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Penentuan Urutan Protein, Situs Pemotongan

Protease, Ukuran Protein, dan Muatan Protein

Meilia suherman

Proteins large biomolecules, or macromolecules,

consisting of one or more long chains of amino acid

Proteins perform a vast array of

functions within organisms,
catalysing metabolic
DNA replication,
responding to stimuli, and
transporting molecules from
one location to another.
Protein sequencing

Technique to find out the sequence of amino acids in

a protein

Sequencing methods
N-terminal sequencing (Edman degradation)
C-terminal sequencing
Prediction from DNA sequence
N-terminal sequencing

Edman degradation

Protein purification
Protein denaturation
Protein digestion
N-terminal labeling
Separation of labeled amino acid by chromatography
Detection through mass spectrometry
Data analysis
Protein isolation(purification)

(sodium dodecyl sulfate-poly
acryl amide gel)
2-Two dimensional gels

Protein of interest is
immobilized by being
absorbed onto a chemically
modified glass or by electro
blotting onto a porous
polyvinylidene fluoride (PVDF)
Protein hydrolysis(denaturation)

by heating a sample of the

protein in 6 Molar HCL up to
100-110 degrees Celsius for
24 hours or longer
It may degrade some amino
To avoid this
Thiol reagents or phenol are
- Performic acid for intra chain
or inter chain S-S bonds
Protein digestion

Use Endoproteinase Lys-C, CNBr, Pepsin or trypsin to

digest proteins into a population of peptides
Other enzymes include Glu-C and chymotrypsin
Add enzyme at 1:20 enzyme: protein ratio
incubate at room temperature for 6-9hrs
For better results use mixture of enzymes
N-terminal labeling

The Edman reagent, phenylisothiocyanate (PTC), is added to

the adsorbed peptide, together with a mildly basic buffer
solution of 12% trimethylamine
This reacts with the amine group of the N-terminal amino acid
The terminal amino acid can then be selectively detached by
the addition of anhydrous acid
The derivative then isomerises to give a substituted
phenylthiohydantoin which can be washed off and identified
by chromatography, and the cycle can be repeated
Chromatography is a technique
in which molecules are
separated based on volatility
and bond characteristics when
subjected to a carrier
Derivatives of amino acid can
be separated by
2-Gas chromatography
In gas chromatography (GC),
the mobile phase is an inert
gas such as helium

Mass spectrometry (MS) is an analytical technique that

measures the mass-to-charge ratio of charged particles
The MS principle consists of ionizing chemical
compounds to generate charged molecules or molecule
fragments and measuring their mass-to-charge ratios
Separated amino acid derivatives are analyzed by mass
MS procedure

A sample is loaded onto the MS instrument, and

undergoes vaporization
The components of the sample are ionized by one of a
variety of methods (e.g., by impacting them with an
electron beam), which results in the formation of
charged particles (ions)
The ions are separated according to their mass-to-
charge ratio in an analyzer by electromagnetic fields
The ions are detected, usually by a quantitative method
The ion signal is processed into mass spectra
Mass spectrometer
MS data analysis

first strategy for

identifying an unknown
compound is to compare
its experimental mass
spectrum against a library
of mass spectra
Standard solutions of
amino acids are also used
and the resulting pattern
is compared with standard
Limitations of Edman degradation

Need Pure Samples of Peptides

Requires 40-60 min / Amino Acid
Cant Analyze N-Terminally Modified Peptides

Most Reliable Sequencing Technique
C-terminal sequencing

The number of methods available for C-terminal

amino acid analysis is much smaller than the number
of available methods of N-terminal analysis. The
most common method is to add carboxypeptidases
to a solution of the protein, take samples at regular
intervals, and determine the terminal amino acid by
analyzing a plot of amino acid concentrations against
Prediction from DNA sequence

Protein sequence can also be determined indirectly

from the mRNA or, in organisms that do not have
introns (e.g. prokaryotes)
Sequence a short section, perhaps 15 amino acids
long, of the protein
Design primers from the amino acid sequence and
amplify the gene, sequence the gene and determine
the amino acid sequence of protein
Automatic protein sequencers

Automatic protein
sequencers are
designed that perform
the 3 steps(labeling,
separation and analysis
of amino acids) at a
time and analyze data
and give results
Applications of protein sequencing

