Protein Sequencing
Edman Degradation
• Phenyl isothiocyanate is reacted with the N-terminal amino acid. Unreacted phenyl
isothiocyanate is removed and the peptide bond is broken. The derivative of the first
amino acid is converted into a more stable form and identified by chromatography.
H+
N C
R1 O
S
H 2N C C
R2 O
R3 O
R4 O
N C C N
C C N C C
H
H H
H
R1 O
H+ R2 O
R3 O
N C C N
C C N C C
S H H
H H
H
S
R2 O
O H N C C N
2
H
N C N C C
N C
HN
H
H
H
H
H H
R4 O
H H
R3 O
R4 O
C C N C C
H
H H
R1
• The reaction cycle is repeated with the shortened polypeptide and the derivative of the
second amino acid is identified by chromatography.
H+
N C
R2 O
S
R3 O
R4 O
N C C N
C C
H
H H
H
R2 O
H+ R3 O
R4 O
N C C N
C C
S H H
H H
H
S
R3 O
O H N C C N
2
H 2N C C
H
N C N C C
N C
HN
H
H
H
H
R4 O
C C
H
R2
• By repeating the reaction cycle N-terminal amino acid sequences of up to fifty residues
can be determined.
Biochemistry Handout 09.1
Use of Peptide Fragments in Sequencing
• The information below can be used to generate the complete amino acid sequence of
the polypeptide chain.
Entire Polypeptide
1: AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHDQHIQLQLSAESVGE…
CnBr Fragments
1: AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHDQHIQLQLSAESVGE…
2: DTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSC…
NTCB Fragments
1: CSNGGHDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEE…
2: CLFLERLEENHYNTYISKKHAEKNWFVGLKKNGS
3: AEGEITTFTALTEKFNLPPGNYKKPKLLY
4: CKRGPRTHYGQKAILFLPLPVSSD
Answer:
Determining Evolutionary Relationships from Protein Sequences
• The information below can be used to determine the evolutionary relationships of
species A, B, and C.
Species
Amino Acid Sequence
A
Ala-Met-Phe-Gly-Asp-Leu-Gln-Arg-Thr-Pro
B
Ala-Met-Val-Gly-Asp-Ile-Gln-Arg-Thr-Pro
C
Ala-Met-Val-Ala-Glu-Leu-Gln-Arg-Arg-Pro
Answer:
Biochemistry Handout 09.2

Allegato 3 – Struttura Screening Neonatale Valori di riferimento

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