Protein Definition

Proteins are large biological molecules consisting of one or more chains of amino acids. Proteins perform a vast
array of functions within living organisms, including catalyzing metabolic reactions, replication DNA, responding to
stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their
sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results
in folding of the protein into a specific three-dimensional structure that determines its activity.
A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between
the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined
by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard
amino acids; however, in certain organisms the genetic code can include selenocysteine and in certainarchaea
pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified
by posttranslational modification, which alters the physical and chemical properties, folding, stability, activity, and
ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be
called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often
associate to form stable protein complexes.
Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of
organisms
and
participate
in
virtually
every
process
within cells.
Many
proteins
are enzymes that catalyze biochemical reactions and are vital to metabolism. Proteins also have structural or
mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system
of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell
adhesion, and the cell cycle. Proteins are also necessary in animals' diets, since animals cannot synthesize all the
amino acids they need and must obtain essential amino acis from food. Through the process of digestion, animals
break down ingested protein into free amino acids that are then used in metabolism.
Proteins may be purified from other cellular components using a variety of techniques such
as ultracentrifugation, precipitation, electrophoresis, and chromatography; the advent of genetic engineering has
made possible a number of methods to facilitate purification. Methods commonly used to study protein structure and
function include immunehistochemistry, site-directed mutagenesis, nuclear magnetic resonance and mass
spectrometry

protein occurrence
We have used polyclonal antibodies against fusion proteins produced from cDNA fragments of a meiotic
chromosome core protein, Cor1, and a protein present only in the synapsed portions of the cores, Syn1, to detect the
occurrence and the locations of these proteins in rodent meiotic prophase chromosomes. The 234 amino acid Cor1
protein is present in early unpaired cores, in the lateral domains of the synaptonemal complex and in the
chromosome cores when they separate at diplotene. A novel observation showed the presence of Cor1 axial to the
metaphase I chromosomes and substantial amounts of Cor1 in association with pairs of sister centromeres. The
centromere-associated Cor1 protein becomes dissociated from the centromeres at anaphase II and it is not found in
mitotic metaphase centromeres. The extended presence of Cor1 suggests that it may have a role in chromosome
disjunction by fastening chiasmata at metaphase I and by joining sister kinetochores, which ensures co-segregation at
anaphase I. Two-colour immunofluorescence of Cor1 and Syn1 demonstrates that synapsis between homologous
cores is initiated at few sites but advances rapidly relative to the establishment of new initiation sites. If the rapid
1

advance of synapsis deters additional initiation sites between pairs of homologues, it may provide a mechanism for
positive recombination interference. Immunogold epitope mapping of antibodies to four Syn1 fusion proteins places
the amino terminus of Syn1 towards the centre of the synaptonemal complex while the carboxyl terminus extends
well into the lateral domain of the synaptonemal complex. The Syn1 fusion proteins have a non-specific DNA
binding capacity. Immunogold labelling of Cor1 antigens indicates that the lateral domain of the synaptonemal
complex is about twice as wide as the apparent width of lateral elements when stained with electron-dense metal
ions. Electron microscopy of shadow-cast surface-spread SCs confirms the greater width of the lateral domain. The
implication of these dimensions is that the proteins that comprise the synaptic domain overlap with the protein
constituents of the lateral domains of the synaptonemal complex more than was apparent from earlier observations.
This arrangement suggests that direct interactions might be expected between some of the synaptonemal complex
proteins.

Classification of Proteins and Their Functioons

Class of
Protein
Structural

Contractile

Transport

Storage

Hormone

Enzyme

Protection

No

Function in the body

Examples

structura Collagen is in tendons and

cartilage.
.Keratin is in hair, skin, wool,
and nails.
Move muscles.
Myosin and Actin contract
muscle fibers.
Carry essential substances Hemoglobin transports oxygen.
throughout the body.
Lipoproteins transport lipids.
Casein stores protein in milk.
Store nutrients.
Ferritin stores iron in the spleen
and liver
Regulate
body nsulin regulates blood glucose
metabolism and nervous level.
system.
Growth hormone regulates body
growth.
Catalyze
biochemical Sucrase catalyses the hydrolysis
reactions in the cells.
of sucrose. Trypsin catalyses the
hydrolysis of proteins.
stimulate
Recognize and destroy Immunoglobulins
foreign substances.
immune
responses.
Provide
components.

In terms of structure, proteins can also be classified as:

Simple Proteins If they yield only amino acids when they are hydrolyzed.
Conjugated Proteins If they yield amino acids and additional products when hydrolyzed.

On a nutritional basis, proteins are classified as:

Complete If they supply all the essential amino acids.
Incomplete If they are deficient in one or more essential amino acids.

Proteins show four structural levels namely, primary, secondary, tertiary and quaternary.

The linear sequence of amino acids in a polypeptide chain represents the primary structure. The enzyme
ribonuclease and the protein myoglobin function only in their primary structure.

If the polypeptide chain is coiled into a spiral or helix to have a three-dimensional structure, then it is called
secondary structure. e.g., keratin of skin.

Levels of Structure in Proteins

If the helical polypeptide molecule is folded on itself assuming a complex but specific structure such as
spherical or rod like, then it is called tertiary structure. e.g., globulins of blood.

