1. Which of the following amino acids would be first to elute at pH 7 from an anion-exchange column?
a). glutamic acid b). alanine c). lysine d). asparagine e). glycine

2. A mixture of lysine, glycine, alanine, isoleucine and glutamic acid are separated by ionic exchange chromatography. What is the order of elution of these amino acids if :
Use a cation exchange resin?
b) use an anion exchange resin?
Which column would give the best separation?

3. Glycine, alanine, valine and leucine can be successfully separated by ionic exchange
chromatography even though their pKas are almost identical. Explain the behavior of these amino acids.

4. Since these four amino acids can be separated by ion exchange chromatography, but that their pI are virtually identical, another property must be responsible for this behavior.
A close look at the structure of glycine, alanine, valine and leucine reveals a progressive increase in the hydrophobic character of their side chain. We can deduce that these amino acids can establish hydrophobic interactions with the ion exchange resin, allowing their separation.

5. Consider the following peptide:
A-L-K-M-P-E-Y-I-S-T-D-Q-S-N-W-H-H-R
Indicate the fragments generated after the following digestions :
trypsin
chymotrypsin

6. Trypsin hydrolyzes the peptide bond on the carboxyl-side of the basic amino acids lysine and
arginine.
A-L-K M-P-E-Y-I-S-T-D-Q-S-N-W-H-H-R
Chymotrypsin hydrolyzes the peptide bond on the carboxyl-side of the large hydrophobic amino acids F, Y, W, and M
A-L-K-M P-E-Y I-S-T-D-Q-S-N-W H-H-R

7. We load a DEAE-cellulose column adjusted to a pH of 6
We load a DEAE-cellulose column adjusted to a pH of 6.5 with the following mixture of proteins:
ovalbumin (pI = 4.6),
urease (pI = 5.0)
myoglobin (pI = 7.0).
The proteins are eluted first with a buffer of weak ionic strength at a
pH of 6.5, and then the same buffer containing increasing amounts of sodium chloride is used to elute the proteins. What order are the proteins eluted?

8. myoglobin will elute first, since it will not bind to the resin (pH<pI: positive net charge);
the pI of urease (5,4) is closer to the pH of the buffer that is the pI of ovalbumin (4,6): urease
will therefore carry a lower number of negative charges than ovalbumin.
We can therefore predict that urease will elute at a NaCl concentration which will be lower than the one required
to elute ovalbumin.

9. DEAE cellulose columns are rarely used at pH greater than 8.5. Why?

10. 6-phosphogluconate dehydrogenase has a pI of 6
6-phosphogluconate dehydrogenase has a pI of 6. Explain why the buffer used for a chromatography on DEAE-cellulose must have a pH greater than 6 but less than 9 in order to ensure the enzyme is efficiently bound to the column.

11. At a pI above 6, 6-phosphogluconate dehydrogenase
has a net negative charge (pH>pI): it will bind the resin.
At a pH value above 9, the diethylamino group of the resin is deprotonated, preventing any separation of the enzyme as a function of its charge.