Organic Chemistry

Section 23.12 Determining the Primary Structure of a Protein 983

PRIMARY STRUCTURE

AND EVOLUTION

When we examine the primary structures of pro-

teins that carry out the same function in different organisms,

we can relate the number of amino acid differences between

the proteins to the taxonomic differences between the

species. For example, cytochrome c, a protein that transfers

electrons in biological oxidations, has about 100 amino acid

residues. Yeast cytochrome cdiffers by 48 amino acids from

horse cytochrome c, while duck cytochrome cdiffers by only

two amino acids from chicken cytochrome c. Chickens and

turkeys have cytochrome c’s with identical primary struc-

tures. Humans and chimpanzees also have identical cyto-

chrome c’s, differing by one amino acid from the cytochrome

cof the rhesus monkey.

23.12 Determining the Primary Structure of a Protein

The first step in determining the sequence of amino acids in a peptide or a protein is to

reduce any disulfide bridges in the peptide or protein. A commonly used reducing

agent is 2-mercaptoethanol, which is oxidized to a disulfide. Reaction of the protein

thiol groups with iodoacetic acid prevents the disulfide bridges from reforming as a re-

sult of oxidation by

PROBLEM 28

Write the mechanism for the reaction of a cysteine residue with iodoacetic acid.

The next step is to determine the number and kinds of amino acids in the peptide or

protein. To do this, a sample of the peptide or protein is dissolved in 6 N HCl and heat-

ed at for 24 hours. This treatment hydrolyzes all the amide bonds in the pro-

tein, including the amide bonds of asparagine and glutamine.

6 N HCI

100 °C

24 h

protein amino acids

100 °C

ICH 2 COH

cleaving disulfide bridges

O

CH 2

CH 2

S

S

C

O

NHCH

C

O

NHCH

+ 2 HSCH 2 CH 2 OH

CH 2

CH 2

SH

SH

SCH 2 CH 2 OH

SCH 2 CH 2 OH

C

O

NHCH

C

O

NHCH

+

CH 2

CH 2

SCH 2 COH

SCH 2 COH

C

O

O

O

NHCH

C

O

2 HI

NHCH

+

iodoacetic acid

2-mercaptoethanol

O 2.

Insulin was the first protein for which

the primary sequence was deter-

mined. This was done in 1953 by

Frederick Sanger, who received the

1958 Nobel Prize in chemistry for his

work. Sanger was born in England in

1918 and received a Ph.D. from

Cambridge University, where he has

worked for his entire career. He also

received a share of the 1980 Nobel

Prize in chemistry (Section 27.15) for

being the first to sequence a DNA

molecule (with 5375 nucleotide

pairs).

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