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6 Protein Analysis by Reversed Phase HPLC (Dong and Gant 1989)
Protein Purification Techniques
The sample components move through the column, towards the detector, only when
they are in the mobile phase, hence, velocity of migration of the component is a
function of equilibrium distribution between the mobile and stationary phase.
Traditionally, the separation of proteins have been carried out by a wide range of
well-established techniques, such as precipitation procedures, gel chromatography,
ion-exchange chromatography, affinity chromatography and electrophoretic
separations. Although these methods are still frequently and successfully employed,
each has certain disadvantages, e.g. poor resolution, low recovery and long separation time. In recent years, HPLC has been used successfully for the separation of
macromolecules, ionic species, labile natural products and wide variety of other
high-molecular weight and less stable products. HPLC has been found superior to
older methods in separation power, yield, speed and lower level of detectability. It
acts as a combination of two or more classical purification methods, as the HPLC
separations, more obviously, are governed simultaneously by several different
properties of proteins, e.g. both charge and polarity. The unprecedented degree of
resolution which may be obtained by HPLC makes it particularly suitable for purity
control, peptide mapping or search for genetical abnormalities in a protein. HPLC
has, for several reasons, proved to be of special importance when handling minute
amounts of protein material. Thus, fewer purification steps are needed and losses
are accordingly reduced.
In reversed-phase high pressure liquid chromatography (RPHPLC), the packing
is nonpolar and the solvent is polar with respect to the sample. Retention is the
result of the interaction of the nonpolar components of the solutes and the nonpolar
stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy
liquids or bonded hydrocarbons (such as C18, C8, C4, etc.) and the solvents are
polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.
HPLC consists essentially of a high pressure pumping system, relatively narrow
bore column packed with small particle size stationary phase and on line highly
sensitive detectors (Fig. 8.2).
The solvent reservoir: Generally made of glass. Before use it is necessary to degas
the solvent to remove dissolved gases (particularly oxygen), which may interact
either with mobile or stationary phase.
Pumping system: Different types of pumps are used for solvent delivery system,
e.g. constant volume type and constant pressure type.
Flow controller: These consists of flow through pressure transducer, which
measures the flow rate by measuring the pressure drop across restrictor of fixed
value placed at the pump outlet. The flow rate signal is fed back to a control unit
which compares the actual and pre-set flow rate.
Sample injectors: The sample is introduced by syringe through the septum of an
injection port into the system.
8.6 Protein Analysis by Reversed Phase HPLC
Fig. 8.2 Schematic diagram of a HPLC
Detector system: An ideal detector should have a high sensitivity (be able to detect
less than 1 ppm concentration), non-destructive. Most commonly used detector is
UV-visible detector (170–700 nm).
50 mM Tris buffer, pH 8.0
50 mM Tris buffer, pH 8.0 containing 0.5 M NaCl
10 mM Tris buffer, pH 7.5
Preparation of sample
Extract 10 g of seedlings in 50 mL of 50 mM Tris buffer, pH 8.0, by grinding in a
mortar and pestle. Centrifuge it at 15,000 rpm at 4 C for 15 min. Use the supernatant for soluble protein separation. Store at 4 C.
Prior to use, filter the solvent/eluent through 0.5-mm membrane. This can be done
using solvent clarification kit, which removes the particulates very quickly and
easily. Degas the solvent for detector stability by passing through nylon membranes
as they can be used for both hydrophilic and hydrophobic solvents.
Pass the sample through sample filter (0.5-mm nylon membrane) to assure complete
particulate removal before injecting.
Protein Purification Techniques
HPLC system equipped with a variable wavelength UV-VIS detector and a
Rheodyne injector (20 mL loop) and connected to a Datajet reporting integrator.
Stationary phase: Consisted of a Lichrosorb C-18 column (250 Â 4.6 mm i.d.)
Mobile phase: 50 mM Tris buffer, pH 7.5
Flow rate: 1 mL/min
Detector wavelength: 280 nm
Separation of Proteins on C-18 Column
1. Equilibrate the column with 10 mM Tris buffer (pH 7.5) for 3–4 h.
2. Inject the sample using a syringe with blunt tipped needles.
3. Wash the column with the same buffer till the O.D. at 280 nm comes to zero.
Elute the proteins using a linear gradient of 50 mM Tris buffer, pH 7.5 and
4. Collect the fractions of the separated proteins.
5. Calculate the concentration of the protein using the peak area of that protein.
• Degas solvent before use to remove dissolved oxygen.
• Use only blunt tipped needle for sample injection.
