Analytical Ultracentrifuges: A Reinvention

Largely unchanged since the 1960s and virtually ignored by researchers over the past decade, the analytical ultracentrifuge has been the focus of energetic new interest in the past few years. Recently, a redesigned--nearly reinvented--version of the instrument has been released, according to a small, tightly knit group of molecular biologists. The new instrument, intended mainly for characterizing proteins and nucleic acids, has already helped to shatter the working models of some important biological problems and to point research efforts in promising new directions, especially in areas of drug discovery. Reasons given for the resurgence of the analytical ultracentrifuge vary. Some say the field withered because the commercially available technology stagnated almost 30 years ago. Those who wanted to continue working with the tool had to take on many of the upkeep tasks and the needed modifications themselves. But others say the kinds of questions asked in biology changed in the early 1980s, leading researchers away from the analytical ultracentrifuge, and that those questions now have changed again, encouraging a reappraisal of the instrument. "The nature of research changed from a very quantitative period in the '60s and '70s to a very qualitative one," says Howard Schachman, a molecular biologist at the University of California, Berkeley, who has worked with centrifuges since the early 1950s. "[In the '80s], if you isolated a wonderful gene repressor, you just worried about whether it interacted with a specific DNA or it didn't interact. Now, you really want to know how strong that interaction is. And that you can't do by other techniques." For investigators who can't afford a $100,000 analytical ultracentrifuge, National Institutes of Health researcher Allen Minton, with colleague Arun Attri, has developed a much less expensive device to derive much of the same information from a preparative centrifuge. It's called the automated centrifuge tube microfractionator, engineered by Biomed ical Research and Development Laboratories Inc. (BRANDEL), in Gaithersburg, Md., and available from BRANDEL for about $4,200. "The device removes very small fractions sequentially from the top of the [centrifuge] tube," says Minton. Once removed from the tube, the sequential samples can be subjected to various assays--or example, of radioactively labeled molecular species. "Then we plot the relative concentrations as a function of radial distance," Minton says. "That gives us back our original gradient." --F.H. Microfractionator: BRANDEL Inc. 8561 Atlas Dr. Gaithersburg, Md. 20877 (301) 948-6506 Fax: (301) 869-5570 Preparative centrifuges that support the microfractionator: Beckman Instruments Inc. 2500 Harbor Blvd. Fullerton, Calif. 92634 (714) 871-4848 Fax: (714) 773-8898 DuPont Co.(SORVALL) Barley Mill Plaza P.O. Box 80022 Wilmington, Del. 19880-0022 (800) 551-2121 Fax: (302) 892-0719 Optima XL-A analytica ultracentrifuge: Beckman Instruments Inc. Attn: Ken Johnson Mail Stop Y-141050 Page Mill Rd. Palo Alto, Calif. 94304 (415) 859-1501 The old instrument, the Model E ultracentrifuge from Beckman Instruments Inc., Palo Alto, Calif., was based on work done in the 1930s by Sweden's The Svedberg. It was introduced shortly after World War II and was never seriously challenged by competitors in following decades, say researchers who have worked with it. The Model E was called an ultracentrifuge because it was able to spin at extremely high speeds, resulting in forces 400,000 times that of gravity. And it evolved into the analytical ultracentrifuge with the addition of optical systems--such as absorption and interferometry systems--for direct analysis of the molecules during centrifugation. The new instrument, also from Beckman and also without competition currently, is the Optima XL-A analytical ultracentrifuge. It is the result of the efforts of a group of "diehard" researchers faithful to the Model E, according to Ken Johnson, Beckman's product marketing manager for the ultracentrifuge line. "The analytical ultracentrifuge was deemed a dead technique," says Johnson. "But people who wanted to do this work continued to keep the Model E operational, and they pointed out the necessity for that type of instrument to Beckman. They were very helpful to us in advising what the new instrument should look like and working with us to make sure that we did our homework." The outcome is an instrument that is smaller than the old Model E, and reliably capable of high, constant speeds. Its optical system is also extremely stable and precise. But the biggest advance is that computer control and data acquisition have entered the picture. One result is that the power of centrifugation techniques will become available to a wider range of biological researchers. "You don't have to be a centrifuge jock anymore," says Allen Minton, a physical biochemist with the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, Bethesda, Md. "In the past, it was the province of people who committed their whole lives to the care and well-being of an analytical ultracentrifuge--very exotic types." Of course, because the price tag is about $100,000, not every lab will be able to afford one. The main use of the analytical ultracentrifuge is to precisely characterize large biological molecules like proteins--called macromolecules. This is done in two basic ways. "One technique is equilibrium sedimentation, for measuring molecular weight," says Preston Hensley, senior investigator in the department of macromolecular sciences of SmithKline Beecham Pharmaceuticals in King of Prussia, Pa. "Basically, if you spin at intermediate