and amino acids. (Coppens, 2016). Ninhydrin is one of the common reagent to detect -amino acids, free amino acid, and carboxylic acid groups on proteins and peptides. Despite its side effects (i.e. impotence), it is mostly used in tests wherein detection of amino acids are involved. The reaction of ninhydrin with amino acids will form purple color due to the degradation of amino acids into aldehydes, ammonia, and CO2 with series of reactions (see figure 6.4). Eventually, according to Senese (2010), these series of reactions will produce hydrindantin, hen the partially reduced ninhydrin will condense with ammonia that leads to the production of the purple color in the amino acids.
Figure 6.4. Reaction mechanism of Ninhydrin with amino acid until purple colored product.
In running paper chromatography, it is advisable to run standards alongside of the sample to ensure that the tests were run under the same condition. Using of literature Rf value standards can yield error in the totality of the experiment. This is due to the presence of contamination, human error (i.e wrong execution of the method), and the quality of the materials being used. In addition, the immediate visual comparison between the bands in the chromatogram is much easier to distinguish.
In paper chromatography, comparing the Rf of the samples to the amino standards the dipeptide sequence of H1 is Leucine and Valine while H2 consists Glutamic acid and Alanine. Aside from this, we can also concluded that the components in dipeptide sequence or higher number of peptides separate in paper chromatography and each amino acid has its own distinct Rf value and can be determined when compared to the standards. Next in determining the components of the dipeptide sequence, determination of the Nterminal of the dipeptide sequence was also done through TLC. Thin-layer chromatography (TLC) is known as a chromatography technique used in the separation of the non-volatile mixtures. The principle behind this is the use of proper stationary and mobile phase making the different anal ytes ascent in the TLC plate at different rates, resulting to the separation (Lewis & Moody, 1989). The use of TLC also involved the use of correct visualizing agent depending on the purpose. According to Boulander (2012), the examples of visualizing agents use in TLC and its corresponding target group are bromcresol green: carboxylic acids; fluorescamine: primary amine detection; iodine vapour: hydrocarbons; ninhydrin: amino acid; rhodamine 6G: lipids. It varies depending on the type of molecules being analysed in the chromatography. In the second extraction, ether phase was recovered instead of the aqueous phase since the addition of hydrocholoric acid cleaves the N-terminal to the rest of the dipeptide with cation. Then, addition of ether was done in the mixture to remove the non-polar N-terminal DNP-amino acid due to the presence of large hydrophobic group in the DNP (i.e. benzene). The other amino acids in the sequence now contains cation and is available in the aqueous phase of the mixture. In thin layer chromatography, the ether phase was run and separate having different Rf values. The Rf values of DP1 and DP2 were compared to the amino acid standard. DP1 has the closest value to leucine while DP2 to alanine. It can be concluded the N-terminal amino acid of H1 and H2 (result of paper chromatography) are leucine and alanine, respectively. Apart from chromatography, there are also other techniques in determining the protein sequence of dipeptide or polypeptide. It can be chemical techniques or enzymatic processes each serving different cleavage styles and specific recognition of group. Example in chemical techniques are Cyanogen bromide, Sanger s reagent which uses fluorodinitrobenzene and Edman Degradation which utilizes phenyl isothiocyanate. On the other hand, the use of enzymes digest peptide with a higher degree of specificity (see table 6.5). ’
Table 6.5. Different enzymes and its specificity of cleavage procedures for sequence analysis. Enzyme Trypsin Chymotrypsin
Cleavage site Carboxyl side Carboxyl side
Thermolysin
Amino side
Pepsin
Amino side
Aminopeptidase Carboxypeptidase
Carboxyl side Amino side
Recognition site Basic amino acids (arg,lys) Aromatic amino acids and leucine Aromatic amino acids and bulky non-polar side chains amino acids Aromatic amino acids, acidic amino acids, isoleucine N-terminal C-terminal
By the use of these enzymes, cleaving the peptides at specific sites to determine the specific arrangement of amino acids can be done (Touchstone, 1992).
References: Badani, H., Freeman, T. 2011. PepDraw. Wimley Laboratory. Tulane University. New Orleans, Louisiana. United States.
Boulander, W. (2012). Thin Layer Chromatography. School of Chemical Sciences, Roger Adams Lab Penthouse. 600 S. Mathews Avenue, Urbana, IL 61801. Retrieved on http://scs.illinois.edu/hpl/tlc.php. Coppens, T. June 15, 2016. What is Paper Chromatography and How Does it Work? Owlcation. Retrieved from https://owlcation.com/stem/What-is-Paper-Chromatography-andHow-does-it-Work Harry W. Lewis and Christopher J. Moody (1989). Experimental Organic Chemistry: Principles and Practice (Illustrated ed.). WileyBlackwell. pp. 159 –173. Polypeptides and Proteins. [PDF file]. Retrieved from http://www.hull.ac.uk/php/chsanb/PP/POLYPEPTIDES%20AND%20PROTEINS.pdf
Senese, F.(2010). What is a simple test for the presence of amino acids?. Retrieved April 11, 2015. Retrieved from http://antoine.frostburg.edu/chem/senese/101/organic/faq/aminoacid-test.shtml. Touchstone , J.C. 1992. Practice of Thin Layer Chromatography. 3 rd Edition. NY.