• Column should be washed thoroughly after the analysis is over and should be
preserved either in methanol or 0.1% sodium azide to prevent from microbial
Cell Disruption and Fractionation
The living cell contains a number of subcellular fractions. The fractionation of cells
involves two distinct phases: disruption of the tissue or cells in a suitable medium
and the subsequent separation of the subcellular particles, by differential centrifugation which exploits differences in their size and density. The procedure results in
rather crude subcellular fractions which are enriched with one particular component
and are by no means pure. These fractions are then purified by different techniques.
The ideal homogenization medium should be capable of maintaining the morphological and functional integrity of the organelles.
Homogenization media usually has the following composition:
• 0.3 M mannitol so as to be isotonic with the cytosol.
• A buffer of pH 7–8 (often Tris) at about 50 mM concentration to neutralize the
acidic vacuolar contents.
• A sulphydryl compound (dithiothreitol or mercapto-ethanol) at about 10 mM
concentration to minimize the inactivation of enzymes.
• Mg2+ at about 10 mM concentration to keep ribosomes intact.
• Ca2+ at about 1 mM concentration to prevent the clumping of nuclei.
• Polyvinylpyrrolidone and bovine serum albumin (0.1–0.2%) to precipitate out
the tannins and phenolics.
Since the plant cell wall is tough, disruption is done by mechanical means.
The most common method employs a blender whose high-speed blades exert
large shearing forces on the cells. The tissue is immersed in an equal weight of
R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,
DOI 10.1007/978-1-4419-9785-2_9, # Springer Science+Business Media, LLC 2011
Cell Disruption and Fractionation
homogenizing medium and blended for 0.5–2 min at full speed. Unfortunately, this
procedure is far severe for the delicate cellular components and may be partly
damaged. Probably the gentlest procedure of all is hand-grinding of chopped pieces
of sample with a pestle and mortar in an equal volume of homogenizing medium
sometimes with a little acid-washed sand to act as an abrasive. All these cell
disruption procedures are carried out rapidly at 2–4 C to minimize autolytic
Fractionation of Tissue Homogenate
The cell homogenate is fractionated using differential centrifugation into at
least five major fractions namely nuclei, chloroplasts, mitochondria, microsomes
and supernatant. These fractions are however impure and can be purified by a
number of techniques such as centrifugation, phase separation, electrophoresis and
specific adsorption of these components. Centrifugation remains the most generally
used procedure. Again, a number of centrifugation media such as sucrose, ficoll
and metrizamide are used either on a linear or nonlinear density gradients.
A generalized procedure for separating a tissue homogenate into crude fractions
enriched in a particular cell component is given in the flow-sheet (Fig. 9.1).
The particular fraction is resuspended in homogenization medium and then
carefully layered onto the top of a sucrose gradient in a centrifuge tube. The
gradient is made by successively adding layers of sucrose solution of decreasing
concentration to the centrifuge tube so gently that they do not mix to any great
extent. The tube may be allowed to stand at 2–4 C for about 30 min to allow
diffusion to smooth the steps in the gradient. After the resuspended cell fraction has
been added, the tube is centrifuged in a swinging bucket rotor. Depending upon the
nature of gradient and the length of the centrifugation period different particles
separate and band at zones where their density equals that of sucrose solution. After
centrifugation, these bands are carefully pipetted out separately as pure fractions.
Isolation of Mitochondria (Douce et al. 1972)
Due to their crucial role in the processes such as photorespiration and fatty acid
oxidation, they often result in their close proximity to other cellular organelles such
as peroxisomes, glyoxysomes and chloroplasts. Isolated mitochondria show marked
changes following fractionation suggesting some degree of structural damage
during homogenization or from the presence of disruptive enzymes. Most of the
problems involved in the isolation of intact mitochondria occur during the initial
homogenization because the cell wall of plant tissues is a rigid structure and the
high shearing forces necessary to rupture cell walls often have a deleterious effect
on sub-cellular organelles. The fresh tissue is gently homogenized to disrupt the
9.4 Isolation of Mitochondria
Filter through several
Layers of cheese cloth
(suspension of cell components)
centrifuge for 5 min at 200g
Centrifuge for 10 min at 1000g
Centrifuge for 15 min at10,000g
Centrifuge for 120 min at 105,000g
Fig. 9.1 Scheme for the separation of tissue homogenate
cells and release the contents and the mitchondria are pelleted by differential
centrifugation. Further purification is carried out by sucrose gradient centrifugation.
• Isolation medium (pH 7.8) containing 30 mM 3-(N-Morpholino) ethane sulfonic
acid (MOPS), 0.3 M mannitol, 4 mM cysteine, 1 mM EDTA and 0.1% (w/v)
defatted BSA adjusted to pH 7.8. For green leaf tissue, 0.6% (w/v) insoluble
polyvinyl pyrrolidone (acid-washed) is included and the BSA concentration
increased to 0.2% (w/v).
