Protein analysis by capillary electrophoresis with laser-induced fluorescence detection: Some applications of covalent and noncovalent fluorescent probes.

摘要

Capillary electrophoresis (CE) has become a respected analytical technique in the field of separation science. The use of CE has become widespread in the areas of environmental science, biochemistry, clinical diagnostics, and many others. Speed and separation efficiencies far surpassing those of typical HPLC analysis have given CE an added advantage relative to many common separation techniques.; CE, although a powerful analytical tool, has found limited application in undergraduate laboratory study. In an effort to expose freshman and/or sophomore chemistry students to this technique, thereby providing them with practical instrumental experience early in their academic careers, a method using CE to analyze student-synthesized acetylsalicylic acid (ASA), was developed. CE can accomplish this in a short period of time, with minimal disruption to the regular laboratory curriculum.; Determination of proteinases is often difficult due to the presence of interferences in complex biological media and limited sample size. CE with laser-induced fluorescence (LIF) detection can serve as a useful tool for such determinations. The work presented involves protein substrates labeled with BODIPY dye, a relatively pH insensitive, high fluorescence quantum yield dye. Digestion of the BODIPY labeled and quenched protein by an unlabeled enzyme yields smaller peptide fragments in which the fluorescence of associated BODIPY tags is restored. Changes in the fragmentation pattern of BODIPY-labeled casein as a function of incubation time with trypsin, as well as the effect of varying concentrations of trypsin on the BODIPY-casein digest, are presented.; The nature of noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emission of which have been shown to be enhanced upon complexation with various proteins. By using a variety of buffer systems to effect changes in the pHs of the protein-dye mixtures, it was possible to explore the role of electrostatic interactions between dye and protein in bonding. NN127 showed promise as a pseudo-fluorogenic reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.