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6 Protein Analysis by Reversed Phase HPLC (Dong and Gant 1989)

6 Protein Analysis by Reversed Phase HPLC (Dong and Gant 1989)

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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


Gradient Device








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).


Working solutions





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.

Sample filtration

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


Chapter 9

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.


Homogenization Media

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.


Cell Disruption

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


(poorly disrupted

tissue, discarded)


(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




(soluble components)

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

tissue pieces.

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

centrifugal fields.


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.

Chapter 10

Enzymes in Metabolism

Enzymes are the biocatalyst in metabolic processes. The study of the enzymes

involve estimation of activities; isolation, purification and characterization of

different enzymes


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

the pH.

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.





Tertiary amine


Polymerizing system










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

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6 Protein Analysis by Reversed Phase HPLC (Dong and Gant 1989)

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