• Re-suspension medium: As above medium but without 4 mM cysteine.
• Non-linear sucrose gradient: Sucrose solution of 1.8 M (52.1% w/v), 1.5 M
(43.1%), 1.2 m (35.6%) and 0.6 M (19.1%) separately prepared in 10 mM MOPS
or phosphate buffer (pH 7.2), 0.1% (w/v) BSA.
• Tissue homogeniser
1. Collect and chop 100–200 g of fresh tissue into two volumes of chilled isolation
medium (4 C).
2. Disrupt the tissue using either a mixer for 20 s, a waring blender at low speed
for 2–3 s.
Cell Disruption and Fractionation
3. Squeeze the homogenate through six layers of cheese cloth to remove unbroken
4. Centrifuge at 700–1,000 Â g for 10 min to remove cell debris and starch
5. Decant the supernatant taking care to leave the starch pellet undisturbed, and
this is done by leaving 1–2 mL of supernatant with the starch layer.
6. Centrifuge the supernatant fraction at 10,000 Â g for 20 min or alternatively at
39,000 Â g for 5 min and discard the resultant supernatant.
7. Gently disperse the pellet in 40–50 mL of resuspension medium using a widebore 10-mL pipette and further resuspend with a glass homogenizer.
8. Centrifuge the suspension at 250 Â g for 10 min to reduce the levels of
9. Centrifuge the supernatant at 10,000 Â g for 15 min. Suspend the mitochondria
in the pellet in 1–2 mL of resuspension medium. This crude preparation can be
purified by a variety of gradient centrifugation.
10. Prepare step gradients in a suitable centrifuge tube by carefully pipetting 6 mL
1.8 M, 6 mL 1.5 M, 6 mL 1.2 M and 3 mL 0.6 M sucrose solutions respectively,
load 1 mL of crude preparation (40–50 mg protein) onto the gradient.
11. Centrifuge the gradient at 40,000 Â g for 45 min in an ultracentrifuge.
12. The mitochondria band at the 1.5–1.2 M interface.
13. Collect the band by side-puncturing the tube using a hypodermic needle
slightly below the band. Alternatively appropriate fractions in drops can be
collected by injecting 2 M sucrose into the base of the tube.
14. Dilute the gradient fraction containing mitochondria to isotonic conditions
(0.3 M) by slow, careful addition of buffer.
15. Pellet the mitochondria by centrifuging at 10,000 Â g for 15 min and finally
suspend in a small volume of relevant medium.
• The most important step is that the disruption of the cells should be done gently
to avoid damage to the organelle and at the same time ensuring maximum
• Mitochondria from green leaf tissue can be isolated in a similar way described
above. The medium is identical to that used for etiolated tissues except for the
addition of 0.6% (w/v) acid washed insoluble PVP and increasing the defatted
BSA concentration 0.2% (w/v). After filtration through cheese cloth, chloroplasts
sediment at 3,000 Â g for 5 min and the mitochondria are collected from the
supernatant by centrifugation at 12,000 Â g for 20 min. The pellets are
resuspended in approximately 50 mL of medium except for addition of 0.2%
defatted BSA as described previously. Following a low speed centrifugation at
15,000 Â g for 10 min, mitochondria are sedimented from the supernatant by
centrifugation at 11,000 Â g for 15 min.
9.5 Isolation of Chloroplasts
Isolation of Chloroplasts (Walker 1980)
Isolated chloroplasts are required for different studies including the electron transport system of the photosynthetic apparatus. The separation is carried on the basis
that the cell organelles, depending upon their size and weight, sediment at different
1. Isolation medium: Weigh 2.42 g Tris (20 mM); 72.8 g sorbitol (0.4 M); 1.168 g
NaCl (20 mM); 0.61 g MgCl2 Á 6H2O (3 mM) and dissolve in 1 L of distilled
water and adjust the pH to 7.8.
1. Cut 5–10 g of leaf tissues into small bits. Add 20 mL of the prechilled isolation
2. Homogenize with standard homogenizer.
3. Filter the debris through eight-layered cheese cloth.
4. Centrifuge at 3,000 Â g for 2 min.
5. Discard the supernatant and suspend the pellets in the isolation medium.
6. Centrifuge again at 3,000 Â g for 2 min.
7. Discard the supernatant and resuspend the pellet in a small volume of the
grinding medium and store on ice.
8. Since any further advanced study on isolated chloroplasts is expressed on the
basis of chlorophyll, estimate the chlorophyll by diluting 0.1–0.2 mL of chloroplast suspension to a total volume of 4 mL with 80% acetone.
