Choose your own adventure: Purifying invertase and using assays to illustrate principles of enzymes

    

Deema ElRufaei, Spomenka Curic, Jennifer Kearley, Kelly Teal-Dunwald, and Brianne Burkinshaw

Advances in Biology Laboratory Education, 2025, Volume 45

https://doi.org/10.37590/able.v45.art47

Abstract

Students in our second-year biochemistry course complete experiments that complement lecture learning objectives related to protein structure and function, protein purification, enzyme catalysis, and carbohydrate structure. The experiments are completed in two 3-hour laboratory sessions. In the first session, students use affinity chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and a Bradford assay to purify, visualize and quantify the histidine-tagged enzyme invertase. The second laboratory session uses a quantitative reducing sugars assay to detect and measure cleavage of the substrate sucrose by invertase. Variations of this assay test the effect of pH or mutation of a catalytic residue on invertase activity, quantify sucrose in beverages, test the activity of invertase toward a non-caloric sweetener, sucralose, and to build a protein purification table to determine the effectiveness of our purification process. Here we present the assay to compare activity of wild type invertase to that of the catalytic mutant.

Keywords:  biochemistry, enzymes, carbohydrates, protein purification

University of Maryland (2024)