Protein sequence determination

[Ricarda]

How is FDNB used to determine the sequence of a polypeptide? (which is the reaction or what happens to the aminoacids)

[blcr11]

You can't really sequence a protein with FDNB. Sanger's Reagent (FDNB) was useful in the "old" days of protein work because it gives a clean reaction with free primary and secondary amine groups. DNP-amino acids are stable under acid hydrolysis and yield yellow derivatives that are readily identified. You can only identify the N-terminal amino acid with FDNB. Sequential Edman degradations and/or mass spectroscopy are the methods of choice for amino acid sequencing these days. Sanger's Reagent is an historical footnote now.

[blcr11]

Maybe it's not quite fair to say you can't sequence with FDNB. That's true as far as it goes, but with some additional chemistry and ingenuity you can sequence peptides, but you need something more than just FDNB. You need to be able to isolate peptides. You need to be able to do things like tryptic or chymotryptic digestion. Those sorts of things. Then, with some logic and the FDNB reagent you can in principle do a sequence determination. Sanger did that in the early 1950s for insulin. You can work through the sequence determination yourself if you can get hold of a copy of Wood, Wilson, Benbow, and Hood--Chapter 3 Problem 3.14 of the second edition gives you Sanger's data and lets you repeat the sequence determination. It's a useful exercise.