No abstract

Determination of proteinases--enzymes that catalyze the hydrolysis of peptide bonds--is often difficult due to the presence of interferences in complex biological media and limited sample size. Capillary electrophoresis (CE), with laser-induced fluorescence (LIF) can serve as a useful tool for such determinations. LIF detection offers the advantages of increased sensitivity and increased selectivity. However, direct LIF detection requires the proteinase analyte to be fluorescently derivatized prior to analysis. A viable alternative is offered by the present work, in which protein substrates are first labeled with BODIPY dye, a relatively pH-insensitive, high-fluorescence quantum yield dye. Upon binding of some 4-10 molecules of dye to a single protein, the dye is effectively fluorescence-quenched. 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. We will present how the fragmentation pattern of BODIPY-labeled casein changes as a function of incubation time with trypsin, as well as the effect of varying concentrations of trypsin on the BODIPY-casein digest.

Capillary electrophoresis (CE), although a powerful analytical tool, has found only limited application in undergraduate laboratory study. In an effort to expose freshman and sophomore chemistry students to this technique, thereby giving them practical instrumental experience early in their careers, we propose to use CE in the analysis of student-synthesized acetylsalicylic acid (ASA). The synthesis of ASA from salicylic acid (SA) is a routine undergraduate laboratory, although students rarely have the opportunity to test the purity of their product. The CE method described herein provides students with a method to test purity and yield of their product and to determine the effect of aging on their sample. CE can accomplish this in a short period of time, with minimal disruption to the regular laboratory curriculum. Optimized separation conditions, limits of detection, and linear range for ASA and SA are also given.