Bill-
Another option you have is to convert nitrotyrosine to aminotyrosine prior
to hydrolysis or sequencing. Treatment of 3-nitroTyr with sodium
hydrosulfite (Na2S2O4) results in rapid, quantitative conversion into
3-aminoTyr (see Sokolovsky BBRC 27, p 20, 1967). AminoTyr is stable under
conditions of hydrolysis and can be quantitated by AAA and by sequencing,
though where it elutes I do not know. It also has nice absorbtion
spectra, depending upon pH, which is nice for proof of existence (but not
location). Conditions for conversion: treat the protein with 0.05 M
Tris, pH 8.0, 32-fold molar excess of sodium hydrosulfite, 10 minutes.
Tom Andersen
>>> Bill Enslow <wle2@cornell.edu> at Internet-Mail 11/11/99 07:41PM >>>
I have a question for the protein sequencing people,
A customer asked me about identifying 3-nitrotyrosine in a protein
through N-terminal sequencing. I got a reference telling how to
synthesize the PTH derivative (Thanks to Mark Crankshaw!) and successfully
completed the synthesis and purification. However, when the moment of
truth came and I loaded the PTH-3-nitrotyrosine onto my Procise I was
dissappointed. It elutes RIGHT ON TOP of DPTU by our HPLC method. I am
not aware of anything I can do about it, but I know that separation
chemistry can be different from lab to lab. Does anyone out there have a
system where
PTH-3-nitrotyrosine resolves, or would you like to try? I am willing to
send out the standard I made. I am going to be gone for the next week,
so if anyone is interested, please send an e-mail to Ruba Deeb
(rsdeeb@mail.med.cornell.edu).
Thank you,
Bill
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--- Bill Enslow
Protein Sequencing Technician
Cornell BioResource Center
Rm 143 Biotechnology Building
phone: 607-254-4848
fax: 607-254-4847
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