rotor speeds, you get an exponential distribution of protein in the sample cell, and that distribution is proportional to molecular weight. The other technique, velocity sedimentation, gives you shape information. There, you're looking at the hydrodynamic properties of the molecules, which are the result of a combination of molecular weight and shape." Measuring molecular weight is useful in tracking associations. In the simplest case, for example, when the weights of two molecules, A and B, in solution are known, and an amount of a complex at the weight of A plus B is discovered, then inferences about the association of A and B can be made. And the shape of a molecule can be very important in predicting its biological actions. Both characteristics are important in understanding molecular associations, aggregation states, and assembly processes, which, in turn, have implications in drug discovery efforts, for example. One strong point of the analytical ultracentrifuge for biological researchers, says Minton, is its ability to study interactions among molecules in solution at chemical equilibrium. This, he says, is a feature of the equilibrium sedimentation technique. In the case of weaker, reversible associations--such as those involved in control mechanisms in biology--this is especially important. In these instances, any perturbation of the chemical equilibrium--any change in the amount of free or complexed molecules in the solution--will result in further associative changes and, potentially, misinterpretations. "The kinds of interactions we're studying are those where the product and the reactants are in dynamic equilibrium," says Minton. "That is, the complex is constantly dissociating and the reactants associating." There are simpler, less expensive techniques available to measure macromolecular associations than those involving the analytical ultracentrifuge, including polyacrylamide gel electrophoresis and gel permeation chromatography. Their disadvantage, say researchers, is that they are less precise than the analytical ultracentrifuge. This lack of precision, not surprisingly, sometimes leads to approximate results, but it can also cause researchers to construct models of biological problems that are simply inaccurate and, therefore, misleading. "Not only are these methods approximate," says Hensley, "but they have tragic flaws. It's not just that the results are off by 10 percent--the whole vision of the problem can change." For example, says Hensley, his company investigated a molecule called E-selectin, important in the adhesion of white blood cells to the endothelial cells lining the walls of blood vessels. Understanding the binding mechanisms of E-selectin and its receptors might lead to a better understanding of inflammation or the underlying events leading to asthma, the researchers felt. They found initially, by using gel permeation chromatography, that the molecule's weight was 260,000 daltons (a measure of atomic mass). They knew, from amino acid composition studies, that the component monomer involved weighed about 58,000 daltons, so the implication was that the protein was in a high state of aggregation as an oligomer--that four of the 58,000-dalton units were in association. "So, there are, potentially, four binding sites on the protein interacting with four binding sites on the cell," says Hensley, adding that this arrangement, called a multivalent interaction, presents a very complex problem to researchers. "In fact, there is no theory for understanding the affinity of these interactions," he says. After a thermodynamically rigorous molecular weight and shape analysis in the analytical ultracentrifuge, the researchers found that the actual weight of E-selectin was 80,000 daltons, rather than 260,000 daltons, and that the shape of the molecule was more like a cigar than a sphere, which had been a necessary supposition of the first technique. The shape information was especially important, because a cigar-shaped molecule moves through solution very differently from the way a round one does, in this case mimicking a heavier molecule. The new view of the molecule and its binding action, as a one-to- one interaction rather than a four-to-four interaction, allowed the researchers to reshape and greatly simplify their thinking about binding studies involving this protein. Researchers using the new analytical ultracentrifuge are enthusiastic about what it can do and predict a bright future for it in biochemistry laboratories. "We feel like we're part of a renaissance," says Minton at NIH. "The most exciting thing is that it's really becoming a technique that a wide variety of people will feel comfortable using." Also, the resurgence of interest in the analytical ultracentrifuge and a recognition of the importance of its capabilities have helped to encourage the larger pharmaceutical research laboratories to add new research departments, says Hensley. "The drug companies--and the biotech companies, too--have historically had at least two major components," says Hensley. "One is a department of molecular genetics. These are the guys who play with the genes that make proteins. And then there's usually a group doing nuclear magnetic resonance and crystallography. These are the guys doing high-resolution structure determinations. Now, companies are forming departments like ours, macromolecular sciences." "If you are interested in how proteins interact with one another, or with themselves," says Schachman, "this is the tool for that kind of question. And that's what people want to know now."

Analytical Ultracentrifuges: A Reinvention

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