Some proteins have two or more polypeptides, each with primary, secondary and tertiary structures then it is
called quaternary structure. e.g., Insulin and hemoglobin.

protein isolation and purification

The first step in protein purification involves a cell disruption step. The method of choice
depends on the type of cell. In general, animal cells are easier to disrupt than bacteria, yeast orplant cells. The table
below summarizes some of the methods. This list is by no meanscomplete, as there are as many methods of
disruption as there are types of cell.
Cell Type
Bacteria

Method
French press

Bacteria

Sonication

Comment
Shearing forces disrupt cell
wall as the cells are forced
through a small opening under
very high pressure. Not
practical for large volumes
Disruption of cell walls by
shearing and
Cavitation
Cell wall sheared through
abrasion with
glass beads

Bead Mill

Animal Cells

Blender

Plant Cells
Bacteria
Spores

Blender

Bacteria

Lysis

Bacteria
Yeasts

Lysis

Homogenization of tissue or
cells will
disrupt cell walls
Glass beads (100-200 m)
are used to
disrupt some bacteria, plant
cells and
bacterial and fungal spores
Solubilization
of
cell
membranes by
treatment with lysozyme and
EDTA; Grampositive
bacteria are more susceptible
than
Gram-negative bacteria.
Solubilization
of
cell
membranes with an
organic solvent such as
toluene

.
.
.

Concentration and partial fractionation of a protein extract

The salting-out technique of protein purification is mainly dependent on the hydrophobic character of the protein.
The salt is dissolved into the solution containing the protein. Water will solvate the added salt ions,decreasing the
solvation of the protein itself. This decrease in solvation exposes the hydrophobic regions of the protein, which then
interact with each other to form aggregates that will precipitate. In general, higher molecular weight proteins will
precipitate out at lower salt concentrations. For this laboratory experiment, we will use ammonium sulfate. The
optimum concentration of ammonium sulfate required to precipitate the protein of interest is determined by adding
increasing amounts of the ammonium sulfate and saving the precipitate for further analysis. A table is included in
this handout for preparing protein solutions of different concentrations of ammonium sulfate. A disadvantage of this
method is the high amount of salt that must be removed from the precipitate. To remove the salt from the protein
sample, we will use both dialysis and gel filtration chromatography.
Sample: Bovine serum albumin and lysozyme in water
Procedure: Ammonium sulfate precipitation
1.Pipet 3.0 mL of protein solution into a 50 mL centrifuge tube labeled 0-50 (be sure to put your name on the tube as
well).
2. Weigh out the required amount of ammonium sulfate for 50% saturation at 0C (see table).
The initial concentration of ammmonium sulfate is 0%.
3. Add 1/3 of the ammonium sulfate to the 50 mL centrifuge tube, swirl the tube and allow to dissolve. Repeat with
the remaining portions of ammonium sulfate.
5

4. Place the centrifuge tube on ice for 15 minutes.

5. Balance your tube with another students tube. Centrifuge for 10 minutes at 10,000 x gravity.
Remember the position your tube was placed in the rotor.
6. Label a 15 mL tube 50-70. Decant the supernatant from the 50 mL centrifuge tube into the 15 mL tube marked
50-70. Measure the volume of the 50-70 supernatant in the 15 mL tube and save for step .
7. Transfer the supernatant from the 15 mL tube labeled 50-70 into the 50 mL centrifuge tube .
8. Weigh out the required amount of ammonium sulfate for 70% saturation at 0C
Remember the initial concentration of ammonium sulfate is 50%.
9. Add 1/3 of the ammonium sulfate to the 50 mL centrifuge tube, swirl the tube and allow to dissolve. Repeat with
the remaining portions of ammonium sulfate.
10. Repeat steps 4 and 5.
11. Decant the supernatant from the 50 mL centrifuge tube. Discard the supernatant into the waste container.

Generic outline for protein purification

In general, protein purification entails essentially five types of steps: 1) efficient extraction from biological material;
2) separation from non-protein components (nucleic acids and lipids); 3) precipitation steps, initially to recover the
bulk protein from a crude extract, followed by preliminary resolution into manageable fractions; 4) use of ionexchange chromatography/size fractionation or hydrophobic chromatography columns to further separate the target
protein-containing fraction from the bulk protein; 5) a more refined set of steps including an affinity matrix to
enable recovery of the target protein in a highly purified state along with a high yield. A variety of agarose-based
matrices with immobilized reactive dyes, covalently bound nucleotides, metals and numerous other ligands are
commercially available (supplied by Sigma, Amicon, etc.).
In order to evaluate the progress of purification, a convenient assay procedurebased on enzymatic activity or some
other easily monitored property specific to the proteinshould be available. A spectrophotometric or colorimetric
method for enzymatic activity measurement is most convenient and a progressive increase in specific activity (for
enzymes, activity in units /mg protein) is an excellent indicator of the efficacy of the purification step. For proteins
lacking a readily measurable biological activity, it may be feasible to use an immunochemical procedure such as
western blotting or ELISA (Enzyme-Linked-Immuno-sorbent Assay), provided suitable antibodies are available. In
this case, electrophoretic resolution of the protein population in samples at each stage of purification will be
required.

Purification of native proteins

While purification of the native proteins is a challenging exercise, several reliable approaches have stood the test of
time. Compared to soluble proteins, membrane-bound. proteins are more difficult to purify. Solubilization of
membrane proteins can be achieved by the use of detergents but removal of the detergent is necessary for subsequent
analytical manipulations. A detailed treatment of the properties of various detergents and their applications is
available in reference 1. In the following a representative procedure for purification of soluble Neurospora proteins is
outlined.
1. Preparation of crude extracts: Efficient extraction of the total protein from the starting material is vital for success
of any purification procedure. Complete disruption of cells and release of contents from cellular debris is the most
important step in the process. For purification of Neurospora proteins in the native state, the first step involves the
extraction of bulk protein fraction from mycelial cells. All steps in the procedure are carried out at 4C to minimize
protein degradation. Mycelial cultures are grown for 18 to 20 h in a medium conducive to optimal production of the
6

