Re: ProtSeq: 1-iodoiodoxolin-3-one.

Carol Beach (cmbeach@pop.uky.edu)
Tue, 5 Oct 1999 09:55:39 -0400 (EDT)

Hi, Len,
Along with David Schooley, the literature I have regarding cleavage
after tryptophan residues involves o-iodosobenzoic acid, not the para- form.
Check out "Fragmentation of proteins with o-iodosobenzoic acid: Chemical
mechanism and identification of o-iodoxybenzoic acid as a reactive
contaminant that modifies tyrosyl residues", W.C. Mahoney, P.K. Smith and
M.A. Hermodson, Biochemistry 20 (1981) 443-448.
Looking back though my very old notes, I see that I tried using this
method with some solution-phase standard proteins and also with some
PVDF-blotted proteins.
The reaction appears to have worked, so it's not immediately clear why I
decided to abandon it in favor of BNPS-skatole. I was separating fragments
by RPHPLC, so maybe I preferred how I could clean up the BNPS-skatole
reaction with ether extraction. Or perhaps I was just partial to orange.
I have another paper in a pamphlet called "Protein Analysis
Renaissance" distributed by ABI that I picked up at the Protein Society
meeting in 1993: "Sequencing on ProBlott membranes of protein fragments
produced in polyacrylamide gels", P. Johnson and E.F. Saulinskas, pp 29-32.
The authors attempted in-gel chemical cleavage of proteins by CNBr,
BNPS-skatole and o-iodosobenzoic acid. They were able to produce fragments
with CNBr and BNPS-skatole, but not with o-iodosobenzoic acid, even at a
higher concentration and longer reaction time.
Hope this helps.

Carol

At 03:54 PM 10/4/99 +0000, you wrote:
>Dear ABRFers (especially the chemist ones)
>
>In ASMS 97, Ron Beavis had an abstract for a method for cleaving after Trp
>residues. On enquiry that year, he sent the method shown below. (Typically,
>we haven't needed to try it until now!)
>
>I can't find para-iodosobenozoic acid in Aldrich, only the 2- (ortho-) form.
>I also do not understand the mechanism of action and the use of the term
>1-iodoiodoxolin-3-one in Ron's subject line. I have been unable to contact
>him at beavis@proteometrics.com to get clarification and no literature
>searches have helped.
>
>Can anyone help explain the reaction or get me in touch with Ron? Where can
>one buy para-iodosobenozoic acid? Has anyone tried this reaction?
>
>Len
>
>.---------------------------------------------------------------------------
>>Dissolve the following components in 2:1 water:acetonitrile, with 0.1% TFA
>>
>>0.5 millimolar para-iodosobenozoic acid
>>0.1 millimolar sodium iodide
>>
>>Add that to your sample for a final sample concentration of approximately
>>50 micromolar. Allow the reaction to continue for approximately 30 minutes
>>at room temperature. Quench the reaction with DTT.
>>
>>Note: Methionines (+1 oxygen) and cysteines (+2 oxygens) are
>>quantitatively oxidized by this procedure. Cysteines protected by acetate
>>or acetimide groups do not seem to be oxidized, at least the rate is much
>>slower that for methionines in the peptides we have examined. There is
>>some iodination of aromatic groups, but for the reaction time indicated it
>>is minimal.
>>
>>Note: The presence of excess halide ions (such as chloride) slows down the
>>reaction. For best results, the protein sample should be as salt-free as
>>possible.
>>
>>Ron Beavis
>
>*********************************************************************
>Dr Len C. Packman
>Assistant Director of Research
>Protein and Nucleic Acid Chemistry Facility
>Department of Biochemistry
>University of Cambridge
>80 Tennis Court Road
>Old Addenbrookes Site
>Cambridge, CB2 1GA, UK
>Tel: +44 (1223) 333639
>FAX: +44 (1223) 766002
>e-mail: lcp2@mole.bio.cam.ac.uk
>Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
>
>
>
>

Carol Manley Beach, Ph.D.
Protein Sequence Analysis
Macromolecular Structure Analysis Facility
232 Combs Cancer Research Building
University of Kentucky
Lexington, KY 40536-0096

cmbeach@pop.uky.edu
(606)257-4932 (office)
(606)257-4475 (lab)
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