[From CASE Reports, Vol. 11, No. 4, 1996]


Analytical Ultracentrifugation at UConn

National Facility at Storrs Supports Fundamental, Applied Research

It has been said that Theodor Svedberg, the Swedish scientist, developed his idea for an ultracentrifuge from contemplating a dairy cream separator. Soon he had made one which turned enormously faster than any "spinner" known to commerce. With this he succeeded in separating and studying very large molecules and their glue-like agglomerates from aqueous solutions, thus opening to science a new approach to understanding the structural protein building materials of living things. His idea, the machines he built, and their applications won him the 1923 Nobel Prize in Chemistry.

Svedberg was a gifted engineer as well as a chemist and solved many of the problems involved in spinning a massive rotor at high and constant speed without vibration; his designs included arrangements to observe the contents of the rotor as it spun; to maintain the temperature constant by eliminating friction at bearings and having the rotor turn in a vacuum or a hydrogen atmosphere. An American, Jesse Beams of the University of Virginia, also contributed to determining the best materials and shapes for rotors and new designs for fast drives.

A Plateau

It was assumed that the basic, physical design for ultracentrifuges had been brought to its full development by the 1960s. Meanwhile, the more recently developed methods of electrophoresis and chromatography, though much less accurate for determining molecular weights (and unsuited for measuring mass distributions, molecular interaction constants, and other molecular characteristics), were found to be easier, faster in separating species, and used simpler and less costly equipment. This led many to consider these methods preferable to the ultracentrifuge. As a consequence, fewer ultracentrifuges were sold-and fewer molecular biologists were in training to perform analytical ultracentrifugation.

On the other hand, the new, burgeoning research in recombinant DNA technology resulted in the production of large numbers of supramolecular biological objects: virus assembly systems, repressor-DNA interactions, multi-enzyme complexes, and ribosomes, to name a few. The time was ripe for action to simplify and streamline the operations of the analytical ultracentrifuge. And ultracentrifugation was still alive and active in the Biophysical group at the University of Connecticut (UConn), where equipment, faculty, experienced investigators, and quality students were engaged in the work.

A New Horizon in 1986

A workshop on the topic at the 1986 annual meeting of the Biophysical Society set the stage for a new departure. Significantly, a third of the scientists attending it were, or had been, associated with UConn's Biophysics Department. The report of the workshop concluded that there should be foundation support for fundamental research in the field and that centers should be established "to perform analyses routinely, to act as foci for further technique development, and to train persons in the theory and methods of analytical ultracentrifugation."

The NSF Grant

As a result, the UConn Biotechnology Center applied for support of its program and obtained a number of used machines to augment its equipment; this was followed by a major National Science Foundation (NSF) grant to Emory H. Braswell, John Philo, and CASE members Todd M. Schuster and David A. Yphantis as principal investigators to establish and maintain a National Facility for Analytical Ultracentrifugation. The University having supplied faculty salaries as well as laboratory and office space, the objectives to be served by the grant would include: instruments, software engineering, collaborative research, and service and training. Early improvements included a pulsed laser light source, control circuitry to provide improved interference data, multiplexing capability (allowing up to twenty molecular weight determinations in a single run), an automated plate reader for rapid, high-precision reduction of interference patterns to digital form, a high resolution TV camera for real time data acquisition and analysis, and various improvements in optics and sample cells. In addition to software programs, developed largely by Dr. Yphantis and his students, the Facility has been able to make available programs for extracting molecular weight data, non-ideality and interaction coefficients, and other equilibrium parameters for associating systems. Accessory equipment for high precision density and differential refractometry measurements as well as general biophysical and biochemical laboratory facilities are now available to users of the Facility. Analytical Ultracentrifugation (AU) had a new beginning in the early 1990s with the development of better instrumentation, the availability of faster and higher capacity computers, and the development of new software programs for data collection.

At Storrs Today

The facility at Storrs now offers users: three Model E. Beckman Analytical Ultracentrifuges with UV-Visible Scanners and pulsed laser light sources which enable the multiplexing of interference experiments from several cells in a single experiment; three Beckman Optima XL-A ultracentrifuges with absorption optics; and a Beckman Optima XL-I ultracentrifuge which has both absorption and interference optics. In the optical system of the XL-A machines, the light passes through a monochromator so that wave length can be chosen for maximum absorbance or scanned over a range. The interference system is far more precise but may not allow the study of solutions as dilute as the absorption system. Temperature in both ultracentrifuges is controlled by the method known as the Peltier effect (named after the French scientist who in 1834 discovered that, at the junction of two dissimilar metals, an electric current will generate either heat or cold, depending on the direction of current flow). For data acquisition and manipulation there is a Digital Alpha Computer, two older Digital machines and five advanced PCs. In addition, the facility has a Paar six-place density meter.

