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A protein's on/off switch The wild-type green fluorescent protein (GFP) of the jellyfish Aequorea victoria has The switching behavior of a aingle T203F molecule. In "A" frames the sample was pre-illuminated with 488-nm generated much inter- radiation for 90s to prepare the dark ktate, followed by 5 min with no illumination, at which point the tmage was recorded. .n "B" ffames the ere-illumination was followed by 5 min of irradiation at 405 nm, immediately after whicw est as a possible probe the image was recorded. (Adapted with permission. Copyright 1997 Macmillan Magazines.) molecule, but mutant varieties may prove even more exciting. W. E. Moerner and emission. The blinking pattern continued Moerner believes that the blinking co-workers at the University of California- for several minutes. However, after the behavior could pose problems for use of San Diego studied two GFP mutants that emission of about a million photons, the these particular mutants as analytical tools exhibited unusual fluorescence behavior: molecules reached a long-lived dark state. but that the switching behavior could The proteins could be switched between Even after 5 min with no pumping, the prove very useful. The dark state could be fluorescent and dark states. When ningle resumption of 488-nm irradiation proprepared and then selectively turned on in molecules of the mutants designated duced no emission. The emissive state the cellular region of interest. Moerner T203F and T203Y were excited at 488 nm could be recovered by irradiating the mol- sees the possibility of engineering differthe moleculesfluorescedfor several sececule for 5 min with 405-nm light before ent mutants for specific analytical uses. onds followed by several seconds without excitation at 488 nm. {Nature 1997,388, ,35558)
Hybridized DNA characterized by MALDI-TOF The hybridization of oligonucleotides onto planar surfaces has resulted in various synthetic combinatorial products. Characterization of the resulting products is often carried out by radiography or fluorescence analysis. Robert Levis and Gowri Narayanaswami at Wayne State University evaluate MALDI time-of-flight (TOF) MS as an alternative method for assaying DNA from hybridization surfaces. In their experiment, an oligonucleotide containing a known sequence is attached to a planar surface. A second oligonucleotide, complementary to the first and referred to as the target oligonucleotide, is
Formation of a hybridized oligonucleotide on a planar surface.
hybridized with the original from a dilute aqueous solution. A high concentration of the hybridized species gathers at the surface. The resulting product is assayed by MALDI-TOF. 586 A
Electrochemical assay for proteinase inhibitor Human proteinase inhibitors are commonly analyzed by immunological assays or chromatographic methods, both of which require complicated sample preparation procedures. Mark Meyerhoff and co-workers at the University of Michigan have developed a polyion-selective electrode for use in the analysis of trypsin-like proteinase inhibitors. The electrode consists of a polycation-sensitive membrane containing dinonylnaphthalene sulfonate (DNNS), which responds to protamine, a substrate for trypsin-like teinase inhibitors. In a typical assay, solutions containing a mixture of proteinase-antiproteinase, varying in the amount of antiproteinase, are added to the sample. The rate of decrease in membrane re-
Control experiments confirmed that the observed mass spectral signal was because of the hybridization product and not the initial oligonucleotide tethered to the plastic surface. In addition, nonspecific binding was investigated by attempting to bind noncomplementary strands with the original oligonucleotide. In both cases, no mass spectral signal was observed, confirming that the
Analytical Chemistry News & Features, October 1, 1997
sponse is then monitored. The initial rate of loss of protamine depends on the amount of proteinase inhibitor in the sample. Meyerhoff and co-workers tested the method with four trypsin-like proteinase inhibitors. Upon addition of trypsin to the samples, the initial rate of decrease in membrane response was directly proportional to the concentration of inhibitor in the sample. Detection limits were well below the levels found in normal plasma levels. Aprotinin, a nonspecific proteinase inhibitor, was also assayed by this potentiometric detection method to determine which proteinase offers the lowest detection limit for aprotinin. In addition to trypsin kallikrein and plasmin were investigated Trypsin-based inhibition offered the lowest detection limit and greatest sensitivity for the quantification of aprotinin (Anal Biochem .997 250 74481)
signal was the result of the hybridization product. Mass spectral analysis of hybridization arrays provides a second dimension of information. In addition to providing information pertaining to the location of positive binding events, significant diagnostic information can be obtained from the mass spectrum of the hybridization "pixels". (J. Am. .hem. Soc. 1997,119, 6888-90)