target protein, harvested, lyophilized and stored at 70oC. Ten to 20 g of lyophilized mycelial powder is suspended
in 10 volumes of an extraction buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM -mercaptoethanol or
dithiothreitol) and the mixture is stirred for 45 min in the cold room. The presence of EDTA serves to inhibit protease
action and -mercaptoethanol (or DTT) is necessary for maintenance of a reducing environment. This slurry is
homogenized using a glass homogenizer and the homogenate is centrifuged at 12 000 x g for 20 min (to remove
cellular debris) in a refrigerated Centrifuge. The pellet is discarded and the supernatant is used in subsequent steps.
At this stage it may prove helpful to add a mixture of protease inhibitors (Complete cocktail: Roche or Sigma) if the
target protein is suspected to be unstable. [Note: Nucleic acids can be removed from the extract by addition of
protamine sulfate to a final concentration of 0.2%, while stirring. The precipitated nucleic acids are removed by
centrifugation. For most purposes, nucleic acid removal is not necessary; the precipitate may also bind the protein of
interest].
2. Precipitation of proteins: Several methods are available for precipitation of proteins utilizing changes in pH and
temperature, or addition of salts and organic solvents. Ammonium sulfate is the most commonly used precipitant for
salting out of proteins. At saturation (3.9 M at 0oC and 4.04 M at 20oC) it precipitates most proteins and protects
proteins in solution from denaturation and bacterial growth. To the supernatant from step
1, sufficient solid (NH4)2SO4 (Ultrapure reagent or Enzyme grade) is added to achieve 40% saturation [See Ref.1
for Table showing relationship between (NH4)2SO4 concentration and % saturation]. To avoid surface denaturation,
the solution should not be stirred vigorously and (NH4)2SO4 should be added gradually, in small amounts, allowing
each successive batch to dissolve completely before addition of the next. The precipitated protein is removed by
centrifugation at 12 000 x g for 10 min and to the supernatant more (NH4)2SO4 is added to yield 80% saturation.
The fraction of precipitated proteins between 40 and 80% saturation is recovered by centrifugation, resuspended
gently in 5 to 10 ml of a suitable buffer (e.g. 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 10 mM MgCl2) and dialyzed in
the cold room against several, 4-L changes of the same buffer over a 16-h period to remove residual (NH4)2SO4.
The dialyzed suspension is then centrifuged at 12 000 x g for 10 min to remove insoluble particulate matter and the
supernatant is tested for the presence of the target protein (pX).
3. Ion-exchange chromatography: The dialyzed fraction is applied to a 16 mm x 30 cm column packed with an
anion-exchanger, DEAE-cellulose (Sigma Fast Flow Fibrous DEAE Cellulose) or DEAE-Sepharose, previously
equilibrated against the above-mentioned dialysis buffer. The column is connected to a Pharmacia P-1 pump and a
Frac-100 fraction collector and is washed with ~60-100 ml of buffer to remove unbound proteins. The protein
fraction bound to the matrix (including the target protein) is eluted with 150 ml of a linear 0 to 1.75 M NaCl or KCl
gradient, prepared in the same buffer, generated by a Pharmacia GM-1 gradient mixer. [Note: See instructions for
column packing in Ref. 2].
Alternatively, the fraction can be chromatographed by passage through a Mono Q anion-exchange column (HR 5/5)
attached to a Pharmacia Fast Protein Liquid Chromatography (FPLC) system. The sample is clarified by
centrifugation, loaded onto the column and eluted with a discontinuous gradient consisting of steps of 0 to 0.3 M, 0.3
to 44 M and 0.44 to 1.2 M NaCl, as an example. The fractions enriched in pX are pooled and centrifuged at 12 000 x
g for 10 min to remove insoluble material. The supernatant is dialyzed for 4 to 6 h against 20 mM Tris-HCl, pH 7.5
to remove NaCl, brought to 80% (NH4)2SO4 and the precipitated fraction is resuspended in 20 mM Tris-HCl, pH
7.5. If necessary, residual salt can be removed by passage through a small gel filtration column. For applications
requiring cation-exchange columns carboxymethyl (CM)-cellulose or CM-Sepharose can be employed.
4. Separation by hydrophobic interaction: The protein sample from either of the preceding stepsin 30% saturated
(NH4)2SO4can be applied to a column containing a hydrophobic matrix, such as Phenyl- or Octyl Sepharose, preequilibrated with 20 mM Tris-HCl (pH 7.5) containing 30% saturated (NH4)2SO4. The column is washed
successively with buffer containing 25%, 20%, 15%, 10% and 5% saturated (NH4)2SO4 . Finally the protein is
eluted with the original buffer. The fractions containing pX are combined and concentrated using Centricon filter
concentrators (10 or 30 kDa cutoff).
5. Affinity chromatography: An example of affinity chromatography for separation of NAD(P)-binding proteins is
the use of agarose or sepharose-bound reactive dyes. The protein sample is loaded onto a 10 mm x 20 cm column
packed, for instance, with Cibacron Blue-agarose 3GA resin (immobilized on cross-linked 4% beaded agarose, type
3000 CL; Sigma), pre-equilibrated with a buffer. Proteins bound to the matrix are eluted with ~80 ml of a linear 0 to
1.75 M NaCl gradient, fractions with pX are pooled and centrifuged to pellet insoluble proteins. The supernatant is
7

