3-6 SEQUENCING AMINO ACIDS IN PROTEINS
Amino Acid Composition of Proteins

Once a protein has been isolated, its amino acid composition can be determined.

The peptide bonds are cleaved by acid hydrolysis, usually using 6 M _____.

Then amino acid analysis is used – a process that _______________ and_______________ each
amino acid.
o
One method is to treat the
peptidewith phenylisothiocyanate
(_____) at pH of 9.0 to generate
phenylthiocarbamoyl (_____)amino acid derivatives.
o
The PTC-amino acid mixture is
then sent through HPLC to
_______________each amino acid.
o
Unfortunately, acid hydrolysis
can_______________ the side
chains ofseveral amino acids.
o
Other techniques must be applied
to determine the _______________
amino acid composition of a protein.
Determining the Sequence of Amino Acid Residues

The Edman degradation procedure – Peter Edman in 1950 developed a technique that removes
and identifies one residue at a time from the N-terminus of a protein.

A protein can contain _______________ bonds between cystineresidues, and these must be
cleaved to permit the release as PTH-amino acids during Edman degradation. _______________
compounds (like 2-mercaptoethanol) are often used to cleave disulfide bonds. After the thiol
compound separates the disulfide bond, the mixture is treated with an _______________ agent
(like iodoacetate) that prevents the re-formation of disulfide bonds.

The yield of the Edman degradation can approach _____% under carefully controlled conditions!

Many proteins contain __________ __________ residues to be completely sequenced by Edman
degradation. Treatment with cyanogens bromide, trypsin, S. aureus V8 protease, and
chymotrypsin to individual samples of a large protein, one can generate many peptide
_______________ of various sizes. These fragments can then be treated by Edman degradation.
* Read page 76 for a description of how the four methods identified above work.

Frederick Sanger was the first to determine the complete sequence of a protein
(_______________ in 1956), and was awarded the Nobel Prize. He won a second Nobel Prize
for pioneering the sequencing of _______________ __________.
* Read section 3.11 to see how comparing the primary structures of proteins can reveal
evolutionary relationships.