Dennison С. A Guide to Protein Isolation

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28 Chapter 2

motion generates strong shear forces, due to laminar flow, while the

irregular outline of the vessel gives good overall mixing of the solution.

The degree of disruption depends upon the speed of rotation of the

blades. At high speeds, a blendor will disrupt mitochondria and nuclei and

may even denature proteins. it is mostly used with plant and animal

tissues but is less effective with micro

organisms.

Note that although it is a ìblenderî, the trade name is ìWaring

blendorî.

2.4.4 The Polytron/UItra

Turrax

type homogeniser

ìPolytronî and ìUltra

Turraxî are trade names for a type of

homogeniser which consists of a stationary vertical tube, equipped with

serrated teeth and radially distributed holes at its lower edge. Fitting

closely into the stationary tube is a motor

driven tube, also with radially

distributed holes corresponding to those on the stationary tube.

Figure

16. The Polytron/Ultra-Turrax type homogeniser.

Rotation of the inner tube causes the sample to be flung outwards,

through the holes in the tubes. Because the two sets of holes

continuously and rapidly come into and out of register, the sample gets

chopped into small pieces and simultaneously homogenised by shear

between the rotating and stationary tubes. Such homogenisers are very

effective and only a short period of homogenisation is required, the

sample being cooled in an ice bath during this period.

2.4.5 Grinding

Several types of apparatus are available for grinding. in the Edmund

Buhler disintegrator, bacterial cells are vibrated with glass beads in a

Assay, extraction and sub

cellular fractionation

jacketed container. Cells are broken by impact, tearing and maceration

between the hard surfaces. To avoid heating, cooling liquid is circulated

through the jacket.

2.4.6 The Parr bomb

In the Parr bomb, the sample is subjected to nitrogen gas under very

high pressure. Under these conditions, the nitrogen dissolves in the cell

fluids. When the pressure is released, the explosive generation of

nitrogen bubbles causes disruption of the cell, and less frequently of

organelles.

2.4.7 Extrusion under high pressure

In an apparatus such as the French pressure cell (Fig. 17), cells are

broken by extrusion through a narrow orifice at pressures of up to

8,000 p.s.i. Laminar flow causes intense shearing forces which disrupt

the cells as they pass through the narrow orifice of the needle

valve.

Figure 17. A French pressure cell.

Needle valves.

Needle

valves arc devices used to adjust or regulate the

flow of fluids. They consist of a tapering “ needle”, with a round cross

section, which fits into a corresponding round hole, called

“jet”.

When

the needle is retracted slightly from the jet, an annular gap is formed,

between the needle and the jet, and fluid can flow through this gap.

The

cross

sectional area

the annular gap can be altered by adjusting the

degree to which the needle is retracted

from

the jet.

30 Chapter 2

An every

day application of needle

valves is in carburettors, where

they are used to control the flow of petrol and air, to ensure a correct

mixture of the two. Adjusting the needle

valves is one of the steps

involved in “tuning” a carburettor.

2.4.8 Sonication

Application of high frequency sound waves is an effective method of

cell breakage which can be applied to micro

organisms. The mechanism

is thought to involve “micro

cavitation”, i.e. the production of very

local transient pressure differences, which break cell walls. The

efficiency of cell breakage is influenced by the power output of the

instrument, the duration of exposure and the volume of material

processed. In general, the volume which can be treated in a given time is

not great - not as great, for example as that using high pressure extrusion.

Cooling is necessary to prevent the build

up of heat.

Micro

cavitation. The formation of a bubble of vapour in a liquid, due

to a local reduction in pressure to below the vapour pressure of the liquid

at that temperature, is known as “cavitation”. For example, in the case

of a boat propeller, there is a pressure differential on either side of the

blades

low pressure in front and high pressure at the back. If too much

power is applied. the pressure in front becomes too low and the water

vaporises. The propeller then spins in the vapour bubble formed, without

generating thrust. “Micro

cavitation,” is caused by the formation and

collapse of very small bubbles of vapour in the liquid, due to the passage

of sound (pressure) waves. Micro

cavitation can be very corrosive and is

a major cause of the erosion of ships’ propellers, for example.

