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New Procedure Lets Scientists Probe Short-lived Molecules

Date:
March 4, 2003
Source:
National Science Foundation
Summary:
Some of the most important compounds are the shortest lived -- transient molecules that exist for only thousandths of a second or less during chemical reactions. Characterization of such "reaction intermediates" can play a key role in understanding the mechanisms by which molecules change, shedding light on processes ranging from basic chemical reactions to complex diseases such as Alzheimer's. Yet by their very nature, reaction intermediates exist for brief periods too short to be seen by most sensors.

ARLINGTON, Va. – Some of the most important compounds are the shortest lived -- transient molecules that exist for only thousandths of a second or less during chemical reactions. Characterization of such "reaction intermediates" can play a key role in understanding the mechanisms by which molecules change, shedding light on processes ranging from basic chemical reactions to complex diseases such as Alzheimer's. Yet by their very nature, reaction intermediates exist for brief periods too short to be seen by most sensors.

Now, in the March 18 Proceedings of the National Academy of Sciences, NSF CAREER grantee Jason Shear and his graduate student Matthew Plenert describe a method for taking quick, microsecond snapshots of these intermediate molecules before the structures change into their more stable end products.

"This research continues in the tradition of elegant experiments yielding insights about the chemistry of the complex environment of living cells," said Janice Hicks, the NSF program officer who oversees Shear's grant. "This is another excellent example of analytical chemists contributing new methods that foster discovery in the biological realm," she added.

The new method, based on a technique called "capillary electrophoresis," was developed in the Shear research laboratory at the University of Texas at Austin. Unlike some electrophoresis techniques researchers use for DNA sequencing, where an electric field draws charged molecules across a gel plate, capillary electrophoresis is performed in needle-thin, glass-like, fused-silica tubes, where very large electric fields draw the molecules along without the heating that would exist in a gel.

To further increase the electric field in their procedure, Plenert and Shear have altered the capillary, creating a narrowed zone within an hourglass-shaped structure. The researchers perform the analyses at the most narrow region, where the diameter is only 5 micrometers (5 millionths of a meter) and the electric field can be greater than 100,000 volts per centimeter.

The chemicals are separated over a distance of about 10 micrometers (about one tenth the width of a human hair), with smaller, more positively charged pieces moving at faster rates than the larger, more negatively charged ones, thereby isolating the various components.

"Scientists interested in probing transient molecules commonly rely on very fast methods," said Shear, "an approach that can be difficult when analyzing chemical mixtures." And, he adds, while mixtures of stable molecules can be separated into individual components, this strategy can take minutes or longer -- far too long to characterize unstable reaction intermediates.

In this initial experiment, Plenert and Shear examined a solution containing the neurotransmitter serotonin and its metabolic precursor, a compound known as hydroxytryptophan, using the electric field to draw the solution through the capillary.

To produce the short-lived reaction intermediates from the serotonin and hydroxytryptophan, Shear and Plenert hit the mixture with a microsecond blast of laser light when the solution passes through the capillary's hourglass section. Ten micrometers further down the capillary, a probe laser hits these intermediates -- now spatially segregated into two groups according to their molecular parent -- causing them to emit light.

The molecules migrate between the two laser spots approximately 100-million times faster than in conventional separation procedures.

"We think it may be possible to do these analyses in less than a microsecond," said Plenert, "although ultimately there's a limit to how large of an electric field can be used and how small of a distance is adequate to distinguish differences between molecule velocities."

Next, the researchers are interested in applying their technique to study how proteins fold into functional molecules. They hope to determine changes in molecular shape as a protein evolves from an unfolded to a biologically active form.

The intermediates may offer clues into how proteins take shape, a vital area of study for understanding how proteins fold incorrectly in the brain tissue of people stricken with Alzheimer's and other neurodegenerative diseases.

"Understanding a process as complex as protein folding requires application of numerous experimental and theoretical tools," said Shear. "We think that an ability to probe properties of shortlived molecules, potentially in complex mixtures, can play an important role in attacking this problem," he said.

In addition to the National Science Foundation CAREER award, the researchers have developed their techniques using support from the Robert A. Welch Foundation, Eli Lilly, and the Searle Scholars program.


Story Source:

The above story is based on materials provided by National Science Foundation. Note: Materials may be edited for content and length.


Cite This Page:

National Science Foundation. "New Procedure Lets Scientists Probe Short-lived Molecules." ScienceDaily. ScienceDaily, 4 March 2003. <www.sciencedaily.com/releases/2003/03/030304073011.htm>.
National Science Foundation. (2003, March 4). New Procedure Lets Scientists Probe Short-lived Molecules. ScienceDaily. Retrieved October 23, 2014 from www.sciencedaily.com/releases/2003/03/030304073011.htm
National Science Foundation. "New Procedure Lets Scientists Probe Short-lived Molecules." ScienceDaily. www.sciencedaily.com/releases/2003/03/030304073011.htm (accessed October 23, 2014).

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