Biochemical Laboratory Experiments on Polyphenol Oxidase

A common goal of biochemical laboratory courses is to
give students
hands-on experience in enzyme purification and protein characterization
techniques. We present a well-tested module of five linked experiments
centered on the enzyme polyphenol oxidase (PPO) from banana. This
module includes buffer and plant extract preparation, protein and
enzyme activity assays, column chromatography, and sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Our electrophoresis
experiment exploits the fact that PPO is activated by SDS for rapid
and enzyme sensitive gel staining. PPO activity can be measured with
various phenolic substrates. We tested different assay procedures
and recommend dopamine as a substrate in a classroom setting. All
five experiments, including the chromatography experiment for which
we used gravity flow and syringe operated columns, provide hands-on
learning experiences in essential laboratory techniques for undergraduate
Biochemistry students. Our assessment of students’ learning
challenges revealed that most students require assistance in interpreting
and converting enzyme activity units. The use of nonstandard units
in the scientific literature makes this a topic worthy of discussion.