Recombinant protein synthesis

Drugs production
Antibiotic production
Functional genomics
Determine the protein folding patterns
In bioinformatics
It plays vital role in proteomics
Used for the prediction of final structure, function and location of protein
To find out the location of gene coding for that protein
No generalizations can be made about the molecular
weights of biologically active peptides and proteins in
relation to their functions.
Naturally occurring peptides range in length from
two to many thousands of amino acid residues.
Even the smallest peptides can have biologically
important effects.
Many small peptides exert their effects at very low concentrations. For
example, a number of vertebrate hormones (Chapter 23) are small peptides.
These include oxytocin (nine amino acid residues), which is secreted by the
posterior pituitary and stimulates uterine contractions; bradykinin (nine
residues), which inhibits inflammation of tissues; and thyrotropin-releasing
factor (three residues), which is formed in the hypothalamus and stimulates
the release of another hormone, thyrotropin, from the anterior pituitary
gland. Some extremely toxic mushroom poisons, such as amanitin, are also
small peptides, as are many antibiotics. Slightly larger are small polypeptides
and oligopeptides such as the pancreatic hormone insulin, which contains
two polypeptide chains, one having 30 amino acid
residues and the other 21. Glucagon, another pancreatic
hormone, has 29 residues; it opposes the action of insulin.
Corticotropin is a 39-residue hormone of the anterior pituitary
gland that stimulates the adrenal cortex. How long are the
polypeptide chains in proteins? As Table 32 shows, lengths vary
considerably. Human cytochrome c has 104 amino acid residues
linked in a single chain; bovine chymotrypsinogen has 245
residues. At the extreme is titin, a constituent of vertebrate
muscle, which has nearly 27,000 amino acid residues and a
molecular weight of about 3,000,000. The vast majority of
naturally occurring proteins are much smaller than this, containing
fewer than 2,000 amino acid residues. Some proteins consist of a
single polypeptide chain, but others, called multisubunit proteins,
have two or more polypeptides associated noncovalently (Table
The individual polypeptide chains in a multisubunit protein may be identical or
different. If at least two are identical the protein is said to be oligomeric, and the
identical units (consisting of one or more polypeptide chains) are referred to as
protomers. Hemoglobin, for example, has four polypeptide subunits: two identical
chains and two identical chains, all four held together by noncovalent interactions.
Each subunit is paired in an identical way with a subunit within the structure of this
multisubunit protein, so that hemoglobin can be considered either a tetramer of four
polypeptide subunits or a dimer of protomers. A few proteins contain two or more
polypeptide chains linked covalently. For example, the two polypeptide chains of
insulin are linked by disulfide bonds. In such cases, the individual polypeptides are not
considered subunits but are commonly referred to simply as chains. We can calculate
the approximate number of amino acid residues in a simple protein containing no
chemical constituents by dividing its molecular weight by 110.
Although the average molecular weight of the 20 common
amino acids is about 138, the smaller amino acids predominate
in most proteins. If we take into account the proportions in
which the various amino acids occur in proteins (Table 31), the
average molecular weight of protein amino acids is nearer to
128. Because a molecule of water (Mr 18) is removed to create
each peptide bond, the average molecular weight of an amino
acid residue in a protein is about 128 - 18 = 110.
Ukuran Protein
Muatan Protein
In chemistry, a zwitterion (/tsvtr.a.n/ tsvit-r-eye-n; from German
zwitter [tsvt], meaning "hermaphrodite"), formerly called a dipolar ion,
is a neutral molecule with both positive and negative electrical charges.
(In some cases multiple positive and negative charges may be present.)
Zwitterions are sometimes called inner salts.
Amino acids are the best-known examples of zwitterions. These
compounds contain an ammonium and a carboxylate group, and can be
viewed as arising via a kind of intramolecular acidbase reaction: The
amine group deprotonates the carboxylic acid.
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To sequence an entire polypeptide, a chemical method devised by Pehr
Edman is usually employed. The Edman degradation procedure labels and
removes only the amino-terminal residue from a peptide, leaving all other
peptide bonds intact (Fig. 325b). The peptide is reacted with
phenylisothiocyanate under mildly alkaline conditions, which converts the
aminoterminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The
peptide bond next to the PTC adduct is then cleaved in a step carried out
in anhydrous trifluoroacetic acid, with removal of the amino-terminal
amino acid as an anilinothiazolinone derivative. The derivatized amino acid
is extracted with organic solvents, converted to the more stable
phenylthiohydantoin derivative by treatment with aqueous acid, and then
identified. The use of sequential reactions carried out under first basic and
then acidic conditions provides control over
the entire process. Each reaction with the aminoterminal amino
acid can go essentially to completion without affecting any of the
other peptide bonds in the peptide. After removal and
identification of the aminoterminal residue, the new amino-
terminal residue so exposed can be labeled, removed, and
identified through the same series of reactions. This procedure is
repeated until the entire sequence is determined. The Edman
degradation is carried out on a machine, called a sequenator, that
mixes reagents in the proper proportions, separates the products,
identifies them, and records the results. These methods are
extremely sensitive. Often, the complete amino acid sequence can
be determined starting with only a few micrograms of protein.
The length of polypeptide that can be accurately sequenced by the Edman
degradation depends on the efficiency of the individual chemical steps. Consider a
peptide beginning with the sequence GlyProLys at its amino terminus. If glycine
were removed with 97% efficiency, 3% of the polypeptide molecules in the solution
would retain a Gly residue at their amino terminus. In the second Edman cycle, 97%
of the liberated amino acids would be proline, and 3% glycine, while 3% of the
polypeptide molecules would retain Gly (0.1%) or Pro (2.9%) residues at their
amino terminus. At each cycle, peptides that did not react in earlier cycles would
contribute amino acids to an ever-increasing background, eventually making it
impossible to determine which amino acid is next in the original peptide sequence.
Modern sequenators achieve efficiencies of better than 99% per cycle, permitting
the sequencing of more than 50 contiguous amino acid residues in a polypeptide.
The primary structure of insulin, worked out by Sanger and colleagues over a period
of 10 years, could now be completely determined in a day or two.
Edman degradation, developed by Pehr Edman, is a method of
sequencing amino acids in a peptide.[1] In this method, the amino-
terminal residue is labeled and cleaved from the peptide without
disrupting the peptide bonds between other amino acid residues.
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