concentrated and electrophoresed in SDS-polyacrylamide gels to determine the state of purity of the protein. Other
types of affinity matrices, such as ADP-, ATP-agarose or Con A-Sepharose (containing immobilized adenosine
nucleotides or Concanavalin A) can be employed to capture ATP/ADP-binding proteins or glycoproteins,
respectively.
[Note: Selection of the appropriate combination of purification steps will depend on the properties of the target
protein. If it is a known protein, such as an enzyme that has been isolated and characterized from another organism, a
good starting point will be to try out some of the steps in the published procedure.]
The state of purity of the sample is judged by SDS-PAGE; the number of stained polypeptide bands will decrease
with progressive removal of contaminating proteins. Ultimately, a single, stained band should result, indicating a
nearly homogeneous sample. An efficient purification protocol should also result in high enough yield to justify the
effort involved. Ideally, no more than five or six purification steps should be required for purification of a given
protein. The entire process should be completed in four to five days as a small proportion of the target protein is
inevitably lost in the course of each step, due to denaturation, protease action and storage conditions. On starting
with ~20 g of lyophilized mycelium, the yield of inducible enzymes and environmentally-regulated proteins in the
vicinity of 5-10 mg is normal. Higher yields can be attained if losses during individual steps can be kept to a
minimum. The final homogeneous preparation should be stored in a concentrated solution (~ 10 mg/ml) at 20o C or
70oC, along with 30% glycerol for maximum stability. All of the above steps can be scaled-up.
Representative purification procedure:
Isolation of the protein, resulting from fusion of the coding sequence with the metal-binding tag sequence, involves
the following: (1) growth and induction of transformed bacterial cultures; (2) lysis of cells in a suitable buffer
containing a detergent and lysozyme; (3) DNase and RNase treatment for removal of the nucleic acid fraction; (4)
passage of the extract through an affinity resin (Ni+-NTA agarose for His-tagged proteins) and (5) elution of bound
protein. Many of the
so-called one-step purification systems, offered by commercial suppliers, frequently require multiple steps for
recovery of a homogenous product.

Expression and purification of the fusion protein:

The initial inoculum is prepared in SOB-amp medium (20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 0.186 g KCl,
0.01M MgCl2 per litre containing 50 g/ml ampicillin) by addition of 3-5 l of the stock culture and incubation at
35oC for 4-6 h while shaking. Two-ml aliquots of the culture are used to inoculate fresh 50-ml SOB-amp medium
which is incubated at 30oC while shaking for 12 h. For expression, 20 to 30 ml/litre of this culture is used to
inoculate SOB-amp medium, yielding a starting OD600nm of 0.07 to 0.11. Incubation is resumed in the shaker at
30oC for 3.5 to 4.5 h, until OD600 of 0.35 to 0.45 is attained. Addition of 1 2 mM IPTG during the last hour of
growth can, often though not invariably, enhance the yield of the fusion protein. The culture is harvested by
centrifugation and the pellet frozen at 20oC, thawed on ice, resuspended in cold Extraction Buffer (50 mM
phosphate buffer, 300 mM NaCl, pH 8.0, containing 20 mM imidazole and 10 mM -mercaptoethanol) and the cells
are homogenized using a glass homogenizer. A detergent such as Triton X-100 is added to a final concentration of
0.01% followed by freshly prepared lysozyme (100 g/ml) and the mixture shaken at room temperature for 20 min.
Next, DNase (5 g/ml) and RNase (6.25 g/ml) are added and shaking continued for an additional 30 min
and the crude cell extract is centrifuged at 12 000 g for 20 min. The resulting supernatant is clarified by recentrifugation, mixed with 1 to 1.5 ml of the affinity resin (Ni +-NTA agarose; Qiagen), pre-equilibrated with 20 mM
imidazole in phosphate buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The mixture is shaken for 90 min at 4oC
8

to allow binding of the His-tagged protein. The resin is poured into a column and washed in two steps: first with the
above phosphate buffer containing 20 mM imidazole, followed by two washes with 40 mM imidazole to remove the
non-specifically bound protein fraction.
Elution is conducted in a batchwise manner with 2 ml of 150 mM, 3 ml of 200 mM and 3 ml of 250 mM imidazole.
Alternatively, a linear gradient of 100 mM to 400 mM imidazole can be employed if larger volumes of the affinity
resin and the supernatant are to be used. The eluted fractions are monitored for the presence of the fusion protein by
SDS-PAGE. The fractions containing high levels of the fusion protein are pooled and concentrated using Centricon
filters (with a cutoff of 30K or 10K depending on the size of the protein). The filters are washed with 1 ml of
appropriate buffer (e.g. 20 mM Tris-HCl, pH 7.5) to remove residual imidazole. The purity of the protein should be
checked by SDS-PAGE and its identity verified by western blotting, if specific antibodies are available or by
measurements of specific activity if the expressed protein is an enzyme. When contaminating proteins are present,
additional purification steps including size fractionation or ion-exchange chromatography will be necessary.
Problems are often encountered during the initial cell lysis step resulting in incomplete liberation of the expressed
protein from bacterial cell debris. Therefore, it is essential to explore different methods of cell breakage, such as
sonication, freeze-thaw cycles, etc. to optimize the yield. A number of commercial cell-lysis solutionsclaimed to
be suitable for bacterial, yeast or mammalian cells are marketed by various suppliers but home-made mixtures are
just as satisfactory. A sizeable fraction of the protein can also be lost by formation of insoluble aggregates in the socalled inclusion bodies. Some of the aggregated protein may be recoverable by using one or more cycles of
denaturation-renaturation. However, the latter approach can lead to inactivation of the protein and reduced yield.
Low yields may also result from proteolytic degradation of the target protein during one or more of the purification
steps. Inclusion of protease inhibitors in the lysis buffer and/or subsequent steps is helpful.
While many eukaryotic proteins have been successfully expressed in bacterial hosts, recovery of biologically
active proteins is often difficult due to the lack of post-translational modifications that can occur only in a eukaryotic
intracellular milieu. As an alternative, Pichia pastoris cells can be used for expression of fungal proteins. The main
advantage is the ability of P. pastoris cells to perform appropriate post-translational modifications. However, many
proteins are not readily expressed in this system.
hydrolysis of proteins and determination of ammino acid sequence

