A. Heaney and D. L. WeHer University o f Vermont Burlington, Vermont 05401
II
isoelectric pH of Hemoglobin and Cytachrome C by Electrofocusing
Electrophoresis of proteins has been commonly used as an experiment in biochemistry laboratorv courses t o reinforce c o n c e ~ t sof ~ r o t e i n structure and t o give students experience with electrophoresis as a technique for purification of proteins. The isoelectric separation of proteins in p H gradients established during electrolysis of a mixture of low molecular weight ampholytes and protein(s) has been termed electrofocusing (1, 2 ) . We have used this relatively new technique successfully in our biochemistry course to demonstrate the amphoteric nature of proteins. I n this report we present a description of a simple apparatus and procedure developed by us (3) t h a t makes it economically feasible to do electrofocusing experiments in laboratory courses. Results with two colored proteins, hemoglobin and cytochrome C, are presented. These proteins were selected so that the progress of the electrofocusing experiments as well as the end points could be visualized. Electrofocusing was originally developed by Kolin (4) for separation of amphoteric substances. It is based on the observation t h a t electrophoresis of a mixture of ampholytes leads t o the formation of a p H gradient. The ampholyte species stack according to their pI1s and those species that have groups whose wK's are in the vicinitv of their DI'S will buffer and maintain the p H of their domain a t or near their isoelectric pH. If an ampholyte mixture containing species whose pI1s form a graduated series is electrophoresed, the result is a smooth gradient of p H a t equilibrium. Equilibrium is signaled by a fall in the current to near zero because the ampholyte species carrv little current a t their isoelectric wH. A recent technical development has made available commercially mixtures of low molecular weight (
