Ultracentrifugation Ultracent rifugation History Theodor Svedberg 1923 Analytical

Ultracentrifugation Ultracent rifugation

History • Theodor Svedberg (1923) - Analytical ultracentrifuge • Nobel Prize in Chemistry in 1926 • Edward Greydon Pickels - Vacuum ultracentrifuge • Pickel (1946) cofounded Spinco • Spinco (1949) - First preparative ultracentrifuge • In 1954, Beckman Instruments purchased this company.

Ultracentrifugation • Ultracentrifuge is a centrifuge spinning a rotor at very high speeds. - 1, 000 g (9, 800 km/s²) • There are two kinds of ultracentrifuge: Preparative and analytical • Uses in molecular biology, biochemistry and polymer science.

Types of ultracentrifuges Analytical Preparative • Small sample size (< 1 ml) • Built in optical system to analyze progress of molecules during centrifugation • Uses relatively pure sample • Determines sedimentation coefficient and MW of molecules • Beckman Model E is an example • Larger sample size • No optical read-out - collect fractions and analyze after the run • Uses less pure sample can be used • Estimates sedimentation coefficient and MW • Generally used to separate organelles and molecules. • Most widely used • Models L 5 -65 and L 5 -75

Analytical ultracentrifugation • Uses high centrifugal force (typically >> 100, 000 xg) to separate high MW sub-cellular molecules or organelles that differ only slightly in density • Sample is placed in a single sample cell at a right angle to the axis of rotation. • Then placed in a cylindrical hole on the rotor, balanced by a blank cell with the same weight on the opposite side. • Rotor placed on a spindle attached to a motor below the centrifuge

• Rotor chamber is evacuated with a vacuum pump to reduce air friction, and cooled with a cooling jacket to maintain a constant temperature and prevent overheating. • Progress of the experiment is monitored by passing a beam of light (UV - if nucleic acid experiments) through a quartz window in the bottom of the chamber, through transparent glass windows on the top and bottom of the sample cell, and out through another window in the top of the chamber, where it is directed by mirrors to an optical detection device. • The light is always on and a signal is detected only when the sample cell passes through the detection path.

• Solution of molecules introduced into the sample cell along with a solvent while the rotor is spinning. • Solvent in DNA experiments is typically cesium chloride (Cs. Cl), which dissociates into high-density Cs+ ions that migrate outwards in the direction of the centrifugal force, forming a shallow density gradient. • Molecules in solution diffuse centripetally. • For example, there are two molecular species, the lighter of which migrates faster, and the denser of which lags behind. • The discrete change in density at the interface between the two regions bends the light passing through the sample cell at that point, and superimposition of adjacent signals (Schleirin Optical imaging) is seen as a spike at the transition point. • Size and position of the spike are indications of quantity and molecular weight, respectively.

Separation by density gradient

Kinds of Information • • Gross shape of macromolecules Conformational changes in macromolecules Size distributions of macromolecular samples For macromolecules, such as proteins, that exist in chemical equilibrium with different non-covalent complexes, the number and subunit stoichometry of the complexes and equilibrium constant can be studied.

Two kinds of experiments Sedimentation velocity • • Interprets entire time-course of sedimentation Reports on the shape and molar mass of the dissolved macromolecules, as well as their size-distribution. Size resolution depends on particle radii and rotor speed Used to study reversible chemical equilibria between macromolecular species, Sedimentation equilibrium • • Final steady-state Sedimentation is balanced by diffusion opposing the concentration gradients, resulting in a timeindependent concentration profile Sedimentation equilibrium distributions in the centrifugal field are characterized by Boltzmann distributions. Insensitive to the shape of the macromolecule, and directly reports on the molar mass of the macromolecules and, for chemically reacting mixtures, on chemical equilibrium constants.

Zonal analytical ultracentrifugation • Zonal analytical rotor is divided into four quadrants (sectors) • Density-gradient medium is pumped into the sectors while the rotor is turning at low speed. • At high speed, a gradient forms with the same density at the same radius in each sector, in effect creating a series of concentric rings. • Once the gradient is formed, molecules or organelles introduced into the rotor through the hub will migrate to the radius where they have the same density as the gradient. • The distribution in the gradient is photographed with reflected light through the transparent rotor lid.

Analytical separation of three cell components in a sucrose gradient (A) microsomes, (B) mitochondria, and (C) nuclei and membranes.

PREPARATIVE DENSITY- GRADIENT ULTRACENTRIFUGATION

• Under high centrifugal force, a solution of cesium chloride (Cs. Cl) molecules will dissociate, and the heavy Cs+ atoms will be forced towards the outer end of the tube, thus forming a shallow density gradient. • DNA molecules placed in this gradient will migrate to the point where they have the same density as the gradient (neutral buoyancy or isopycnic point). • Gradient is sufficient to separate types of DNA with slight differences in density due to differing [G+C] content, or physical form (e. g. linear versus circular molecules).

• After centrifugation for 10 h at 100, 000 rpm (450, 000 x g), two distinct bands, sheared linear nuclear DNA above and circular mitochondrial DNA below are visible under ultraviolet light. • The DNA has been mixed with the intercalating dye ethidium bromide, which enhances the density difference between the two forms and causes the DNA to fluoresce. • The separate bands are collected by poking a hole in the bottom of the tube. • The intact mt. DNA is available for further biological analysis.

Collection of fractions • After centrifugation, the density gradient and the entangled particle zones are usually removed from the tube (or rotor) and collected as a series of fractions. This achieved in several ways. 1. Puncture the bottom of each centrifuge tube and allow the contents to slowly drip out. 2. Remove gradient by carefully lowering a narrow cannula to the bottom of the tube and withdrawing the gradient by use of a peristaltic pump. • In both procedures the gradient exits the tube dense end first, and thus the separated particles are collected in order of decreasing sedimentation rate or density. 3. Puncture the tube at the location in the gradient of the material of interest and extract it manually.

Collection of fractions

Applications of Preparative Ultracentrifuge • Used in biology for pelleting of fine particulate fractions, such as cellular organelles such as mitochondria, microsomes, ribosomes and viruses. • Used for gradient separations • Gradients of sucrose for separation of cellular organelles. • Gradients of caesium salts for separation of nucleic acids. • After the spun at high speed for sufficient time, allow the rotor to come to a smooth stop • Gradient is gently pumped out of each tube to isolate the separated components.

Applications • To study high polymers, proteins, nucleic acids, viruses, and other macromolecules of biological origin. • To study solution properties of small solutes. • Analytical ultracentrifuge: for accurate determination of sedimentation velocity or equilibrium, . • Preparative ultracentrifuge: to separate solutes on the basis of their sedimentation velocities or buoyant densities.