(See p47, “How hard can one “suck” on water”)

2.4.9 Enzymic digestion

Enzymes provide a very gentle and specific means of disrupting cells

to release their contents. For example, the cell walls of bacteria may be

digested with the enzyme, lysozyme. Similarly plant cell walls may be

digested with cellulases and fungal cell walls with chitinases.

Assay, extraction and sub-cellular fractionation

2.5 Clarification of the extract

The cellular extract prepared by one of the methods described above

may be clarified, by filtration through a nylon mesh or cheese

cloth, to

remove the larger tissue debris, and centrifuged at relatively low speed to

remove insoluble cell components.

2.6 Centrifugal sub

cellular fractionation

Centrifugal = ìfleeing the centreî

The Svedberg Equation. As an introduction to the topic

centrifugation and to gain insight into the forces acting upon a molecule

undergoing centrifugation, it is useful to consider the derivation of the so

called Svedberg equation.

Newton’s law of motion states that any body in motion at a constant

speed in a straight line will continue in that motion unless acted upon by

a force. This force will cause the body to accelerate in the direction of

the force, according to the equation:

Force = Mass x acceleration

e.g. the passengers in a cornering motor car.

Figure 18. The equal and opposite forces acting on a cornering car.

The occupants of the car experience a gravitational field, due to their

centripetal acceleration “a”, described by the equation:-

where, = the angular velocity in radians sec-1 (remember one

revolution = radians)

r = radial distance from axis of rotation.

32 Chapter 2

The centripetal force (F) which the car exerts on the occupants and

the equal

and

opposite centrifugal (inertial) force which the occupants

exert on the car is a function of their mass.

i.e. 2.9

From equation 2.9 we can see why a car will skid if it is turned too

rapidly;

Figure 19.

The effect

increasing the sharpness

a turn.

Increasing the sharpness of a turn increases but decreases r. In

equation 2.9, however, a is squared, while r is linear, so the increase in

has the greater effect. The centrifugal force is thus increased in a sharp

turn and the car is more likely to skid, which it will do when the

centrifugal force becomes greater than the frictional grip of the tyres.

If we now consider molecules of molecular weight M,

undergoing

centrifugation, the centrifugal force acting upon them is given by;

2.10

This Centrifugal force causes the molecules to sediment down the

centrifuge tube. As they start to move, however, they encounter a

frictional resistance to their movement, given by:

2.11

Where = frictional coefficient

= the rate of sedimentation expressed as the

change in radius with time (t).

Assay, extraction and sub

cellular fractionation

The sedimenting molecule must also displace the solvent into which it

sediments and this gives rise to a buoyant force:

2.12

Where = partial specific volume of the molecules (cm

volume increase caused by 1g of solute),

= density of the solution.

Note that the buoyancy force increases with the radius in the same way as

the gravitational force does. To grasp what this means, conduct the

following thought experiment:

Imagine a little ship floating on the

surface of the water in a centrifuge tube, undergoing centrifugation, with

its plimsoll line on the water line. If “r” was increased, by removing

some of the water, the gravitational force would increase but the

ship would not float lower in the water because the buoyancy force

would increase by the same amount (Fig. 20).

Figure 20.

The effect

radius

rotation

gravitational and buoyancy forces.

Equations 2.6 to 2.8 can be combined in the expression:

Centrifugal force = Buoyant force + Frictional force

i.e.

34 Chapter 2

Hence,

2.13

i.e. if all other factors are kept constant, the rate of sedimentation

depends upon the molecular weight.

