Rather than using native sGC heme, which is difficult to isolate in good yields and pure form, the researchers collaborated with Michael Marietta and Yunde Zhao (also at the University of Michigan) to produce a high yield of heme domain by overexpressing the N-terminal fragment of the heme-binding subunit of sGC in Escherichiiaoll "We needed to be able to produce a suitable quantity of [sGC] to actually make the sensors with. We took only the important part of the molecule—the nitric oxide binding part and lost the peripheral protein," explains Barker. Because the heme domain is much smaller (44 kDa) than native heterodimeric sGC (150 kDa) overexpression of the heme domain yielded a greater number of NO binding sites per unit mass The heme domain was labeled with the fluorescent reporter dye Oregon Green 488 carboxylic acid, succinimidyl ester. Oregon Green responds to changes in the protein's conformation after the protein has bound to NO. Gold-coated optical fibers (100-um core diameter) were immersed in the dyelabeled heme domain solution, allowing the peptide to adsorb to the surface of the gold.
Shaping up The physical and chemical properties of nanoparticles are influenced by both the size and the shape of the particles. Size exclusion chromatography (SEC) has already been used to separate nanospheres of different sizes. Although the ability to make nanoparticles of specific shapes has improved, most techniques produce a mixture of shapes. Now GuorTzo Wei, C. R. Chrrs Wang, and du-Ken Iiu of the National Chung-Cheng University (Taiwan) hope to use SEC to separate nanoparticles of different shapes (Anall Chem. 1999 71, 2085-91). One of the problems with trying to analyze nanoparticles by SEC is the irreversible adsorption of the particles on the stationary phase caused by the large surface area of the packing material. The authors had previously found that the addition of the surfactant sodium dodecyl sulfate (SDS) to the mobile phase decreased the amount of adsorption. When they used water as the eluent to separate a mixture of differently shaped nanoparticles (rods and spheres) by SEC, the adsorption was so severe that no sig-
Detection of nitric oxide produced by macrophages with an optical sensor based on the heme domain of soluble guanylate cyclase.
Fluorescent polystyrene reference spheres coated with a hydrophilic polymer, which contained carboxylic acid groups, were then added to the fiber. The sensor response is based on the ratio of the fluorescence intensity of the dye to the fluorescence intensity of the reference spheres. By using this ratiometric approach rather than measuring absolute intensities, signal changes due to optical variations or light scattering can be distinguished from changes in analyte concen-
tration. "Changes in the alignment of the laser or the light setup of the microscope don't matter, because they affect the reference signal and the analyte signal equally," explains Kopelman. The sensors were used to measure extracellular NO released by BALB/c mouse macrophages. In essence, these white blood cells use NO as a chemical weapon against pathogens, says Kopelman. Minimal NO was released from untreated cells; whereas high levels of NO (111 uM) were
nal was observed at all, indicattng that even nanoparticles that have been stabilized by the addition of cationic surfactant can encounter problems with irreversible adsorption. Although adding SDS to the eluent reduced the adsorption problem, it had little effect on the separation. The authors found that adding the nonionic surfactant Brij-35 with the SDS allowed nanoparticles of different shapes to be observed, but the resolution was very low.
'With the addition of Brij-35, we apparently induce surface adsorption [to a certain extent], depending on the concentration of Brij-35 being added," says Wei. "With SDS and Brij-35, the separation is a combination of steric effects and adsorption effects. SDS is required to eliminate the surface adsorption and Brij-35 is added to improve the shape separation by introducing an adsorption factor that is shape-dependent in the separation." The separation can be optimized byfixingthe SDS concentration at 40 mM nmd sdjustinff the Brij-35 conhigh as possible without causing baseline deterioration With a mixture of 40 mM SDS and 30 mM Brij-35, the difference in the retention times was 0.50 min. The rods and spheres were not baseline-resolved, but the detector was a UV-vis diode array, which allowed a spectrum to be collected that assisted in interpreting the chromatogram. Wei suggests that the SEC was inadequate because the gross size difference between the rods and spheres was insufficient for them to be separated effectively. Celia Henry
A transmission electron micrograph of a mixture of nanorods and nanospheres.
Analytical Chemistry News & Features, June 1, 1999 3 7 3 A