Hydrolysis
Hydrolysis is done by heating a sample of the protein in 6 M hydrochloric acid to 100110 C for 24 hours or longer.
Proteins with many bulky hydrophobic groups may require longer heating periods. However, these conditions are so
vigorous that some amino acids (serine, threonine, tyrosine, tryptophan, glutamine and cystine) are degraded. To
circumvent this problem, Biochemistry Online suggests heating separate samples for different times, analysing each
resulting solution, and extrapolating back to zero hydrolysis time. Rastall suggests a variety of reagents to prevent or
reduce degradation, such as thiol reagents or phenol to protect tryptophan and tyrosine from attack by chlorine, and
pre-oxidising cysteine. He also suggests measuring the quantity of ammonia evolved to determine the extent
of amide hydrolysis.

Separation

The amino acids can be separated by ion-exchange chromatography or hydrophobic interaction chromatography. An
example of the former is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in
acid solution and passing a buffer of steadily increasing pH through the column. Amino acids will be eluted when the
pH reaches their respective isoelectric points. The latter technique may be employed through the use of reversed
phase chromatography. Many commercially available C8 and C18 silica columns have demonstrated successful
separation of amino acids in solution in less than 40 minutes through the use of an optimised elution gradient.

Quantitative analysis
Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will
form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol, ninhydrin can be used for this - it
gives a yellow colour when reacted with proline, and a vivid purple with other amino acids. The concentration of
amino acid is proportional to the absorbance of the resulting solution. With very small quantities, down to 10
pmol, fluorescamine can be used as a marker: this forms a fluorescent derivative on reacting with an amino acid.

10

Protein sequencing is a technique to determine the amino acid sequence of a protein, as well as which conformation
the protein adopts and the extent to which it is complexed with any non-peptide molecules. Discovering the
structures and functions of proteins in living organisms is an important tool for understanding cellular processes, and
allows drugs that target specific metabolic pathways to be invented more easily.

The two major direct methods of protein sequencing are mass spectrometry and the Edman degradation reaction. It is
also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is
known. However, there are a number of other reactions which can be used to gain more limited information about
protein sequences and can be used as preliminaries to the aforementioned methods of sequencing or to overcome
specific inadequacies within them.

Determining amino acid composition

11

It is often desirable to know the unordered amino acid composition of a protein prior to attempting to find the
ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to
distinguish between ambiguous results. Knowledge of the frequency of certain amino acids may also be used to
choose which protease to use for digestion of the protein. A generalized method often referred to as amino acid
analysis for determining amino acid frequency is as follows:
1. Hydrolyse a known quantity of protein into its constituent amino acids.
2. Separate the amino acids in some way.
N-terminal amino acid analysis

Sanger's method of peptide end-group analysis: A derivatization

reagent (DNFB), B total acid hydrolysis of the dinitrophenyl peptide

of N-terminal

end

with Sanger's

Determining which amino acid forms the N-terminus of a peptide chain is useful for two reasons: to aid the ordering
of individual peptide fragments' sequences into a whole chain, and because the first round of Edman degradation is
often contaminated by impurities and therefore does not give an accurate determination of the N-terminal amino
acid. A generalised method for N-terminal amino acid analysis follows:
1. React the peptide with a reagent which will selectively label the terminal amino acid.
2. Hydrolyse the protein.
3. Determine the amino acid by chromatography and comparison with standards.
There are many different reagents which can be used to label terminal amino acids. They all react with amine groups
and will therefore also bind to amine groups in the side chains of amino acids such as lysine - for this reason it is
necessary to be careful in interpreting chromatograms to ensure that the right spot is chosen. Two of the more
12

common reagents are Sanger's reagent (1-fluoro-2,4-dinitrobenzene) and dansyl derivatives such as dansyl
chloride. Phenylisothiocyanate, the reagent for the Edman degradation, can also be used. The same questions apply
here as in the determination of amino acid composition, with the exception that no stain is needed, as the reagents
produce coloured derivatives and only qualitative analysis is required, so the amino acid does not have to be eluted
from the chromatography column, just compared with a standard. Another consideration to take into account is that,
since any amine groups will have reacted with the labelling reagent, ion exchange chromatography cannot be used,
and thin layer chromatography or high pressure liquid chromatography should be used instead.

C-terminal amino acid analysis

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 addcarboxypeptidases to a solution of the protein,
take samples at regular intervals, and determine the terminal amino acid by analysing a plot of amino acid
concentrations against time.

Predicting protein sequence from DNA/RNA sequences

In organisms that do not have introns (e.g. prokaryotes) the amino acid sequence of a protein can also be determined
indirectly from the mRNA or the DNA that codes for the protein. If the sequence of the gene is already known, then
this is all very easy. However, it is rare that the DNA sequence of a newly isolated protein will be known, and so if
this method is to be used, it has to be found in some way. One way that this can be done is to sequence a short
section, perhaps 15 amino acids long, of the protein by one of the above methods, and then use this sequence to
generate a complementary marker for the protein's RNA. This can then be used to isolate the mRNA coding for the
protein, which can then be replicated in a polymerase chain reaction to yield a significant amount of DNA, which
can then be sequenced relatively easily. The amino acid sequence of the protein can then be deduced from this.
However, it is necessary to take into account the possibility of amino acids being removed after the mRNA has
been translated.

13

Reactions
As amino acids have both a primary amine group and a primary carboxyl group, these chemicals can undergo most
of the reactions associated with these functional groups. These includenucleophilic addition, amide bond formation
and imine formation for the amine group and esterification, amide bond formation and decarboxylation for the
carboxylic acid group.The combination of these functional groups allow amino acids to be effective polydentate
ligands for metal-amino acid chelates The multiple side-chains of amino acids can also undergo chemical
reactions.The types of these reactions are determined by the groups on these side-chains and are, therefore, different
between the various types of amino acid.

