Passage IV

Paper electrophoresis can be used to separate and identify amino acids:

1. A strip of paper is wetted with a solution of known pH, and a mixture of amino acids is applied as a spot to the middle of the strip.

2. Each end of the strip is immersed in the solution of known pH, and an electric field is applied to the strip such that one end is positive and the other end is negative.

3. Different amino acids travel along the strip different dis­tances and/or in different directions.

Figure 1 illustrates this process.

Figure 1 adapted from William B. Wood et al., Biochemistry: A Problems Approach. @1981 by Benjamin/Cummings Publishing Company, Inc.

The net charge of an amino acid varies with the pH of the solution. The isoelectric point, pl, of an amino acid is the pH at which the amino acid has no net charge. At all other pH values, the amino acid does have a net charge, and, consequently, will travel along the strip. The direction of travel depends on the sign of the net charge (see Table 1).

Experiment 1

In each of 5 trials (Trials 1-5), paper electrophoresis was performed on leucine (an amino acid) using a solution of known pH. The greater the difference between the pH and leucine's pl, the farther leucine traveled. From the results (see Figure 2), leucine's pl was determined.

The pl of each of 3 additional amino acids was like­wise determined (see Table 2).

Experiment 2

In each of 4 trials (Trials 6-9), paper electrophoresis was performed on a mixture of 2 or more of the amino acids studied in Experiment 1 using a solution of known pH. The distance traveled by an amino acid depended only on the difference between the pH and the amino acid's pl: the greater the difference, the farther the amino acid trav­eled (see Figure 3).