Problem Set
Protein Purification
Problem 1
Hair grows at a rate of 15 to 20 cm/yr. All this growth is
concentrated at the base of the hair fiber, where a-keratin
filaments are synthesized inside living epidermal cells and
assembled into ropelike structures (see Fig. 4–10). The
fundamental structural element of a-keratin is the a helix, which
has 3.6 amino acid residues per turn and a rise of 5.4 Å per turn
(see Fig. 4–4a). Assuming that the biosynthesis of a-helical keratin
chains is the rate-limiting factor in the growth of hair, calculate the
rate at which peptide bonds of a-keratin chains must be
synthesized (peptide bonds per second) to account for the
observed yearly growth of hair.
Solution
Because there are 3.6 amino acids (AAs) per turn and the rise is
5.4 Å /turn, the length per AA of the a helix is
A growth rate of 20 cm/yr is equivalent to
Thus, the rate at which amino acids are added is
42 peptide bonds per second
Clostripain, also known as Endoproteinase Arg-C, is a proteinase
that cleaves proteins on the carboxyl peptide bond of arginine.
Problem 2
Amino acid analysis of an oligopeptide seven residues long gave
Asp Leu Lys Met Phe Tyr
The following facts were observed:
a. Trypsin treatment had no apparent effect.
b. The phenylthiohydantoin released by Edman degradation was
c. Brief chymotrypsin treatment yielded several products, including a
dipeptide and a tetrapeptide. The amino acid composition of the
tetrapeptide was Leu, Lys, and Met.
d. Cyanogen bromide treatment yielded a dipeptide, a tetrapeptide, and
free Lys.
What is the amino acid sequence of this heptapeptide?
Problem 3
Amino acid analysis of a decapeptide revealed the presence of the following products:
NH4+ Asp Glu Tyr Arg
Met Pro Lys Ser Phe
The following facts were observed:
a. Neither carboxypeptidase A or B treatment of the decapeptide had any effect.
b. Trypsin treatment yielded two tetrapeptides and free Lys.
c. Clostripain treatment yielded a tetrapeptide and a hexapeptide.
d. Cyanogen bromide treatment yielded an octapeptide and a dipeptide of sequence NP
(using the one-letter codes).
e. Chymotrypsin treatment yielded two tripeptides and a tetrapeptide. The N-terminal
chymotryptic peptide had a net charge of -1 at neutral pH and a net charge of -3 at pH 12.
f. One cycle of Edman degradation gave the PTH derivative
What is the amino acid sequence of this decapeptide?
Problem 4
An octapeptide consisting of
2 Gly, 1 Lys, 1 Met, 1 Pro, 1 Arg, 1 Trp, and 1 Tyr
Was subjected to sequence studies. The following was found:
a. Edman degradation yielded
b. Upon treatment with carboxypeptidases A, B, and C, only carboxypeptidase C had
any effect.
c. Trypsin treatment gave two tripeptides and a dipeptide.
d. Chymotrypsin treatment gave two tripeptides and a dipeptide. Acid hydrolysis of
the dipeptide yielded only Gly.
e. Cyanogen bromide treatment yielded two tetrapeptides.
f. Clostripain treatment gave a pentapeptide and a tripeptide.
What is the amino acid sequence of this octapeptide?
Problem 5
Amino acid analysis of an oligopeptide containing nine residues revealed the presence of the
following amino acids:
Arg Cys Gly Leu Met Pro Tyr Val
The following was found:
a. Carboxypeptidase A treatment yielded no free amino acid.
b. Edman analysis of the intact oligopeptide released
c. Neither trypsin nor chymotrypsin treatment of the nonapeptide released smaller fragments.
However, combined trypsin and chymotrypsin treatment liberated free Arg.
d. CNBr treatment of the eight-residue fragment left after combined trypsin and chymotrypsin
action yielded a six-residue fragment containing Cys, Gly, Pro, Tyr, and Val; and a dipeptide.
e. Treatment of the six-residue fragment with b-mercaptoethanol yielded two tripeptides.
Brief
Edman analysis of the tripeptide mixture yielded only PTH-Cys.
(The sequence of each tripeptide, as read from the N-terminal end, is alphabetical if the one-
letter designation for amino acids is used.)
What is the amino acid sequence of this nonapeptide?
Problem 6
Describe the synthesis of the dipeptide Lys-
Ala by Merrifield’s solid phase chemical
method of peptide synthesis.
Solutions
Prob 2:
Phe-Met-Tyr-(Leu-Asp)-Met-Lys
Prob 3