A new beta-elimination procedure has been introduced to cleave O-linked oligosaccharides from low- to sub-microgram amounts of glycoproteins prior to analysis by mass spectrometry. Borane-ammonia complex in aqueous ammonia is used as a cleaving solution alternative to the sodium borohydride/sodium hydroxide medium conventionally used in beta-elimination. The procedure results in minimum sample purification, leading to minimal sample loss and consequently an overall enhancement in sensitivity. It was applied successfully in the analysis of bovine fetuin and submaxillary mucin, as well as to a complex bile-salt-stimulated lipase glycoprotein isolated from human milk.
The first and second reduction (E1⁄2.1red, E1⁄2.2red) and the first oxidation (E1⁄2.1oxd) standard potentials of benzenoid alternant hydrocarbons (BAH) were experimentally determined by means of cyclic voltammetry (CV) in nonaqueous solvents. The second standard oxidation potential (E1⁄2.2oxd) was however, estimated by checking the scan rate dependence of the irreversible CV curve. The equations pertinent to these potentials and their mutual relations were formulated from the points of view of the Born–Haber-type thermodynamic energy cycle and SCFMO calculations. Of the SCFMO calculations, the MO-paring property established in the PPP-type π-electron theory was very successful in the discussion of the equations given above. Under the acceptable assumptions that the solvation energies due to mono- and dications are put equal to those of the mono- and dianions respectively, and using the MO-pairing property, the equation of (E1⁄2.1oxd+E1⁄2.1red)=(E1⁄2.2oxd+E1⁄2.2red) was derived. The experimental results were well described by this equation. The solvationenergy values were evaluated by applying the experimentally determined reduction or oxidation potentials to the theoretical equations. An examination of the solvation energies has shown that these values can be interpreted by means of the Born-type equation.
Different macroporous, monolithic capillary columns were prepared to separate various bile acid mixtures through capillary electrochromatography (CEC) at high efficiency. These columns are shown to be ideally suitable for coupling to an electrospray ionization/ion trap mass spectrometer. Detection and structural identification of different bile acid derivatives in either the positive- or negative-ion mode necessitated column technologies with different polarities and the capabilities of a reversed electroosmotic flow. High column efficiencies (610,000 theoretical plates/meter for glycocholic acid in normal-phase separation) were preserved in the coupling to mass spectrometry (MS), with the detection limits of approximately 40 femtomole (for cholic acid) and identification through CEC/MS/MS.
The electrochemical behavior of adriamycin has been studied by means of cyclic d.c. and a.c. voltammetry with a hanging mercury drop electrode, quinizarin being used as a model compound. Both adriamycin and quinizarin are strongly adsorbed on the mercury electrode with their aromatic ring planes oriented parallel to the electrode surface, and give two sets of reduction waves. The first wave, due to the quinone moieties of the adsorbed adriamycin and quinizarin, has been explained on terms of a two-step one-electron surface redox reaction. The formal standard redox potentials, semiquinone formation constants, and charge transfer rate constants of the surface redox reaction of the quinone moieties have been determined. Although the reduced form of adriamycin is not very stable chemically, it is converted to a stable and electrochemically active form for a few minutes at pH 4.54. The second wave appearing at more negative potential would be due to a kinetic or catalytic process.
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