The Strecker amino acid synthesis

Chemical synthesis
Several methods exist to synthesize amino acids. One of the oldest methods begins with the bromination at the carbon of a carboxylic acid. Nucleophilic substitution with ammonia then converts the alkyl bromide to the amino
acid In alternative fashion, the Strecker amino acid synthesis involves the treatment of an aldehyde with potassium
cyanide and ammonia, this produces an -amino nitrile as an intermediate. Hydrolysis of the nitrile in acid then
yields a -amino acid.Using ammonia or ammonium salts in this reaction gives unsubstituted amino acids, while
substituting primary and secondary amines will yield substituted amino acids.Likewise, using ketones, instead of
aldehydes, gives ,-disubstituted amino acids.The classical synthesis gives racemic mixtures of -amino acids as
products, but several alternative procedures using asymmetric auxiliaries or asymmetric catalysts have been
developed.
14

At the current time, the most-adopted method is an automated synthesis on a solid support (e.g., polystyrene beads),
using protecting groups (e.g., Fmoc and t-Boc) and activating groups (e.g.,DCC and DIC).

Peptide bond formation

15

The condensation of two amino acids to form a dipeptide through a peptide bond

As both the amine and carboxylic acid groups of amino acids can react to form amide bonds, one amino acid
molecule can react with another and become joined through an amide linkage. This polymerization of amino acids is
what creates proteins. This condensation reaction yields the newly formed peptide bond and a molecule of water. In
cells, this reaction does not occur directly; instead the amino acid is first activated by attachment to a transfer
RNA molecule through anester bond. This aminoacyl-tRNA is produced in an ATP dependent reaction carried out by
an aminoacyl tRNA synthetase.This aminoacyl-tRNA is then a substrate for the ribosome, which catalyzes the attack
of the amino group of the elongating protein chain on the ester bond.As a result of this mechanism, all proteins made
by ribosomes are synthesized starting at their N-terminus and moving towards their C-terminus.
However, not all peptide bonds are formed in this way. In a few cases, peptides are synthesized by specific enzymes.
For example, the tripeptide glutathione is an essential part of the defenses of cells against oxidative stress. This
peptide is synthesized in two steps from free amino acids.In the first step gamma-glutamylcysteine
synthetase condensescysteine and glutamic acid through a peptide bond formed between the side-chain carboxyl of
the glutamate (the gamma carbon of this side-chain) and the amino group of the cysteine. This dipeptide is then
condensed with glycine byglutathione synthetase to form glutathione.
In chemistry, peptides are synthesized by a variety of reactions. One of the most-used in solid-phase peptide
synthesis uses the aromatic oxime derivatives of amino acids as activated units. These are added in sequence onto the
growing peptide chain, which is attached to a solid resin support.The ability to easily synthesize vast numbers of
different peptides by varying the types and order of amino acids (using combinatorial chemistry) has made peptide
synthesis particularly important in creating libraries of peptides for use in drug discovery through high-throughput
screening.

16

Colour reaction of proteins

Terms

Definitions

Biuret
(procedure)

Test Add 5 drops of 1% CuSO4 to 4 ml of 5% NaOH. Divide into two

portions. Add 2ml of distilled water to one and 2ml of protein solution to
the other.

Biuret
(principle)

Test In alkaline medium copper from CuSO4 will form a coordination

complex with the peptide bond. A minimum of 3 peptides are required to
answer this test. Biuret test is not performed on urine because of presence
of peptide like linkage.

Ninhydrin
(procedure)

Test Boil 1ml of protein solution with 5 drops of ninhydrin reagent.

Ninhydrin
(principle)

Test This test is given by the free amino acids, small peptides and protein will
react to give purple color. Ninhydrin reacts with amino acids to form
hyrindantin and then it further forms Ruheuman's purple by reacting with
amonia and another ninhydrin. Imino acids give yellow color owing to
absence of alpha amino acids.

Xanthoproteic
Test (procedure)

To 1ml of protein solution add 1ml of concentrated HNO3. Boil for 30

seconds. Coo under tap water and add 2-3 ml of 40% NaOH.

Xanthoproteic
Test (principle)

This test is answered by aromatic amino acids like tyrosine and

tryptophan. Phenyl alanine gives a weak positive reaction. When a
preotein solution is heated with concentrated nitric acid, the benzene ring
under goes nitration to form yellow nitro derivatives. When treated with
NaOH the sodium salt formed is tense orange in color.

Millon's
(procedure)

Test Add 2-3 drops of mercuric sulphate in 10% H2SO4 to 1ml of protein
solution. Boil gently of flame for 30 seconds. Add 2-3 drops of 1%
sodium nitrate solution.

Millon's

Test Specific to phenolic group of thyrosine. The hydroxy benzene group of

17

(principle)

thyrosine reacts with Millon's reagent to form red colored complex.

Aldehyde
Reaction
(procedure)

Add 1 drop of 10% mercuric sulphate in 10%H2SO4 to 1 ml of protein

solution and 1 drop of formaline. Mix well. Add 2 ml of concentrated
H2SO4 along sides of test tube.

Aldehyde
Reaction
(principle)

The test indicates presence of indole group containing amino acid. The
oxidizing agents are HgSO4 and H2SO4 which oxidize indole nucleus of
tryptophan. This oxidized indole ring reacts with formaldehyde to give the
violet colored ring at the junction of two liquids.

Sakaguchi's Test To 3ml of protein solution and add 2 drops of Molisch's reagent. Then add
(procedure)
6 drops of alkaline hypobromite.

