Matching amide bands with conformation
map contained cross peaks of out-ofphase vibrations. Because the amide protons of the various conformations are not exchanged at the same rate, the conformational contributions of the amide I and II bands could be separated. Myoglobin was the example, because the secondary structure consists mainly of a-helical and random coil structures, which give overlapping spectral contributions in the amide I region, and (3-turns. They determined that at least four conformations contribute to the amide I band of myoglobin—a-helical, intermolecular (3-sheet, (J-turn, and random coil. lAppl. Spectrosc. 1997,51,466-69)
The various amide bands in the IR spectra of proteins are sensitive to the secondary structure, but conclusive conformation assignments are difficult to make because amides with different conformations have bands that strongly overlap. Anne Nabet and Michel Pezolet of Universite Laval (Canada) used 2-D FT-IR spectroscopy to enhance the spectral resolution of the amide I and II regions. In this work, hydrogen-deuterium exchange of the amide protons provided the external perturbation to generate 2-D synchronous and asynchronous maps. The proteinfilmswere studied by attenuated total reflectance, and the H-D exchange was induced by a nitrogen stream that contained D20. The synchronous map gave autopeaks and cross (A) Synchronous and (B) asynchronous 2-D FT-IR spectra of peaks for in-phase myoglobin recorded 1 h after the beginning of the H-D vibrations whereas exchange. (Adapted with permission from the Society for the asynchronous Applied Spectroscopy.)
In vivo steroid measurements Steroid levels are usually measured in blood or urine samples by immunoassay, but this method involves a delay from sample to analysis. Immunoelectrochemical sensors combine the specificity and sensitivity of immunoassays with an easy and rapid analytical procedure. Christian J. Cook of the Meat Industry Institute of New Zealand and HortResearch described a sensor that combines an immunoenzyme electrode with a dialysate membrane for real-time in vivo measurement of corticosteroids. The detection system is based on a competitive reaction of natural corticosteroids and peroxidase-corticosteroid conjugates with antibodies immobilized on a platinum electrode After the steroids bind to the surface the peroxidase reaction is initiated and the substrate oxidation is monitored Surrounding the
electrode with a dialysis membrane allowed continuous measurement and eliminated the need for sample removal. The sensor was used to measure Cortisol and corticosterone. The concentration and probe potential were inversely related for both steroids, and potential changes predicted free, bound, and mixed corticosteroid solutions. Higher antibody concentrations extended the range of applicable steroid concentrations and increased the number of measurements that could be made with no drop in sensitivity. The sensor was inserted into the jugular vein to make in vivo measurements over 48-h periods of corticosteroids in cattle, sheep, and rats. The in vivo levels correlated well with radioimmunoassay measurements of plasma samples. Cook is now seeking permission for human trials using the immunochemical sensor. (Nature Biotechnology 1997,15, 467-71)
How does Ru(phen)2+3 interact with DNA? Many therapeutic agents target DNA, binding along the major or minor groove or intercalating between base pairs. Understanding the DNA-ligand interaction mechanism at the molecular level would facilitate drug design. However, determining the binding mode is not always straightforward: Detailed memods such as X-ray diffraction and NMR are often precluded, and other methods are indirect Lawrence A Bottomley, Loren Dean Williams, and co-workers at the Georgia Institute of Technology developed a scanning force microscopy assay that measures the length of individual DNA molecules immobilized on a two-dimensional surface and determined the mode of interaction with the premise that intercalating ligands necessarily lengthen DNA whereas groove-binding ligands do not They applied the SFM assay to determine the binding mode of the low bindingaffinity ligand tris-(o-phenanthroline) ruthenium (II), which has remained ambiguous. Some studies suggest that both the A- and A-ruthenium-phenanthroline complexes bind to DNA via intercalation; other studies suggest that the A form prefers intercalation but that the A form prefers surface binding. Still other results indicate that neither form intercalates. The Georgia Tech researchers applied their method to answer this stubborn question. DNA samples incubated at three relatively high Ru(phen)|+ concentrations did not exhibit lengthening in comparison to control samples, indicating that, surprisingly, neither enantiomer of the ligand intercalates. (J. Am. Chem. Soc. 1997, 119, 3792-96)
Representative SFM image of a DNA molecule.
Analytical Chemistry News & Features, July 1, 1997 3 9 7 A
