Organic Chemistry

984 CHAPTER 23 Amino Acids, Peptides, and Proteins

The mixture of amino acids is then passed through an amino acid analyzer to deter-

mine the number and kind of each amino acid in the peptide or protein (Section 23.5).

Because all the asparagine and glutamine residues have been hydrolyzed to aspar-

tate and glutamate residues, the number of aspartate or glutamate residues in the

amino acid mixture tells us the number of aspartate plus asparagine—or glutamate

plus glutamine—residues in the original protein. A separate technique must be used to

distinguish between aspartate and asparagine or between glutamate and glutamine in

the original protein.

The strongly acidic conditions used for hydrolysis destroy all the tryptophan

residues because the indole ring is unstable in acid (Section 21.9). The tryptophan

content of the protein can be determined by hydroxide-ion-promoted hydrolysis of the

protein. This is not a general method for peptide bond hydrolysis because the strongly

basic conditions destroy several other amino acid residues.

There are several ways to identify the N-terminal amino acid of a peptide or pro-

tein. One of the most widely used methods is to treat the protein with phenyl isothio-

cyanate (PITC), more commonly known as Edman’s reagent. This reagent reacts

with the N-terminal amino group, and the resulting thiazolinone derivative is cleaved

from the protein under mildly acidic conditions. The thiazolinone derivative is extract-

ed into an organic solvent and in the presence of acid, rearranges to a more stable

phenylthiohydantoin (PTH).

Because each amino acid has a different substituent (R), each amino acid forms a

different PTH. The particular PTH can be identified by chromatography using known

standards. Several successive Edman degradations can be carried out on a protein.

The entire primary sequence cannot be determined in this way, however, because side

O

HC

HN

HN

R

S

HCl

thiazolinone

derivative

O

H 3 NCHC

R′

O

NHCHC

R′′

O

HC

HN

S

R

N

+

+

+

PTH–amino acid

peptide without the original

N-terminal amino acid

phenyl isothiocyanate

PITC

Edman's reagent

NHC 2 NCHC NHCHC NHCHC

OOO

R R′ R′′

S

N

H

C NHCHC NHCHC NHCHC

O OO

NHCHC

O

S R R′ R′′

N

H

H

N R

C

S

CH H F

C

O− R′

NHCHC

O

R′′

HF

+

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