Sakaguchi's Test Test is answered by arganine. The guanidine group reacts with alpha
(principle)
napthol and sodium hypobromite to form deep red color.

Alkali
Labile To 3ml of protein solution in a boiling test tube. Add equal volume of
Reaction
40% NaOH. Boil on direct flame for at least two minutes. Then add 3-4
(procedure)
drops of lead acetate.

Alkali
Labile When a protein solution is boiled with an alkali, the sulfur of cysteine and
Reaction
cystine splits to form sodium sulfide. This forms black lead sulfate on
(principle)
acting
with
lead
acetate.
This test is not answered by methionine because the sulphur is involved in
a thioether linkage. This is not split by boiling with strong alkali.

Pauly's
(procedure)

Test To 2ml of protein solution, add diazotized sulphanilic acid followed by

1ml of 20% Na2CO3.

Pauly's
(principle)

Test Imidazole group reacts with diazotized aulphanilic acid to form highly
colored azocompouds. In alkaline medium this is red in color. Diazotized
sulphanilic acid is also called Van Den Berg's reaction.

18

Colour reaction of amino acid

Common amino acids have the general structure . They contain in common a central alpha carbon to which a
carboxylic acid group (-COOH) and amino group (-NH2) and a hydrogen atom are covalently bonded. In addition
the alpha carbon atom is bound to a specific chemical group, designated R and called the side chain, that uniquely
defines each of the 20 common amino acids. In solution at about pH 7,0 the amino group is protonated and is in its
ammonium ion form (-NH3+), carboxylic group is in its unprotonated or carboxylate ion form ( negatively charged
-COO-). Optical activity. The alpha carbon atoms of the amino acids, except glycine, are each linked to four different
chemical groups. This is the characteristic of an asymmetric carbon atom (chiral). This amino acids exist as
stereoisomeric pairs called enantiomers ( D and L ).However only L isomers are found in proteins. Amphoteric
properties. Amino acids are amphoteric molecule (dipolar ions), that is they have both basic and acidic groups.
1. Monoamino-monocaboxylic amino acids exist in aequeous solution as dipolar molecules, which means they
have both positive and negative charges and the overall molecules is electrically neutral.
2. At low pH, the carboxyl group accepts a proton and become uncharged, so that the overall charge on the
molecule is positive.
3. At high pH, the amino group loses its proton and becomes uncharged (neutral) , thus the overall charge on the
molecule is negative.pH at which an amino acid is electrically neutral is known as the isoelectric point for the
molecule and the symbol is pI.
In proteinj almost of the carboxyl and amino groups are combined on peptide linkage and are not available for
chemical reactions. Thus it is nature of the side chains that ultimately dictates the role an amino acid plays in
protein. It is useful to classify amino acids according to the properties of their side chain that is , whether are
nonpolar or polar uncharged, acidic or basic.

The chemical reactions of the amino acids are relatively numerous because of the different reactive groups that
are present in the same molecule. Aliphatic monoamino monocarboxylic acids give all reactons expected for
carboxyl and amino groups. The other amino acids give these reactions and in addition those reactions

19

characteristic of additional groups that may be present. Many of these reactions are also given by protein because
they contain these amino acids.

Reaction with Ninhydrin.

Amino acids react with ninhydrin ( triketohydrindene hydrate) to yield carbon dioxide, ammonia and aldehyde
containing one less carbon than the amino acid.
The reaction also yields a blue or purple color , which is useful for the colorimetric quantitative estimation of
amino acids . Proline, an imino acid, also react with ninhydrin, but gives a yellow color.
The reaction of ninhydrin with alpha- amino acids is as follows:

Millon reaction.
A red color is obtained when phenole compounds are heated with Hg(NO3)2
In nitric acid containing a trace of nitrous acid. Protein containing Tyrosine give this reaction:

20

Xanthoprotein reaction.
Aromatic amino acids (Phenylaalanine, Tyrosine, Tryptophan ) reacts with nitric acid on heating to give a yellow
and in alkaline solution orange compound.

Sakaguchi reaction.
Guanidines in alkaline solutions give a red color with reagent which contains alpha-naphtol and sodium
hypobromite. The reaction is given by Arginine and by proteins that contain this amino acids.
21

Adamkiewicz- Hopkins reaction.

With glioksalic acid in sulfuric acid a red violet color is obtained with Tryptophan in proteins.

Cystine and its reduction product, the sulfhydryl containing Cysteine after heating in alkaline solution give th a
brown precipitate ( PbS) with reagent which contain Pb2+.
Amino acids can be linked together in amide bonds through the carboxyl group of one and the amino group of
another to form a substituted amide bond which is termed a peptide bond or linkage.

22

The biuret assay.

When substances containing two or more peptide bonds react with alkaline copper sulfate a violet complex is
formed. The colored product is the result of coordination of peptide nitrogen atoms with Cu 2+.The amount of
product formal depends on the concentration of peptides.

Importance of isoelectric point in proteins:

The isoelectric point of a protein is the pH where the OVERALL charge on the protein is neutral. In isoelectric
focusing gel electrophoresis allows for the separation of protein purely on the basis of their charge characteristics. A
polyampholyte will migrate in an electric field like other proteins , if the protein have a net electric charge. If the gel
with a stable pH gradient covering a wide pH range, each polyampholyte molecule migrates to the position of its
isoelectric point and accumulates there. You can establish such a gradient by using a mixture of lowmolecular
ampholytes as the gel buffer. This method produces distinct bands of accumulated polyampholyte and can resolve
molecules with very small differences in isoelectric points.

