lecture 10 - Edman degradation Flashcards
is Edman degradation repetitive?
yes
where does Edman degradation start?
N-term
what is the Edman reagent? what does it react with?
- phenylisothiocyanate (PITC) (benzene-N=C=S)
- primary amino acids (N-term and Lys side chain)
- secondary amino acids (Pro at N-term)
what is the reaction for Edman degradation?
- amino acid reacts with the reagent (alkaline)
- results in cyclization (acidic) and rearrangment
- rearrangment breaks the peptide bond between the N-term aa and the following aas
- next PTHaa is extracted (new complex of PITC and N-term amino acid)
- this leaves the remainder of the protein in the aqueous solution (ready for next cycle - addition of more PITC in alkaline conditions)
How is PTHaa identified?
by chromatography due to different “R” group
how many PTHaa identified per cycle?
1
how many times can the cycle for Edman degradation be repeated?
~40
can the Edman degradation process be automated?
yes
simplify the Edman degradation process
- starting vs end products
- steps
- how classify end products
- have: PITC and (1)-(2)-(3)-(4)-(5)… (amino acid)
- cyclization, rearrangment, cleavage, extraction
- repeat many times
- now have: PTHaa-(1), PTHaa-(2), etc. etc.
- indentify each specific PTHaa-(#) by their elution time and an HPLC chromatography column.
- sensetive and accurate!!
list the problems with Edman degradation
(1) can only be repeated ~40 times so need to break larger proteins into smaller fragments
(2) some proteins have a blocked N-term (most commonly by acetylation - e.g. N-acetyl-ala-val… so no free amino grp = no reaction)
(3) disulphide bonds can complicate analysis (typically reduce and block disulphides to give free cys residues)
(4) multiple subunits - if heteromultimer get ambiguous results
explain the problem of using edman degradation for multiple subunits when stoichiometry is 1:1
if have subunit 1 = VLSIP, subunit 2 = ALFIS result would be:
cycle 1: PTHaa-A,V
cycle 2: PTHaa-L
cycle 3: PTHaa-F,S
cycle 4: PTHaa-I
cycle 5:PTHaa-P,S
-cannot identify which aa is connected to which aa within each polypeptide IF stoichiometry is 1:1
explain the problem of using edman degradation for multiple subunits when stoichiometry is not 1:1
-if subunit 1 is in a 2:1 ratio and have subunit 1 = VLSIP, subunit 2 = ALFIS result would be:
cycle 1: PTHaa- V : A
cycle 2: PTHaa- L
cycle 3: PTHaa- S : F
cycle 4: PTHaa- I
cycle 5:PTHaa- P : S
-detect the ratio, so could sequence both polypeptides simultaneously
what else is the Edman reagent used for? how?
- aa composition analysis
- hydrolyze protiens to get free aa
- react free aa with PITC to identify PITC-aa
how does hydrolysis for aa composition analysis work? what is the result?
- harsh conditions
- boil in 6M HCL for 24 hours
- hydrolyzies all amide bonds
- all asn –> asp = asx, all gln –> glu = glx
- also destroys all trp so cannot identify amount of Trp by this method
- result = ala 5%, arg 1%, asx 3%, gly 3%, etc. of total aa in a protein
