Organic Chemistry

Section 23.12 Determining the Primary Structure of a Protein 985

products accumulate that interfere with the results. An automated instrument known

as a sequenatorallows about 50 successive Edman degradations to be carried out on

a protein.

The C-terminal amino acid of the peptide or protein can be identified by treating the

protein with carboxypeptidase A. Carboxypeptidase A cleaves off the C-terminal

amino acid as long as it is notarginine or lysine (Section 24.9). On the other hand, car-

boxypeptidase B cleaves off the C-terminal amino acid onlyif it is arginine or lysine.

Carboxypeptidases are exopeptidases. An exopeptidaseis an enzyme that catalyzes

the hydrolysis of a peptide bond at the end of a peptide chain.

Once the N-terminal and C-terminal amino acids have been identified, a sample of

the protein is hydrolyzed with dilute acid. This treatment, called partial hydrolysis,

hydrolyzes only some of the peptide bonds. The resulting fragments are separated, and

the amino acid composition of each is determined. The N-terminal and C-terminal

amino acids of each fragment can also be identified. The sequence of the original

protein can then be determined by lining up the peptides and looking for points

of overlap.

PROBLEM-SOLVING STRATEGY

A nonapeptide undergoes partial hydrolysis to give peptides whose amino acid composi-

tions are shown. Reaction of the intact nonapeptide with Edman’s reagent releases

PTH-Leu. What is the sequence of the nonapeptide?

a. Pro, Ser c. Met, Ala, Leu e. Glu, Ser, Val, Pro g. Met, Leu

b. Gly, Glu d. Gly, Ala f. Glu, Pro, Gly h. His, Val

Let’s start with the N-terminal amino acid. We know that it is Leu. Now we need to look

for a fragment that contains Leu. Fragment (g) tells us that Met is next to Leu and fragment

(c) tells us that Ala is next to Met. Now we look for a fragment that contains Ala. Fragment

(d) contains Ala and tells us that Gly is next to Ala. From fragment (b), we know that Glu

comes next. Glu is in both fragments (e) and (f). Fragment (e) has two amino acids we have

yet to place in the growing peptide, but fragment (f) has only one, so from fragment (f), we

know that Pro is the next amino acid. Now we can use fragment (e). Fragment (e) tells us

that the next amino acid is Val, and fragment (h) tells us that His is the last (C-terminal)

amino acid. Thus, the amino acid sequence of the nonapeptide is

Leu-Met-Ala-Gly-Glu-Pro-Ser-Val-His

Now continue on to Problem 29.

PROBLEM 29

A decapeptide undergoes partial hydrolysis to give peptides whose amino acid composi-

tions are shown. Reaction of the intact decapeptide with Edman’s reagent releases

PTH-Gly. What is the sequence of the decapeptide?

a. Ala, Trp c. Pro, Val e. Trp, Ala, Arg g. Glu, Ala, Leu

b. Val, Pro, Asp d. Ala, Glu f. Arg, Gly h. Met, Pro, Leu, Glu

The peptide or protein can also be partially hydrolyzed using endopeptidases. An

endopeptidaseis an enzyme that catalyzes the hydrolysis of a peptide bond that is not

at the end of a peptide chain. Trypsin, chymotrypsin, and elastase are endopeptidases

R R′ R′′

NHCHCO−

OOO

NHCHC NHCHC

site where carboxypeptidase cleaves

BRUI23-959-998r2 29-03-2003 1:36 PM Page 985