A new term, “s”, the sedimentation coefficient, can be defined:

(i.e. it is the rate of sedimentation per unit of gravitational field)

Hence,

2.14

can be determined from

the diffusion coefficient (D), measured by

molecular exclusion chromatography, immunodiffusion or ultra

centrifugation:



and, therefore

Substituting into equation 2.10, gives the so

called Svedberg equation

named in honour of Professor The Svedberg, of Sweden:

2.15

Note: “s” has the range 1 x 10

-13

to 500 10

-13

Svedberg”, abbreviated “S”, can be defined to obviate the (x 10

-13

A new unit, “the

i.e. S = s x 10

Fractionation of sub

cellular organelles is usually effected by

centrifugation. Assuming one is dealing with rigid, spherical particles, the

time required to sediment a specific particle, subjected to centrifugation,

is given by the equation

;

Assay, extraction and sub

cellular fractionation

2.16

where, = time required for particle to sediment from

meniscus to bottom of the tube,

= viscosity of the medium,

= angular velocity,

= radius of sedimenting particle,

= density of sedimenting particle,

= density of medium,

= radius from centre of rotation to solution

meniscus,

= radius from centre of rotation to the bottom of

the tube.

If the experimental set

up is established and the angular velocity is

kept constant, Eqn 2.16 can be simplified to:

2.17

Note that if the angular velocity is not constant, equation 2.17 must be

modified to:

2.18

So, for a given particle in a given system:

2.19

Hence, if we wish to change the time of sedimentation, the term

must be kept constant,

i.e. specified time x (specified rpm)

= new time x (new rpm)

In the case of a sub

cellular fractionation, most particles are of about

the same density and so equation 2.17 may be reduced to:

36 Chapter 2

2.20

i.e. the time taken to sediment a particle is inversely related to the square

of its radius. This fact underlies the technique of fractionation by

differential centrifugation, such as the example shown in Fig. 21. In a

differential centrifugation fractionation, the “pellet” is unlikely to be

pure because, initially, all particles of the homogenate are distributed

evenly throughout the centrifuge tube. Upon sedimentation, the heaviest

particles sediment first but other less dense material is dragged along and

that originally near the bottom of the tube is co

precipitated.

Repeated

“washings” can improve the purity.

Figure 21. An example of sub

cellular fractionation by differential centrifugation.

2.6.1 Density gradient centrifugation

consider equation 2.17, i.e.:-

If,

and if

2.17

and particle will not sediment,

and particle will have negative

sedimentation in real time, i.e. it

will rise to the top,

and particle will sediment.

Assay, extraction and sub

cellular ,fractionation

Using this information, one of two strategies for isopycnic (equal

density) centrifugation can be adopted to separate particles by virtue of

their differences in density:

 The sample can be suspended in a solution having a density equal to

that of the sample protein of interest. Proteins that are more dense

or less dense will sediment or float to the top of the solution,

respectively, leaving the protein of interest in solution. A difficulty

with this approach, however, is that often the density of the protein

of interest is not known beforehand.

 The sample can be layered on top of a continuous density gradient. If

the gradient extends to densities exceeding the density of the sample

proteins, these will sediment through the gradient until they reach

points where their density is equal to that in the gradient, at which

point sedimentation ceases, i.e. the proteins become focused at their

isopycnic points.

Density gradients may be generated using sucrose dissolved in buffer.

Disadvantages of sucrose are:

 It interferes with the Lowry and Bradford protein assays, though less

with the latter.



It can penetrate cells. This, of course, is a problem which only applies

to the fractionation of cells.

Advantages of sucrose are:

 It is biologically inert.

• It is inexpensive.

• It is dialysable and is thus easy to separate from the sample proteins.

“Ficoll”, a Pharmacia product, consists of sucrose crosslinked with

epichlorhydrin, and has a molecular weight of about 400,000. Like

sucrose it is biologically inert, but in some respects it is opposite to

sucrose, e.g. it cannot penetrate cells and so is suitable for fractionation

of cells, but it is not dialysable and so is difficult to separate from

proteins.

With sucrose or Ficoll, a density gradient can be generated using a two

chamber gradient generator, with an insert in the low density chamber to

compensate for the lower density. To generate a linear gradient, the

solutions in the two chambers must have the same volume and they must

be in hydrostatic equilibrium, i.e. when there is no flow out of the

apparatus, there must be no tendency for fluid to flow from one

compartment to the other.