Christine H. Scaman

Analysis by capillary electrophoresis-laser-induced fluorescence detection of oligosaccharides produced from enzyme reactions.

Six structurally similar, fluorescently labeled oligosaccharides were baseline resolved by capillary electrophoresis (CE); laser induced fluorescence (LIF) detection gave detection limits of 50 molecules for the oligosaccharides. A simple design of the LIF detector that incorporates the advantages of high sensitivity, stability and ease of operation is described. The system was used to monitor enzyme products formed during the incubation of yeast cells with alpha-D-Glc(1-->2)alpha-D-Glc(1-->3)alpha-D-glc-O(CH2)8CONHCH2CH2NHCO - tetramethylrhodamine. This fluorescent trisaccharide is enzymatically hydrolyzed to fluorescent disaccharide, monosaccharide and the free linker arm that is used to conjugate the saccharides with the fluorophore tetramethylrhodamine.

Synthesis of α-d-Glcp-(1 → 2)-α-d-Glcp-(1 → 3)-α-d-Glcp-O-(CH2)8COOCH3 for use in the assay of α-glucosidase I activity

The chemical synthesis of alpha-D-Glcp-(1-->2)-alpha-D-Glcp p-(1-->3) -alpha-D-Glcp-O-(CH2)8 COOCH3 (9), a substrate specific for alpha-glucosidase I, is reported. This enzyme removes the terminal alpha-D-Glcp unit to produce alpha-D-Glcp-(1-->3)-alpha-D-Glcp-O-(CH2)8 COOCH3 (10). This is the first synthetic substrate described for glucosidase I that allows kinetic evaluation of substrates and inhibitors of this enzyme. Tetramethylrhodamine was coupled to 9 through an ethylenediamine linker to produce a brilliant red derivative. Addition of this fluorescent dye did not affect enzyme binding to the substrate, as determined by a comparison of the Km value (1.3 mM). The fluorescent label allows visual detection of 2-3 pmol of product by TLC.

Chapter 4 Stereochemistry and cofactor identity status of semicarbazide-sensitive amine oxidases

The relationship between the soluble copper topaquinone amine oxidases, the membrane bound semicarbazide-sensitive amine oxidases and lysyl oxidase remains unclear. The stereochemical course of substrate oxidation has been determined for each enzyme type and these studies suggest that SSAO and lysyl oxidase are closely related mechanistically, and that they are distinct from the copper amine oxidases. Both lysyl oxidase and SSAO catalyze the oxidation of tyramine with removal of the pro-S hydrogen from C-1 of this substrate. The copper amine oxidase enzymes that react with abstraction of the pro-S hydrogen from C-1 of substrates do not exhibit a solvent exchange pathway. In contrast, this exchange occurs in lysyl oxidase and SSAO reactions. The organic cofactor in all three enzyme types is a quinone; however, the spectral features of phenylhydrazine and p-nitrophenylhydrazine-derivatized SSAO differ from those reported for all known topaquinone-containing enzymes. Cofactor identification is further complicated by the lack of the characteristic topa motif, Asn-Tyr-Asp/Glu, in lysyl oxidase and the absence of any sequence information for SSAO.