Terminal residue analysis the sanger method frederick

This preview shows 3 out of 5 pages.

Terminal Residue Analysis The Sanger Method (Frederick Sanger, Cambridge University, Nobel Prize in Chemistry 1958) This method identifies the N-terminal amino acid in a polypeptide by attaching a spectroscopic label prior to complete hydrolysis. The reaction of attaching the 2,4-dinitrofluorobenzene label is run under slightly basic conditions so the N-terminal group is in its free base form, as required for nucleophilic substitution of the aryl fluoride. F NO 2 NO 2 + H 2 NCHC-NHCHC : O = O = R R' polypeptide aq NaHCO 3 nucleophilic aromatic substitution NHCHC-NHCHC O = O = R R' NO 2 O 2 N labeled polypeptide NHCHC-NHCHC O = O = R R' NO 2 O 2 N H 3 O + hydrolysis NHCHCOOH R NO 2 O 2 N + H 3 NCHCOOH R' + + other amino acids labeled N-terminal amino acid The hydrolysis mixture is separated and the N-terminal residue is identified by spectroscopic analysis.
Image of page 3

Subscribe to view the full document.

The Edman Method (Pehr Edman, University of Lund, Sweden) Pehr Edman developed a chemical labeling method for identifying the N-terminal amino acid in 1950. This method has the advantage that the labeled amino acid can be cleaved off leaving a new N-terminal amino acid. N=C=S phenyl isothiocyanate + H 2 NCHC-NHCHC : O = O = R R' polypeptide pH 9 aq NaOH nucleophilic addition NHCHC-NHCHC O = O = R R' labeled polypeptide NHC S = NHCHC-NHCHC O = O = R R' labeled polypeptide NHC S = H 3 O + cyclization to thiohydantoin with cleavage identifies amino acid + H 3 NCHC O = R'
Image of page 4
Image of page 5
You've reached the end of this preview.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern