Re: From maria Lorenzi (Italy )
Dan Brune (DBrune@asu.edu)
Wed, 04 Nov 1998 15:00:50 -0700 (MST)
Dear Maria,
I don't have an answer to your question about the breakdown
products that you observed on the cycle where you expected to see Trp, but
have a suggestion. I once sequenced a similarly sized protein (a
rubredoxin containing 52 amino acids), and when the N-terminal sequence
died out after residue 48, I used digestion with carboxypeptidase Y to get
the last 3 residues. It helps to have a matrix-assisted laser desorption
time of flight (MALDI-TOF) mass spectrometer for this work, but that should
not be essential. Previously it has been possible to do periodic amino
acid analyses on a reaction mixture of the protein plus carboxypeptidase in
order to follow the release of amino acids from the carboxy terminus. My
experiments are described in Lee et al. (1995) Arch. Biochem. Biophys. 318,
80-88, and that paper also contains a couple of references to earlier work
that used amino acid analysis only (i.e. no mass spectrometry). You should
also be able to confirm the presence of a Trp (as opposed to a non-standard
amino acid) near the C-terminus of the polypeptide by measuring its
extinction coefficient. There is a formula given by Pace et al. (1995)
Protein Sci. 4, 2411-2423 that states that the molar extinction coefficient
of the protein is equal to: (no. of Trp residues) x 5,500 + (no. of Tyr
residues) x 1,490 + (no. of cystine residues) x 125. Since you have the
complete sequence through residue 48, you will already know the 280 nm
extinction coefficient of that part of the protein, and it should not be
too hard to see if additional 280 absorbance from a Trp near the C-terminus
is required to account for the measured extinction coefficient. (I am
assuming here that you have a way to determine the concentration of the
protein in solution independently of the abosrbance measurement - amino
acid analysis might also be useful for this purpose.)
Good luck,
Dan Brune