Calculate the chlorophyll content as follow:
ð12:7 Â A663 Þ Àð2:69 Â A645 ị ẳ chl.a mg=mLị;
22:9 A645 ị 4:68 A663 ị ẳ chl.a mg=mLị;
20:2 A645 ị 8:02 A663 ị ẳ total chl: mg=mLị:
Calculate the chlorophyll concentration of the stock chloroplast suspension.
Most leaves yield better chloroplasts if freshly harvested except spinach, which can
be stored for 4 weeks in cold for better yield. If leaves are brightly illuminated for
20–30 min prior to grinding, the chloroplast yield is increased.
Enzymes in Metabolism
Enzymes are the biocatalyst in metabolic processes. The study of the enzymes
involve estimation of activities; isolation, purification and characterization of
Main Steps During Enzyme Purification
During the first step of purification, the tissue is usually homogenized in buffer at
0–4 C, if mitochondria or particles are to be isolated, an isotonic or hypertonic
solution is employed, namely 0.25–0.8 M sucrose, with a suitable buffer to control
1. Tissue homogenization: The under given scheme can be applied for the separation of particulate systems.
2. Centrifugation: Centrifugation of the homogenate is the next step during protein
3. Fractional precipitation with ammonium sulphate: A homogenate, a soluble
protein extract, or an acetone powder extract can be used for a series of
standard purification procedures. By the addition of a saturated solution
of ammonium sulphate, proteins will be salted out and separated by centrifugation. If conditions are kept constant, remarkable reproducibility can be
4. Selective adsorption and elution on calcium phosphate gels: Proteins are readily
adsorbed on these gels and then are differentially eluted by increasing salt
5. Ion exchange and gel permeation chromatography: The gels, such as
carboxymethyl cellulose (CMC) or DEAE, are extremely useful in purification
procedures. Sephadex columns – gel filtration techniques using unmodified gels
or gels with DEAE or CM side chains on the polysaccharide molecule – are
widely employed for enzyme purification. These methods are the general
R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,
DOI 10.1007/978-1-4419-9785-2_10, # Springer Science+Business Media, LLC 2011
Enzymes in Metabolism
approaches to enzyme purification. All steps must be checked for enzyme units,
specific activities, yields, and recoveries.
6. Purity of enzyme: In order to examine detailed structures of complex proteins, it
is mandatory to have proteins that are homogeneous entities. Over a period of
years techniques have therefore been developed to analyze protein solutions for
Gel electrophoresis of proteins: Since proteins are polyelectrolytes with their
charges dependent on the pH of the surrounding medium, electrophoresis
techniques have been developed which can separate a mixture of proteins in an
electric field. The mobility of a protein in an electric field depends on the number of
charges on the protein, the sign of the net charges, the degree of dissociation which
is a function of pH, and the magnitude of the electrical field potential. An opposing
resistance against the mobility of the protein molecule relates to the size and shape
of the ion, viscosity of the medium, concentration of the ion, solubility of the
protein, and adsorptive properties of the support medium.
The most widespread and useful support medium presently employed for
electrophoresis is a polymer of acrylamide cross-linked with N,N-dimethyl-bisacrylamide.
The advantage of the gel electrophoresis method is that the “pore size”, that is,
the sieving action of the gel, is directly related to the concentration of the gel. Thus,
by increasing the range of gel concentration from 3 to about 9–10%, the pore size
decreases and the proteins move more slowly. By this simple variant, one can alter
the mobility of charged proteins and thus study a wide range of protein sizes.
The apparatus involves a direct-current power supply, and upper and lower reservoir buffer systems, the proteins sample, a stacking gel (2.5% gel), and the running gel
(about 6–7%). The gel is prepared by mixing acrylamide with the cross-linking
component, methylene-bis-acrylamide and the polymerizing initiator, and ammonium
per sulphate. With the running gel in place, the stacking gel is prepared above it, the
gel cassette is appropriately mounted in the apparatus, the protein solution is added
above the stacking gel, and current is turned on. Frequently, a tracking dye is added
with the protein mixture to serve as an indicator of the front of the moving zone as it
descends down the tube. When the tracking dye moves to the bottom of the gel
column, the current is turned off, and the gel tube is removed, stained with an
appropriate dye, and inspected for the number of protein components.
By minor modifications, namely running an unknown protein and a known
protein at different gel concentrations and plotting their log Rm against gel
concentration and in turn the slopes of each curve against the molecular weight,
an accurate molecular weight of the unknown protein can be determined. Thus, one
can ascertain the purity as well as the molecular weight by gel electrophoresis