Today, a typical titanium rotor used in late model ultracentrifuges, employed at the UConn's National Facility for Analytical Centrifugation rotates safely and quietly (and for extended periods) at speeds up to 60,000 rpm. This generates a centrifugal field about 250,000 times as strong as the gravitational field at the Earth's surface, so that a mass of one gram in it experiences an apparent weight of a quarter-ton!

Not Just Molecular Weights

The accurate determination of molecular weights of large molecules in solution is one of the objectives for which the AU has proven an important tool. The justification for this application comes from the following analysis based on a simple mechanical model of sedimentation.

In the centrifuge a solute particle is subject to a sedimenting ("gravitational") force Fs proportional to the mass of the particle and the acceleration; in a spinning rotor the latter is determined by the distance of the particle from the axis of rotation and the square of the angular velocity. The sedimenting force is quickly balanced by a combination of buoyant and frictional forces (Fb and Ff) as the particle moves in the viscous solvent. By assuming equilibrium (at which the particle has no vertical velocity), it is possible to combine the defining equations for these forces to derive a term known as the sedimentation coefficient, which can be used to calculate the molecular weight of the solute.

The molecular weight is probably only the first measurement in an AU study, since that will probably deal with a system less simple than the "single" particle model described above. A case in point is a study by two members of the UConn staff in which the solute was a pure sample of a polypeptide (proline-proline-glycine)10 which was equilibrated at 52000 RPM at 10°c and at 30°c. Analysis of these runs showed that going from the higher to the lower temperature, the apparent molecular weight rose greatly. A test of the proposition that the sample was now a mixture of monomer and trimer yielded the following: that the monomer -> trimer association constant was 0.14 at the higher temperature and 187 at the lower. This "melting" and "freezing" phenomenon is very clear in the automatically produced graphical results.

Collaborative Research

As the hardware and software projects have approached completion, the emphasis of the Facility has shifted increasingly to a collaborative research program under the direction of Emory J. Braswell. Among scientists who have recently made use of the facility are individuals from 11 industrial firms, such as Gillette, Hoffman-LaRoche, Pfizer, Schering-Plough, and DuPont. Industrial research is conducted at the expense of the firm using the facility, and the results are the property of the industry that paid for them. Collaborative research projects have been pursued recently with experimenters from Rutgers, Princeton, Washington University, the University of Alabama, Northeastern University, Rockefeller University, the University of Massachusetts Medical Center, and Wayne State University. The Facility also holds a three-day AU workshop in the spring of each year conducted by Todd M. Schuster in which the latest laboratory techniques and analyses are taught. This is followed by a one-day symposium in which leading scientists in the field discuss the latest theoretical and experimental developments.

In addition to these and additional collaborative research efforts, the NSF grant has also resulted in twenty-one publications in ten journals, including Biochemistry, the Journal of Protein Chemistry, the Journal of Biological Chemistry, the International Journal of Peptide and Protein Research, the Journal of Molecular Biology, Protein Science, Pharmaceutical Research, the European Journal of Biochemistry, Proceedings of the National Academy of Sciences (USA), and a section of a book entitled Modern Analytical Ultracentrifugation.

In sum, it would appear that the vigorous rebirth of interest in ultracentrifugation, as reflected in the success of and ongoing support for UConn's national research facility, has been energized by a number of disparate factors, including the following:

With respect to the last factor, there are now several sites on the Internet which act as mail-servers offering downloadable software and even tutorials on methods of interpretation of data. Interested readers can learn more about the National Facility for Analytical Ultracentrifuga-tion at the University of Connecticut at the facility's Web site: http://www.ucc.uconn.edu/~wwwbiotc/uaf.html. In addition, the Beckman Home Page, located at http://www.beckman.com, might be of interest for its lists of scientific papers and descriptions of techniques.--Arthur Ross, freelance science writer.

 

Some of the substances which have been studied at the University of Connecticut's National Facility for Analytical Ultracentrifugation include the following:


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