Amino acids as ampholytes :

Ampholytes are molecules containing both acidic and basic groups.
All of the common amino acids found in proteins (Figure 5.3) are ampholytes because they contain a carboxyl group
(-COOH) that acts as an acid and an amino group (-NH2) that acts as a base.
23

As free amino acids, each amino acid has at least two pKa values (some have more because they have additional
acidic or basic groups).

The titration of an ampholyte generates a more complex plot of pH versus moles of acid (or base) added than are
obtained for a simple buffer with only a single ionizing species because the ionization of each acidic and basic group
of the ampholyte is represented by a step in the titration curve. In Figure 2.18, for example, there are two steps in the
titration curve of the ampholyte glycine, whereas in Figure 2.17 the titration curves of NH4+ and HCOOH have only
one step each. Figure 2.19 shows the fraction of each molecular species of glycine present in the solution as a
function of pH.

The presence of both acidic and basic groups in a single molecule means the molecule may exist in several different
charged states. For example, glycine can have a charge of +1, 0, or -1, depending on the pH of the solution in which
it is dissolved. The state at a net charge of zero arises when the basic amine group is charged +1 and the acidic
carboxyl group is charged -1.
Molecules containing a mixture of charges that result in the molecule having an overall charge of 0 are called
Zwitterions. The zwitterion form of the amino acid, glycine, is as follows:

The use of carrier ampholyte-free IEF (CAF-IEF) with ITP mobilization and conductivity detection in ITP
mode for preconcentration and analysis of amino acids is demonstrated. The analytical procedure
consists of three subsequent steps. In the first step, amino acids are continuously dosed from an
infinite volume reservoir by electromigration to the column, where a sharp, stationary neutralization
reaction boundary (NRB) is created in between acidic and basic primary electrolyte. Here, amino
acids are selectively focused (trapped), if their pI falls to the pH difference on both sides of the NRB
(pH gap). Amino acids create sharp rectangular zones, arranged according to their pI values. In the
second step, focused zones are mobilized. After accumulation of the detectable amount of amino
acids, dosing electrolyte in the infinite volume reservoir is changed for the mobilizing electrolyte. The
migration mode is changed from CAF-IEF to ITP and substances start to migrate toward the analytical
capillary. In the third step, analytes are transferred into the analytical column equipped with a
conductivity detector and are detected in the new leading electrolyte in an ITP migration mode. The
presented CAF-IEF-ITP-ITP with time-dependent accumulation of the large-volume sample enables to
achieve in a reasonable time a 100 times lower c-LOD (here in orders of nmol/L), than can be reached
by conventional hyphenated ITP-ITP.

24

Levels of protein structure

There are four distinct levels of protein structure.

Protein structure, from primary to quaternary structure.

Primary structure
The primary structure refers to amino acid linear sequence of the polypeptide chain. The primary structure is held
together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. The
two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (Nterminus) based on the nature of the free group on each extremity. Counting of residues always starts at the Nterminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond. The primary
structure of a protein is determined by the gene corresponding to the protein. A specific sequence
of nucleotides in DNA is transcribed into mRNA, which is read by the ribosome in a process called translation. The
sequence of a protein is unique to that protein, and defines the structure and function of the protein. The sequence of
a protein can be determined by methods such as Edman degradation or tandem mass spectrometry. Often however, it
is read directly from the sequence of the gene using the genetic code. We know that there are over 10,000 proteins in
our body which are composed of different arrangements of 20 types of amino acid residues (it is strictly
recommended to use the word "amino acid residues" as when peptide bond is formed a water molecule is lost so,
protein is made up of amino acid residues). Post-translational modifications such as disulfide formation,
phosphorylations and glycosylations are usually also considered a part of the primary structure, and cannot be read
from the gene.

25

Amino acid residues

Each -amino acid consists of a backbone part that is present in all the amino acid types, and a side chain that is
unique to each type of residue. An exception from this rule is proline. Because the carbon atom is bound to four
different groups it is chiral, however only one of the isomers occur in biological proteins. Glycine however, is not
chiral since its side chain is a hydrogen atom. A simple mnemonic for correct L-form is "CORN": when the C atom
is viewed with the H in front, the residues read "CO-R-N" in a clockwise direction.

Secondary structure

26

An alpha-helix with hydrogen bonds (yellow dots)

Secondary structure refers to highly regular local sub-structures. Two main types of secondary structure, the alpha
helix and the beta strand or beta sheets, were suggested in 1951 by Linus Pauling and coworkers These secondary
structures are defined by patterns of hydrogen bonds between the main-chain peptide groups. They have a regular
geometry, being constrained to specific values of the dihedral angles and on the Ramachandran plot. Both the
alpha helix and the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in the
peptide backbone. Some parts of the protein are ordered but do not form any regular structures. They should not be
confused with random coil, an unfolded polypeptide chain lacking any fixed three-dimensional structure. Several
sequential secondary structures may form a "supersecondary unit".

Tertiary structure
Tertiary structure refers to three-dimensional structure of a single protein molecule. The alpha-helices and betasheets are folded into a compact globule. The folding is driven by the non-specific hydrophobic interactions (the
burial of hydrophobic residues from water), but the structure is stable only when the parts of a protein domain are
locked into place by specific tertiary interactions, such as salt bridges, hydrogen bonds, and the tight packing of side
chains and disulfide bonds. The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol is generally
a reducing environment.

27

Quaternary structure
Quaternary structure is the three-dimensional structure of a multi-subunit protein and how the subunits fit together.
In this context, the quaternary structure is stabilized by the same non-covalent interactions and disulfide bonds as the
tertiary structure. Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically
it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it
contains four subunits. The subunits are frequently related to one another by symmetry operations, such as a 2-fold
axis in a dimer. Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a
homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-" (e.g. a
heterotetramer, such as the two alpha and two beta chains of hemoglobin).

28

29