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| Protein biochemistry: Test tubes go microscopic
Microscopic 'test tubes' have permitted researchers to accurately quantify the efficiency of single enzyme molecules. Researchers in Japan have created arrays of microscopic 'test tubes' that permitted them to accurately quantify the efficiency of single enzyme molecules. Scientists seem to be obsessed with performing experiments at increasingly smaller scale in an effort to probe a system's innermost workings or reduce reagent usage in high-throughput experiments. Many of us have experienced the agony of trying to pipet ever-shrinking sample volumes into arrays of test tubes. Now, in the ultimate pipetting nightmare, researchers at the University of Tokyo report in Nature Biotechnology the creation of arrays containing hundreds of microchambers, or test tubes, that are measured not in microliters or even nanoliters, but in femtoliters (Rondelez et al., 2005a). Because of the many favorable characteristics of poly-dimethylsiloxane (PDMS), Noji and colleagues chose this widely used material to create these femtoliter chambers. Using technology similar to that used to make microchips, the researchers prepared a silicon wafer mold with arrays of micron-sized cylindrical protrusions. They used this as a template to mold sheets of PDMS containing hundreds of micron-sized chambers that hold as little as 1.4 femtoliters of liquid when filled. Fortunately for researchers interested in using this technology, it's not necessary to pipet into these microscopic 'test tubes'. All you have to do to fill them is place a drop of your sample on a glass slide, lay the patterned PDMS sheet upside-down on the slide and press it down. They discovered that this alone is enough to effectively seal the liquid into the individual chambers and prevent cross-contamination. These microchambers quickly became handy in Noji's lab, which is interested in studying the properties of single enzymes and how these properties influence function. So far, when examining single enzyme molecules, it had been impossible to study product accumulation because the product could not attain concentrations high enough to be measured. But Noji and colleagues reasoned that with femtoliter chambers this would be possible, and to test this, they added a low concentration of the enzyme
F1-ATPase is a tiny rotary enzyme, which produces ATP that serves as a major store of potential chemical energy in many biological reactions. But the efficiency with which this enzyme produces ATP from ADP has never been accurately measured. Noji and colleagues used the microchambers to confine single F1-ATPase molecules and rotated the enzyme with magnetic tweezers to produce ATP (Rondelez et al., 2005b)*. Because the ATP remained in the microchamber with the enzyme, once they stopped rotating the enzyme, it used the ATP to rotate in the opposite direction (). By comparing the number of rotations in each direction, they were able to accurately measure the efficiency of this enzyme for the very first time. This clever experiment demonstrates quite clearly the potential of these tiny 'test tubes' to examine the function of individual enzymes. Daniel Evanko Footnotes*
NOTE: In the version of this article originally published, page numbers for the second reference were listed incorrectly. The correct reference is: Rondelez, Y. et al. Highly coupled ATP synthesis by F1-ATPase single molecules. Nature 433, 773-777 (2005b). The corrected verison of this article is available in the HTML and PDF format. An erratum will be published in the May issue of the print version of the journal. We regret these errors. References | |
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-galactosidase to the chambers along with a substrate that becomes fluorescent when cleaved by the enzyme. Within 60 seconds they could see a discrete level of fluorescence in each of the chambers. When they plotted the increase in fluorescence over time for each chamber, it became apparent that each one contained zero, one, two or three enzymes. It was clear that these chambers would be ideal for studying their enzyme of choice, F1-ATPase.