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Primary Protein Sequence Determination

This document discusses various reagents used for protein sequence determination: 1. Ninhydrin reacts quantitatively with amino acids and is used to quantify proteins. 2. Edman degradation reacts with the amino terminus of proteins/peptides and reduces them by one residue to determine the sequence. 3. N-terminal analysis reagents like FDNB, dansyl chloride, fluorescamine, and o-phthalaldehyde react with the amino terminus but require complete hydrolysis, and yield fluorescent products that allow detection of small amounts of protein.

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50 views9 pages

Primary Protein Sequence Determination

This document discusses various reagents used for protein sequence determination: 1. Ninhydrin reacts quantitatively with amino acids and is used to quantify proteins. 2. Edman degradation reacts with the amino terminus of proteins/peptides and reduces them by one residue to determine the sequence. 3. N-terminal analysis reagents like FDNB, dansyl chloride, fluorescamine, and o-phthalaldehyde react with the amino terminus but require complete hydrolysis, and yield fluorescent products that allow detection of small amounts of protein.

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sourcandy4242
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© © All Rights Reserved
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Reagents

 for  Protein  
Sequence  Determina4on  
1.  Ninhydrin: for quantitative reaction with amino groups of any amino
acid
2.  Edman Degradation: for reaction with amino terminus of proteins/
peptides and reduction of peptide by one residue
3.  N-terminal analysis: for reaction with amino terminus of proteins/
peptides, but complete hydrolysis of peptide required
a.  FDNB
b.  Dansyl chloride
c.  Fluorescamine
d.  o-phthalaldehyde
Ninhydrin  
The ninhydrin reaction quantitatively reacts with amino
acids in a complex reaction that produces ammonia
from the amine, carbon dioxide from the carboxylate,
and the Ca becomes an aldehyde of the R-group. The
key is the reaction of the intermediate hydrindantin with
another mole of ninhydrin and the ammonia to produce
the large molecule, called Ruhemann’s purple, which is
a deep purple and easily quantified by absorbance at
570 nm.

Mechanism:
Edman  Degrada4on  

OR  
Biochemical  Reac4ons  for  
N-­‐terminal  analysis  
Sanger  Reagent  
(fluorodinitrobenzene-­‐FDNB;  aka  
dinitrofluorbenzene-­‐DNFB)  
Dansyl  chloride  

The dansyl choride reagent


reacts with amino terminal
amino acid. Subsequence acid
hydrolysis yields all the amino
acids plus the N-terminal one
modified by the dansyl group.
This modified amino acid is
highly fluorescent and allows
detection from very small
amounts of protein
Dansyl  chloride  
Another  depic4on:  
Fluorescamine  &  o-­‐Phthalaldehyde  
Fluorescamine  [I]  reacts  readily  with  primary  
amino  groups  to  form  highly  fluorescent  
compounds  [II].    Even  though  fluorescamine  
itself  is  nonfluorescent.  The  fluorescent  
products  have  an  excita4on  maximum  at  390  
nm  and  an  emission  maximum  at  475  nm.  
These  proper4es  make  fluorescamine  ideal  
for  detec4ng  amino  groups,  especially  in  
proteins,  pep4des,  and  amino  acids.  It  is  10  to  
100  4mes  more  sensi4ve  in  detec4ng  primary  
amino  groups  than  the  ninhydrin  reac4on.    
Because  fluorescamine  has  low  solubility  and  stability  in  water,  o-­‐phthalaldehyde  [III]  (OPA),  which  reacts  similarly  with  
primary  amino  groups  and  gives  highly  fluorescent  products  [IV].    OPA  is  some4mes  subs4tuted  for  fluorescamine:    
 

These reactions need to include either a thiol


(β-mercaptoethanol depicted) or cyanide
anion.

[what-­‐when-­‐how]  
Example  of  
primary-­‐
structure  
determina4on  

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