Lecture 5
Bio 451 Molecular Biology Techniques
A brief history:
Centrifuges similar to our bench top centrifuges have been in use since the mid-1800’s. These centrifuges were capable of speed of about 3000 rpm. The early ones were hand driven; after 1912, the centrifuges were electrically driven. First applications were non-biological, such as separation of milk and collection of precipitates.
Miescher (1872) attempted to separate macromolecules – credited with the discovery of nucleic acids.
The development of the ultracentrifuge is generally credited to Svedberg who worked in the 1920’s and 1930’s. Svedberg coined the term “ultracentrifuge.” Svedberg was a colloid chemist - he studied the structure of proteins (at that time all proteins were considered to be colloids). His group used the ultracentrifuges to determine the MW and subunit structure of hemoglobin, studies which changed the ideas concerning the structure of proteins. Svedberg’s group developed a number of ultracentrifuges. Some of the early models were capable of reaching 900,000xg, but the rotors were very small. For routine work they used larger rotors which could reach 260,000xg.
The first commercial ultracentrifuge was produced in 1940 by SPINCO (not Beckman Coulter).
Improvements leading to modern ultracentrifuges:
High speed drive systems – the motor.
Methods to reduce friction generated at high speeds – development of vacuum systems. Vacuum systems also enabled the maintenance of constant temperature.
Improvements in rotor material to withstand high centrifugal forces. Svedberg’s group used high tensile steel. Now aluminum alloys, titanium are used.
Improvements in metjods to examine the particles being centrifuged – better optical methods.
Also important was the development of techniques such as electro microscopy which enabled the early workers to see the organelles that they were separating with the ultracentrifuge – able to formulate structure-function relationships.
Two types of ultracentrifuges developed: analytical and preparative.
Analytical
· Uses small sample size (less than 1 ml)
· Built in optical system to analyze progress of molecules during centrifugation
· Uses relatively pure sample
· Used to precisely determine sedimentation coefficient and MW of molecules
· Beckman Model E is an example of centrifuge used for these purposes.
Preparative
· Larger sample size can be used
· No optical read-out – collect fractions and analyze them after the run
· Lass pure sample can be used
· Can be used to estimate sedimentation coefficient and MW
· Generally used to separate organelles and molecules. Most centrifugation work done using preparative ultracentrifuge
· Several models available, including L5-65 and L5-75 used for preparative purposes.
· Start with a uniform mixture of sample – molecules or organelles in buffer.
· Centrifuge to obtain pellet of heavier molecules, and supernatant
· Because all sized particles are evenly distributed in the beginning, the pellet will contain primarily large sized particles, but will also contain some of the other sized particles. Can re-suspend the pellet and re-centrifuge (2-3 times) to get fairly pure prep of heaviest particles, but 100% separation is not possible by this method.
· Can take supernatant and re-centrifuge to pellet the next largest particle successive centrifugation at increasingly higher centrifugal forcers (g forces) can partially purify the different sized particles. To obtain good separation, size and density of the particles must differ by several orders of magnitude.
· Commonly used for separation of organelles.
· Usually use a fixed angle rotor.
Density gradient centrifugation: Rate zonal (also termed sedimentation velocity, zone centrifugation)
· Use a continuos density gradient of solvent such as sucrose. Desnsity increases toward the bottom of tube. Sample is layered on top. Centrifuge until molecules form discrete bands depending upon their sedimentation values. Generally stop the run before any of the molecules reaches the bottom. 100% separation is possible with a small sample.
· Separates molecules based on size (MW).
· Used to separate various types of macromolecules – mixture of proteins; different types of RNA; separate DNA from RNA, or proteins; ribosomal subunits; polyribosomes.
· Swinging bucket rotor always used.
Density gradient centrifugation: Isopycnic centrifugation (also termed sedimentation equilibrium centrifugation)
· Mix gradient material (such as cesium chloride- CsCl) with sample molecules.
· During the centrifugation, the CsCl generates a gradient (“self-generating gradient”), and the molecules move to the position in the gadient where their density is the same as the gradient material. Isopycnic means “same density,” so the molecules move to their “isopycnic position.”
· In order to generate a gradient, you select a CsCl concentration that, when redistributed during centrifugation to form a gradient, will give you a range of densities tha includes the densities of all the molecules you want to separate. CsCl and other salts of alkali metals (Cs sulfate) are used because you can generate high densities with these materials.
· Requires long centrifugation time to form a gradient (36-48 hrs). Can reduce the time needed to form a gradient (by at leat 1/2) by preparing step (discontinous) gradients before centrifugation.
· Separates molecules based on their density – may have the same size.
· Often used to separate various types of DNA – circular vs linear, double stranded vs single stranded, DNA from RNA (RNA pellets), highly repetitive DNA (satellite DNA) from other Dna in the cell, can be used for separation of lipoproteins and cell organelles.
· Semiconservative replication of DNA was demonstrated using isopycnic centrifugation.
· Can use swinging bucket or fixed angle rotor.
· Properties of the molecules (size, shape, density)
· Properties of the solvent, or gradient material (density, viscosity, temperature)
· Interactions between the solute molecules and the solvent gradient material
As the rotor spins in a centrifuge, centrifugal force is applied to each molecule in the sample:
Centrifugal force = Mw2r
Where M- mass (particle weight, or molecular weight); w (omega) – angular velocity (radieas/sec); r – distance from the axis of rotation.
This equation says that the larger the molecule, or the faster the centrifugation, or the longer the axis of rotation, the greater the centrifugal force and the rate of sedimentation.
Two forces act to counteract the centrifugal force – the two counteracting forces are the buoyant force (or displacement force) and the frictional force.
Bouyant force = Mw2r Vr
Where V – partial specific volume of the solute 9molecules)- the volume displaced by the molecule. Definitions: the ml of solution volume increase caused by addition of 1 g of solute; volume occupied when 1 g of solute is added to a large volume of solution.
r (rho) – density of the solvent (g/ml).
The dissolved or suspended particles (molecules) also generate friction as they migrate through the solution:
Frictional force = f (v) = f (dr/dt)
F – frictional coefficient unique to the molecules in question.
dr/dt – rate of sedimentation expressed as a change in the axis of rotation with time
For a spherical molecule, f = 6ph rm
h– viscosity of the medium in poises (g/cm sec)
rm – radius of particle (molecule)
So f, the frictional coefficient, depends upon the size and shape of the molecule, and on the viscosity of the gradient material.
A sedimenting molecule moves faster and faster in a centrifugal field until the centrifugal force equals the conteracting buoyant and frictional forces:
This occurs because the frictional force increases with increased rate of sedimentation whereas centrifugal force and buoyant force are constant for a particular molecule and rotor speed. In practice, this balancing of forces occurs quickly with the result that a molecules sediments at a constant rate (dr/dt).
If we substitute the above equations into this relationship…
Centrifugal force = buoyant force + frictional force
Mw2r = Mw2r Vr + f(dr/dt)
By rearranging…
Mw2r - Mw2r Vr = f (dr/dt)
M (1 – Vr) w2r = f (dr/dt)
M = (f/1 – Vr) [(dr/dt)/w2r]
Sedimentation coefficient (s)
A new term the sedimentation coefficient (s) describes the velocity attained by a particle (molecule) for a given angular acceleration:
S = v/w2r, where v is velocity, or s = (dr/dt)/ w2r
dr/dt is the change in axis of rotation over time.
If we substitute “s” in the above equation:
M = (f)(s)/1-Vr
Or
S = M (1-Vr)/f
The frictional coefficient f, can be evaluated through an experimentally determined diffusion constant, D, where
D = RT/f
Or
f=RT/C
R= gas constant; T= absolute temperature.
Substituting the the above equation..
M= RTs/D(1-Vr)
s=M(1-Vr)D/RT
These are the equations that are used to convert M (molecular weight) of a molecule or particle to s, its sedimentation coefficient (or vice versa).
Sedimentation coefficients (s) for biological molecules fall between 1 and 500 x 10-13 seconds. We avoid the unit of 10-13 seconds by defining one Svedberg unit, or Svedbers (S as 1 x 10-13 sec. If we measure the sedimentation coefficient of a molecule as 10-12 sec, or 10x10-13 sec, this molecule has a sedimentation value of 10S. Sedimentation coefficients are corrected to a reference solvent having the viscosity and density of water at 20oC – termed s20,w
Centrifugation:
Centrifugation is based on the fact that any object moving in a circle at a steady angular velocity is subject to an outward directed force, F. The magnitude of this force depends on the angular velocity in radians, w, and the radius of rotation, r, in centimeters.
F= w 2r
F is frequently expressed in terms of the earth’s gravitational force and is then referred to as the relative centrifugal force, RCF, or more commonly as the “number times g.” Where the earth’s gravitational field (g=980 cm/s-2).
