Isolation and Functional Analysis of a Pancreatic Enzyme in an Introductory Student Lab

Structure and function are correlated at all levels of biology. This topic is typically addressed early in an undergraduate class in general or molecular biology before students have gained much skill or knowledge in molecular biology. However, an understanding of the chemical bonds involved in forming and maintaining the structure of proteins is critical to understanding how enzymes function and how their activity can be regulated. Here, a laboratory activity is described that is suitable for undergraduate biology students. This activity examines the activity of carboxypeptidase A (CPA), an abundant pancreatic enzyme with a rich history in enzymology and structural biology. The abundance of CPA in pancreatic tissue allows for a series of common biochemical techniques to be easily performed under the constraints of an undergraduate teaching lab, including the separation of proteins by simple precipitation methods, the examination of resulting proteins by SDS-PAGE and Coomassie staining, and the analysis of enzyme function through the determination of constants such as Vmax and Km. These steps illustrate the importance of noncovalent bonds in protein structure and the use of common biochemical instruments in the lab, while providing students with an opportunity to hone analysis skills in their consideration of the resulting data. Finally, this lab may be modified in many ways to make it suitable for upper division classes, CURE approaches to the undergraduate lab, and even to the pre-college classroom.