We often had the same experience with digestion of peptides/proteins
containing several disulphide bridges. This is especially true for specif=
ic
enzymes, more unspecific enzymes like thermolysin or pepsin might give a
better clevage efficiency but also more difficult fragment patterns.
Another approach is to partially hydrolyse the protein by oxalic acid (lo=
ok
for example FEBS Lett. 416 (1997) 197-202 or directly contact Juan Calvet=
e
in Valencia at jcalvete@ibv.csic.es).
Good luck
Michael
----------------------------------------
Dr. Michael Schrader
BioVisioN GmbH & Co. KG
Feodor-Lynen-Str. 5
D-30625 Hannover
GERMANY
Phone +49-511-53 88 96-12
Fax +49-511-53 88 96-66
Internet www.biovision.de
E-mail m.schrader@biovision.de
-----Urspr=FCngliche Nachricht-----
Von: Maria Lorenzi <mlorenzi@ascu.unian.it>
An: Recipients of ABRF List <abrf@aecom.yu.edu>
Datum: Mittwoch, 3. M=E4rz 1999 21:25
Betreff: Digestion of Proteins
>Hi,
>I'm Maria Lorenzi.
>I'm trying to digest my protein with trypsin but I'm not successfull.
>I digested it with trypsin, under non denaturating conditions, after
>reducing and alkylating my protein but it seems to be very hard to diges=
t
>it, using the same conditions of digestion, without any reduction and
>alkylation.
>I tried the digestion of the toxin, without reduction of it, under
>denaturating conditions with urea but it didn't work.
>My toxin has six cysteines and so probably there is a lot of disulfide
>bridges which make the protein very closed and so the trypsin difficult =
to
>work. When the disulfide bridges are broken the digestion is more easy
>because the more accessibility of the protein to the trypsin.
>Please, can you give to me some advises about this subject? About the
>proteolitic enzyme to use and about the more appropriate procedure of
digestion?
>Thank you very much
>Maria
>
>
>
>
>
>
>
>
>
> "Donde hay Fe hay Amor, Donde hay Amor hay Paz
> Donde hay Paz esta Dios y donde esta Dios
> no falta nada"
>
>************************************************************************=
**
>Dott.ssa Maria Lorenzi
>Dottoranda in Biochimica e Biofisica
>Istituto di Biochimica
>Facolt=E0 di Medicina e Chirurgia
>Facolt=E0 di Agraria
>Universit=E0 degli Studi di Ancona
>Via Ranieri 60131 Ancona, Italia
> Tel.:+39 71 220 4677
> +39 71 220 4681
> Fax: +39 71 280 2117
> e-mail: mlorenzi@ascu.unian.i=
t
>
>