4202 Summer 13
Assignment 5
NAME________________________________
Question 1 requires the use of the proteins you used in homework 2. Questions 2-4 are three different peptides for
which you need to determine the sequence. For some of the questions, you will need to either look up information on
structures, or remember your organic chemistry. Think about what each statement means with relation to what we
have talked about in class. You only need to give me the sequence of the amino acids and not the structure of the
unknown peptide. Each sequencing question is worth 5 pts.
1. The proteins that you used in homework assignment 2 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 (you can only pick 1 pH to work at). You may use either technique,
both techniques, or even the same technique more than once. If you use size exclusion the proteins must have a
difference in number of amino acids of at least 800 to be separated. If you use ion-exchange the proteins must have
a difference in pI of a least 1.5 pH units to be separated. Clearly articulate your answers for complete credit. (10 pts)
2. Analysis of the blood of a catatonic football fan revealed large concentrations of a psychotoxic octapeptide. Amino
acid analysis of this octapeptide gave the following results:
2 Ala
1 Arg
1 Asp
1 Met 2 Tyr
1 Val
1 NH4+
The following facts were observed:
a. One round of Edman degradation yields one mole of Ala.
b. Partial acid hydrolysis of the octapeptide yielded a dipeptide of the structure:
c. Chymotrypsin treatment of the octapeptide yielded two tetrapeptides, each containing an alanine residue.
d. Trypsin treatment of one of the tetrapeptides yielded two dipeptides.
e. Cyanogen bromide treatment of another sample of the same tetrapeptide yielded a tripeptide and free Tyr.
f. End-group analysis of the other tetrapeptide gave Asp.
4202 Summer 13
Assignment 5
NAME________________________________
3. Amino acid analysis of an octapeptide revealed the following composition:
2 Arg
1 Gly
1 Met 1 Trp
1 Try
1 Phe 1 Lys
The following facts were observed:
a. Edman degradation gave
b. CNBr treatment yielded a pentapeptide and a tripeptide containing phenylalanine
c. Chymotrypsin treatment yielded a tetrapeptide containing a C-terminal indole amino acid and two
dipeptides.
d. Trypsin treatment yielded a tetrapeptide , a dipeptide, and free lys and Phe.
e. Clostripain (cleaves at arginine residues only) yielded a pentapeptide, a dipeptide, and free Phe.
4202 Summer 13
Assignment 5
NAME________________________________
4. Amino acid analysis of an octapeptide gave the following results:
1 Ala
1 Arg
1 Asp
1 Gly
3 Ile
1 Val
1 NH4+
The following facts were observed:
a.
b.
c.
d.
e.
f.
One round of Edman degradation yielded one mole of Val.
Trypsin treatment yielded a pentapeptide and a tripeptide.
One round of Edman degradation of the pentapeptide yielded one mole of Gly.
Chemical reduction of the free α-COOH and subsequent acid hydrolysis yielded 2-aminopropanol.
Partial acid hydrolysis of the tryptic pentapeptide yielded, among other products, two dipeptides, each of
which contained C-terminal isoleucine. One of the dipeptides migrated as an anionic species upon
electrophoresis at neutral pH.
The tryptic tripeptide was degraded in an Edman sequenator yielding first A, the B:
A:
B: