The virtual laboratory: Sequencing a small peptide
copyright © 1982 - 2006 David A Bender
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If you had discovered a small biologically active peptide, one of the first problems would be to determine its amino acid sequence, so that you could synthesise enough to be able to study its mechanism of action, develop ligand-binding assays, and investigate possible agonists and antagonists.
This program will allow you to perform the various studies needed to determine the sequence of a small peptide eight amino acids, by a combination of methods:
total acid hydrolysis and hplc to determine the amino acid content
enzymic cleavage into smaller fragments
Identifying the terminal amino acids
Each time you run the program you generate a new random peptide. When the peptide is generated, you are shown a sequence of 8 triplets of numbers. You should make a note of this sequence when it is displayed on the screen, in case you want to generate the same peptide again, to perform additional studies.
When you have deduced the sequence of your peptide, you should think about how you would go about ensuring that the sequence you have deduced is indeed that of the peptide you thought you were working with.
Total amino acid composition of the peptide
The first step in determining the amino acid sequence of a peptide is to find out which amino acids are present, and how many of each.
This is achieved by hydrolysing the peptide, then subjecting the hydrolysate to high pressure liquid chromatography to identify (and quantify) the amino acids present.
Peptide hydrolysis and sample preparation
The peptide is subjected to complete acid hydrolysis by heating overnight at 105C in 10 mol/L hydrochloric acid, in a sealed tube. This hydrolyses all peptide bonds, releasing free amino acids. However, it also results in destruction of tryptophan, so if you suspect that your peptide contains tryptophan (e.g. from its absorption spectrum) then you may need to use alkaline or enzymic hydrolysis.
The hydrolysate is then neutralised, and reacted with o-phthaldialdehyde and mercaptoethanol to form fluorescent iso-indole derivatives, before being injected onto an hplc column.
High-pressure liquid chromatography
Chromatography
depends on the partition of solutes between a stationary phase (usually a solid)
and a mobile phase (usually a liquid, but sometimes a gas). Solutes that are
more tightly adsorbed onto the stationary phase travel more slowly, and therefore
in column chromatography are eluted from the column later than solutes that
are less tightly adsorbed by the stationary phase. The time at which a compound
is eluted from the column is its retention time. The separation of different
solutes depends on their chemistry, the nature of the stationary phase and the
composition of the mobile phase.
High pressure liquid chromatography (hplc) is an extremely sensitive column method, using very small volumes of sample, and columns that are typically some 150 mm long and 4 - 5 mm in diameter. The stationary phase is in the form of very uniform spheres, typically some 5 µm in diameter, packed into the column. The mobile phase is pumped through the column under high pressure, and 5 - 10 µL of the sample is injected into the system just above the column. Sometimes the mobile phase has a constant composition; at other times its composition is varied though the elution period by mixing two different solutions to provide a gradient of mobile phase composition.
In some hplc systems the sample is derivatised before chromatography (as in this case, where it is the OPT-derivatives of the amino acids that are subjected to hplc); in other cases the eluate is derivatised (post-column derivatisation) to form coloured or fluorescent derivatives. In some cases the detection system is such that no derivatisation is required before or after chromatography.
The eluate then passes through a detection system, which may be electrochemical, or may rely on absorption of light, of fluorescence. In the case case of OPT-derivatives of amino acids, the detection system is fluorimetric.
The hplc that is used in this simulation uses a reverse phase (C18) column, with a 12.5 mmol/L sodium phosphate buffer at pH 7.2, and a gradient of 10 - 50% acetonitrile. The eluate is monitored using a fluorescence detector with excitation at 340 nm and emission at 445 nm.
The figure below shows typical traces for a mixture of standard amino acids (left) and a peptide hydrolysate (right), so that it is possible to identify which amino acids are present in the peptide.
Fluorescence versus absorption spectrophotometry
In absorption
spectrophotometry, light of s defined wavelength is shone through the sample,
and some is absorbed. The absorption of light is determined by the concentration
of solute and the path length. What is measured is the light that is transmitted
through the solution. This means that at low concentrations of solute, sensitivity
is low, since there is little difference between the intensity of the incident
light (that shone into the sample) and that of the transmitted light, because
little has been absorbed.
The light energy is used to excite electrons in the solute (this is why the wavelength is more or less specific for the compound under consideration). These electrons normally return to the resting state in a series of small jumps, losing the energy of excitation as heat, as shown on the left in the diagram below.
In some compounds, the energy of excitation is lost in a single quantum jump, as light, as shown on the right in the diagram below. Such compounds are said to be fluorescent - if they are excited with light of an appropriate wavelength, they will emit light of a longer wavelength (lower energy).
The light is emitted in all directions, and in a spectrophotofluorimeter what is measured is the light emitted at right angles to the incident light. This permits considerably greater sensitivity than absorption spectrophotometry, since, rather than looking for a small diminution in the intensity of the incident light, we are measuring light emitted without interference from the transmitted light. At least in theory, photomultipliers can detect a single photon.
