Steven Pirie-Shepherd wrote as:
>>I am going to perfomr a reductive deamination on a protein which I
>suspect has multiple O-linked glycans. I want to then separate said
>glycans from protein and chromatograph them on monoQ (I suspect that the
>glycans are sialylated.). I notice that sialic acid absorbs at 280nm , is
>this just free SA, or will SA in linkage to gal also absrob at 280? Can I
>use a simple 280nm detector to pixckj up sialo-glycans on monoQ?
> More questions. When I perform the reductive deamination, will
>this preocedure (45'C and 4M BA Borohydride for 16 hours under N2)
>destroy my protein? I dont care if it does, but I am looking for a way to
>separate glycanns from other junk in the experiment. if the protein
>remains >20kDa then I can use gel filtration. Further, I have never used
>Na borohydride, but it comes in a big metal case!!!!! What precautions
>must i take if I subject a 4M sol'n of this stuff to 45'C under N2 (I was
>going to flush 45mL reaction tubes with N2 and place all the reactants
>into a water bath at 45'C in the flushed tube, is this safe?).
>To answer to the above querry:
(1) I am not sure whether you mean reductive b(beta)-elimination: if that
is the case I can give you the following suggestions:
Reductive b-elimination is a procedure used to release the O-linked glycans
from glycoproteins. This procedure do not release N-glycans. However this
procedure randomly cleave the protein backbone. Hence if you want to save
your protein backbone, do not use this procedure.
(2) For this procedure, NaBH4 alone do not works. NaBH4 is being added to
avoid peeling reaction during the action of alkali. Hence alkali is the one
that liberates O-glycans and NaBH4 reduces GalNAc to GalNAcitol. If you
need procedure for reductive b-elimination, please refer recent vol. 230,
Methods in Enzymology.
(3) Sialic acid do not absorbs at 280 nm. Only proteins containing aromatic
amino acids absorbs at this wavelength. However the amide group of sialic
acid gives absorption at 230 and/or 214 nm and people does use these
wavelengths to detect the glycans.
Hope this helps