4202 Spring 15
Assignment 2
NAME________________________________
Question 1 requires the use of the proteins you used in homework 1. Please use the four from question 5.
1. The proteins that you used in homework assignment 1 need to be purified. You goal is to purify as many individual
proteins as possible. What steps (exact order) would you use to purify the proteins? When would the proteins elute
from what types of columns? How many different proteins can you purify? You can use either Size exclusion
chromatography or ion-exchange chromatography. Size exclusion must have a difference in number of amino acids
of at least 800 to be separated. Ion-exchange must have a difference in pI of a least 1.5 pH units to be separated.
Clearly articulate your answers for complete credit. (7 pts)
2. Three polypeptides, are in a mixture (the three below).
1) ATKNRASCLVPKHGALMFWRHKQLVSDPILQKRQHILVCRNAAG
2) GPYFGDEPLDVHDEPEEG
3) PHLLSAWKGMEGVGKSQSFAALIVILA
Answer the following questions about various purification methods:
a) If the three peptides where placed in an ion-exchange resin that was positively charged, what order would the
peptides elute, why? (3 pts)
b) If the three peptides where placed in an ion-exchange resin that was negatively charged, what order would the
peptides elute, why? (3 pts)
c) If the three peptides where placed in gel filtration column, what order would the peptides elute, why? (3 pts)
3. Determine the sequence of a peptide consisting of 14 amino acids based on the following information: (3 pts)
Amino acid composition (4S, 2L, F, G, I K, M, T, W, Y)
N-terminal analysis (S)
Carboxypeptidase digestion (L)
Trypsin digestion (3S, 2L, F, I, M, T, W) (G, K, S, Y)
Chymotrypsin digestion (F, I, S) (G, K, L) (L, S) (M, T) (S, W) (S, Y)
N-terminal analysis of (F, I, S) fragment from chymotrypsin digestion (S)
Cyanogen bromide treatment (2S, F, G< I, K, L, T, Y, homoserine lactone) (2S, L, W)
4. A protein has the following sequence: ALKJSCLKEPINWDVKLNCSLKRFILQMNLKCVGILKVN
The sequence contains three amino acids that can form a disulfide bond. However, only two of them actually make a
disulfide bond in the protein. Propose a series of experiments that would allow you to determine between which two
amino acids the disulfide bond occurs. You only need to use information that we have learned so far this semester.
HINT: do not overcomplicate the question. (6 pts)