Spectrophotofluorimetry also permits a higher degree of specificity than absorption spectrophotometry, since diffraction gratings can be used to select not only the wavelength of the incident light, but also that of the emitted light.
Cleavage into small peptide fragments
The enzymic method that will be used to determine the amino and carboxyl terminal amino acids in this simulation can only determine the terminal and penultimate amino acids with accuracy, so we need to break the peptide into smaller fragments that can be analysed.
Two enzymes can be used to cleave the peptide, and you should use both, to give you two sets of peptide fragments:
trypsin hydrolyses esters of basic amino acids (lysine and arginine)
chymotrypsin hydrolyses esters of aromatic amino acids (phenylalanine, tyrosine and tryptophan)
You then separate the peptide fragments by electrophoresis.
At any given pH, the side-chains of some amino acids will be charged, and others not.
This
means that the overall charge on a peptide varies with pH, and compounds can
be separated on the basis of their charge by the rate at which they migrate
in an electric field. This is the technique of electrophoresis.
In these studies the peptides to be separated are applied to the origin at the centre of a cellulose acetate strip that is then moistened with buffer and placed between electrodes with a 150 volt potential difference for 1 hour. After this the strip is dried and reacted with ninhydrin to stain the bands that contain the peptides. Selecting the appropriate pH to separate unknown peptides is a matter of trial and error, and in this exercise you may repeat the electrophoresis as often as you wish, with different buffers, until you achieve a separation. You could then separate large quantities of the peptides by electrophoresis, and use them for further studies.
Trypsin and chymotrypsin
Trypsin,
chymotrypsin and elastase are closely related enzymes. The peptide substrate
sits in a groove in the enzyme surface, with the peptide bond that is to be
hydrolysed over the catalytic site (shown here as a red circle).
The amino acid providing the carboxyl group of the bond to be cleaved sits in a pocket below the catalytic site.
In trypsin, which catalyses the hydrolysis of the esters of basic amino acids, the base of this pocket contains an acidic amino acid, aspartate, so providing a negative charge to attract in a basic amino acid side chain.
In chymotrypsin, which catalyses the hydrolysis of the esters of aromatic amino aids, the pocket is lined with small neutral amino acids, leaving room for a bulky aromatic side chain to enter.
In elastase, which catalyses hydrolysis of the esters of small neutral amino acids, the pocket contains two large neutral amino acids, so only a small side chain can fit in.
Identifying the terminal amino acids
Aminopeptidase hydrolyses the peptide bond of the amino acid at the amino terminal of a protein or peptide, releasing a free amino acid.
Carboxypeptidase hydrolyses the peptide bond of the amino acid at the carboxyl terminal of a protein or peptide, again releasing a free amino acid.
As the enzymes act, so they expose a new terminal that can act as a substrate. This means that a relatively brief incubation of a peptide one of the enzymes will result in release of mainly the terminal amino acid, with a small amount of the penultimate amino acid. A somewhat longer incubation results in the release of a large amount of the penultimate amino acid. Unfortunately, if incubations continue too long the results become very unclear, so this method can only be used for the terminal and penultimate amino acids.
The amino acids that are released by the action of aminopeptidase and carboxypeptidase action can be separated and identified by thin layer chromatography. Chromatography depends on the partition of solutes between a stationary phase (in this case a thin layer of silica gel) and a mobile phase (in this case a liquid). Solutes that are more tightly adsorbed onto the stationary phase travel more slowly; the distance they travel, relative to the solvent front (the Rf value) depends on the chemistry of the solutes, the nature of the stationary phase and the composition of the mobile phase.
In these studies you will be using silica gel thin layer plates. The samples are applied to origins about 1 cm above the base of the plate, and the chromatogram is developed by placing the thin layer plate in a tank containing solvent at the bottom ( in equilibrium with solvent vapour above), and allowing the solvent to rise up the plate by capillary action. With a 10 cm thin layer plate the solvent front rises to near the top of the plate within about an hour. After drying the plate it is sprayed with ninhydrin solution; the ninhydrin reacts with amino acids to yield a purple derivative.
In these studies you will determine the amino acids released from the original peptide, and the fragments produced by trypsin and chymotrypsin action, by incubation with aminopeptidase and carboxypeptidase, each acting for both 5 minutes (when mainly the terminal amino acid is released) and 10 minutes (when the penultimate amino acid is also released).
You will need to decide which standard amino acids to spot onto the chromatography plate in order to identify the terminal and penultimate amino acids - your results from hplc of the total hydrolysate should tell you which standards to use.
Some pairs of amino acids do not separate well in some solvents. There are 5 different solvents available for the chromatography exercise; if the first one you select does not give clear results, try another, until you are confident of your identification of the terminal and penultimate amino acids.
You should now be able to work out the amino acid sequence of the peptide fragments produced by trypsin and chymotrypsin action, and hence the amino acid sequence of the complete peptide.
