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Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

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C. Larry Bering and Jennifer J. Kuhns

Department of Chemistry, Clarion University, 840 Wood St., Clarion, PA 16214

Roger Rowlett

Department of Chemistry, Colgate University, 13 Oak Drive, Hamilton, NY 13346

J. Chem. Educ., 1998, 75 (8), p 1021

DOI: 10.1021/ed075p1021

Publication Date (Web): August 1, 1998

Section:

History, Education, and Documentation

Abstract

We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.

Keywords (Audience):

Upper-Division Undergraduate

Keywords (Domain):

Laboratory Instruction

Keywords (Pedagogy):

Hands-On Learning / Manipulatives

Keywords (Subject):

Enzymes

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