Graduation Date

5-2013

Document Type

Master's Thesis

Degree Name

Master of Science

Department or Program

Biological Sciences

Department or Program Chair

Kiowa Bower, Ph.D.

First Reader

Erno Pungor, Ph.D.

Second Reader

Randall Hall, Ph.D.

Abstract

This project seeks to explain the enzyme kinetics of recombinant human Arylsulfate sulfatase B (rhASB) by using an in vitro bioassay and direct hydrolysis substrate assay to approach the problem of substrate limitation in establishing a representative kcat for rhASB. The project also explores the makeup of a possible ASB complex within the cell. Previous published studies have sought to establish a turnover rate for rhASB, but have run into the problem of substrate limitation. Using a direct monosaccharide hydrolysis assay in conjunction with the same natural substrate bioassay, this project will attempt to provide insight to the kcat, the turnover number, or number of reactions an enzyme can process, Km, the substrate concentration at half enzyme saturation, and Vmax , the maximum rate of the enzymatic reaction, for rhASB. ASB deficient cells accumulate Chondroitin Sulfate and Dermatan Sulfate (CS/DS) products, the natural substrate of ASB. Incubating these cells and digesting the lysate with a CS/DS-specific lyase allows quantitation of CS/DS through fluorescent labeling and by capillary electrophoresis. In addition to a direct hydrolysis assay analyzed by High Pressure Liquid Chromatography (HPLC), this gives two potential methods of examining the kinetics of rhASB with both a monosaccharide substrate and the natural intracellular substrate

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