Grant: Discovery Grants Program - Individual

Measurements made on individual molecules of a given enzyme have shown that the different individual molecules have different properties. The phenomenon has been termed enzyme heterogeneity. Several different experimental approaches have been used on several different enzymes. In all cases heterogeneity has been observed. Reported heterogeneous properties include catalytic rate, activation energy of catalysis and electrophoretic mobility. The long term goal of the research in the applicants laboratory is to understand the cause and biological role of this heterogeneity. Most work in the applicants laboratory has been done using beta-galactosidase or alkaline phosphatase as model enzymes, although recently this work has been expanded to the enzyme beta-glucuronidase. These assays are performed using a capillary electrophoresis-based approach. This proposal is comprised of three short term goals that fit into our longer term goal. The first short term objective of this study is to expand our repertoire of enzyme molecules which can be assayed at the single molecule level using capillary electrophoresis. Initial work will focus on improving and expanding our assay for beta-glucuronidase and developing an assay for peroxidase and proteases. Additionally, we intend to develop a method for the separation and detection of metabolites that will be useful in single cell metabolomics. The second goal is to determine the effect of post-translational glycosylation on enzyme heterogeneity using bovine alkaline phosphatase, which is a glycosylated enzyme, as the model enzyme. Initially glycosylation states will be altered using non-denaturation deglycosylation kits targeting different sugar moieties and samples assayed to determine the effect on the distributions of catalytic rates, electrophoretic mobility and activation energy of catalysis. The bovine enzyme will then be expressed in P. pastoris and mutagenesis be performed to determine the effect of selective removal of glycosylation sites. Free zone capillary electrophoresis is a commonly used technique for analytical protein separations. Our research has shown that enzyme molecules are inherently electrophoretically heterogeneous. This represents a fundamental limit to resolution in protein separations using free zone capillary electrophoresis. The third short term goal of this proposal is to determine the factors that cause electrophoretic heterogeneity in order to predict which type of protein structures can be separated with high resolution and which types may show lower resolution using this technique.