RCF= w 2r /980
To be of use, however, these relationships must be expressed in terms of “revolutions per minute,” rpm, the common way in which the operating speed of a centrifuge is expressed. Since rpm values may be converted to radians using the equation
w= p (rpm)/30
then
RCF=
RCF = 11.17r (RPM/1000)2 where r = radius in cm
RCF = 28.38R (RPM/1000)2 where R = radius in inches
Where r varies from the top to the bottom of the sample holder. The centrifugal force exerted at the top and bottom of the sample tube differs by nearly twofold. To account for this, RCF values may be expressed as an average RCF value (RCFave), which is the numerical average of the values exerted at the top and bottom of the same chamber. The important consideration, then, is to clearly define the value of r.
· Desk top clinical centrifuges. The maximum speed of most desk model centrifuges is below 3000 rpm and all of them operate at ambient temperature.
· Highspeed centrifuges are those operating up to speeds of 20,000 to 25,000 rpm.
· The ultracentrifuge has the ability to attain centrifugal forces in excess of 5000xg (75,000, r=8cm). The development of the ultracentrifuge permitted the fractionation of subcellular organelles previously observed only in electron micrograph; this in turn permitted assay of their enzymatic constituents, providing insights into structure-function relationships. The contemporary ultracentrifuges consist of four principal parts: (1) drive and speed control, (2) temperature control, (3) vacuum system, and (4) rotors.
· Drive and speed control. The drive shaft itself is only about 3/16 in in diameter. The samll diameter of the shaft allows it to flex during rotation, thus accommodating a small degree of rotor imbala ce without vibration or spindle damage. An overspeed system was developed to prevent operation of a rotor above its maximum rated speed. Such operation results in the rotor being torn apart or exploding. It is for this reason that the rotor chamber is always enclosed in heavy armor plate capable of containing any such explosion. The over speed system consists of (1) a ring of alternating reflecting and non-reflecting surfaces attached to the bottom of the rotor., (2) a small but intense point source of light, and (3) a photocell. The passing of reflecting and nonreflecting surfaces throught the light beam as a consequence of rotor rotation chaps the light and establishes a pulsing signal in the photocell output circuitry. The frequency of this signal, which is a function of the speed of rotation, is compared to a standard reference signal. If the rotor-generated signal frequency surpasses that of the reference, the instrument is automatically shut down.
· Temperature control in highspeed instruments involves placing a thermocouple in the rotor chamber, and monitoring only the rotor chamber temperature. In the case of an ultracentrifuge and infrared radiometric sensor placed beneath the rotor continuously monitors the rotor temperature directly, ensuring more accurate and responsive temperature control.
· Vacuum System. At speeds below 15,000 to 20,000 rpm only small amounts of heat are generated by friction between air and the spinning rotor. At greater speeds, however, air friction is significant and becomes severe above 40,000 rpm. To eliminate this source of heating, the rotor chamber is sealed and evacuated by two pumping systems operating in tandem. The first system is a mechanical vacuum pump similar to those in normal laboratory use, which can establish a vacuum down to 100 to 50u. Once the pressure in the chamber has decreased below 250u, a water-cooled diffusion pump is also brought into operation. Using both pumps it is possible to attain and hold vacuums of 1 to 2 u. As would be expected, temperature control is significantly improved when the rotor chamber is evacuated.
· A large variety of rotors are available for use in modern ultracentrifuges, and fall into two classes: angle and swinging bucket. Both types are constructed from either aluminum alloys for low to moderate speeds or titanium for highspeed operation. Angle rotors consist of a solid piece of metal with 6 to 12 holes machined at an angle between 20o and 45 o. These rotors are most often used for applications involving total sedimentation or “pelleting” of a constituent. Swinging bucket rotors consists of a rotor from which hang three to six free moving buckets. These buckets hang vertically when the rotor is at rest and swing 90 o to a horizontal position, under the influence of centrifugal force, as the rotor attains a speed of 200 to 800 rpm. This type of rotor was designed principally for incomplete sedimentation of a sample through some sort of gradient. In this position material sedimented to different areas of the tube appearing as bands running across the tube rather than at an angle as in an angle rotor.
· To avoid overstressing the rotor and ensure its continued safe operation an accurate record should be kept of its total usage, that is, the number of runs (at any speed up to its naximum rpm) and the time of each run, so that the rotor can either be derated after a certain number of runs (e.g. 1000 runs) or hours of centrifugation (e.g. 2500 hours) or replaced after a set period of time as specified by the manufacturer.
· Molecules or particles spinning around an axis are subjected to a centrifugal force, F. Under the influence of this force they sediment toward the bottom of the centrifuge tube at a velocity, v, described by the equation
· Where r is the distance (cm) from the axis or rotation to the sedimenting particle or molecule, o is the volume of the particle (cm3), rp is the density of the particle (g/cm3), rm is the density of the medium (g/cm3), f is the frictional coefficient (g/sec), and v is the radial velocity of sedimentation of the particle (cm/sec). A more common form of this equation is expressed in terms of the sedimentation coefficient, s, of the sedimenting particle, that is, its velocity of sedimentation per unit or force field, F.
· The unit of S are seconds, and since many biologically significant molecules possess sedimentation coefficients greater that 10-13 seconds, this quantity is defined as a Svedberg unit (S) in honor of Svedberg, the originator of this type of analysis. Therefore, ribosomal subunits or other particles possessing a sedimentation coefficient of 18x10-13 seconds are said to be 18S.
· The frictional coefficient of a molecules (f) depends on its size, shape, and the viscosity of the medium through which it is sedimenting.
· In a preparative ultracentrifuge the smooth migration of particles through a homogenous solution is disturbed by mechanical vibration, thermal gradients, and convection. These disturbances can be eliminated or largely alleviated by forming a gradient of some rapidly diffusing substance in the centrifuge tube. A wide variety of materials can be used, including sucrose, glycerol, cesium chloride, cesium sufate, as well as more unusual compounds like ficoll and metrizamide. Two major types of techniques in which gradients are employed are in zone or sedimentation velocity centrifugation and isopycnic or sedimentation equilibrium centrifugation.
A useful relationship that may be derived from a linear time dependence of sedimentation relates, the speed and time of centrifugation. These two parameters may be varied as long as the product of the force and its duration are constant
t1 rpm12=t2rpm22
· Once a set of molecular species or subcellular organelles have been separated on a density gradient it is important to recover the separated components. One method involves puncturing the bottom of the centrifuge tube with a needle and allowing the contents to drip out as a result of gravity. Another method involves inserting a cover into the top of the centrifuge tube. A small diameter glass or plastic tube is inserted through this cap to the bottom of the tube. A solution with a density greater than those used to make the gradient in the tube is then pumped into the bottom of the tube, forcing the contents out a hole in the cap. Another approach is to aspirate the gradient solution into a tube that is connected to a spectrophotometer.
· In addition to component identification, a second important piece of information that can be obtained for each fraction is its average density. This is particularly important in the case of isopycnic gradient centrifugation because these methods separate particles or molecules on the basis of their density. The concentrations and therefore densities of most solutions used to establish gradients are proportional to their refractive indexes, a parameter that can be easily determined using a refractometer.
· The material most often used to establish a gradient is sucrose, which is cheap, available in high purity, and yields solutions with a density up to 1.28 g/cm3. Glycerol is another compound that possesses quite similar characteristics but can be used only at densities less than 1.15 g/cm3. Both of these compounds suffer two major disadvantages: they are very viscous at densities greater than 1.10 to 1.15 g/cm3 and exert very high osmotic effects even at very low concentrations. Thus, other substitute compounds have emerged that yield a similar density range, and are also inert and nonionized, and possess low viscosity and osmotic effects. Five such materials are meglumine diatrizoate or Renografin, Urograffin, Ludox, Ficoll, and Methizamide. The latter three compounds are used most often today. Ludox is a trade name for colloidal silica produced by Du Pont. A 40% solution of Ludox is often combined with polysaccharides such as dextran to form gradients that are used to isolate various types of whole cells. Ficoll is the trade name for a high molecular weight polymer of sucrose and epichlorhydrin produced by Pharmacia. Metrizamide can readily generate densities up to 1.45 g/cm3. Although these compounds are adequate for gradients involving protein and whole cells, they are of little value when it comes to the separation of nucleic acids or organisms, such as viruses, that are composed predominantly of nucleic acids. Cesium chloride can be used to establish gradients with densities ranging up to1.70 g/cm3. Cesium chloride and more recently cesium sulfate linear gradients are almost always produced by centrifuging to equilibrium a uniform solution of the salt and sample to be resolved. During centrifugation the gradient forms by diffusion and the the sedimenting species either sediment or “float” to level within the gradient that has a density equivalent to their own.