You might also take a look at Gerd Reuter and Roland Schauer's chapter
Methods in Enzymology Vol.230 (1994) pp168-197. Schauer has published
extensively on the chemistry and biochem of sialic acids. This chapter
has procedures for microscale detection of total and free sialic acids
colorimetrically. I use their orcinol assay (see p. 179) routinely--in
fact I just ran one today. I've adapted it to use with
microplates--basically you reduce the amount of the organic solvent
(which doesn't dissolve polystyrene, BTW) for extracting the chromogen
and transfer samples to your wells to read them. This assay also works
great for pentose detection--just leave out the isoamyl alcohol, double
the amounts of sample and reagent, and read at 450-490nm (depending on
what filters you have in your reader--if you have a variable wavelength
reader you're way ahead). On p.180 you'll find Schauer and Reuter's
microscale adaptation of Warren's periodic acid-TBA assay for free
sialic acids (or total after hydrolysis). I don't know what time frame
you're monitoring, but these assays would work best if you can quench
the reaction you're studying, since they require some time to set up,
run, and read (~1.5-2h, incubation times for orcinol of 15-30 min).
Incidentally, I've also used Prof. Robyt's assays that Greg Cote
mentions below. The BCA assay is a great replacement for Park-Johnson as
an reducing-end-group assay--the results are very comparable. This assay
would also work well for your liberated hexoses, with the appropriate
standard(s). I've been somewhat reluctant to run PhOH-H2SO4 assays
entirely in a microplate as described by Robyt. I scale up the amounts
2-fold and run the samples in Eppendorf tubes, and then transfer the
samples to the plate with glass pipettes.
North American Vaccine, Inc.
a_j_d at ix.netcom.com
(G. Cote) wrote:
>> In article <961105151311.ZM77841 at ether70mac-102.gene.com>, sraju at gene.COM> says...
> >Raymond McGuirk wrote as:
> >Dear Glycoscientists,
> >I am looking for a simple, sensitive and reproducable microplate
> >assay for free or bound hexoses and/or sialic acids. I am trying to
> >quantify the amount of carbohydrate being removed from a glycoprotein
> >with endo- and exo- glycosidase enzymes over time. References would
> >be much appreciated.
> >Thanking you in advance,
> >Raymond McGuirk
> >Department of Biochemistry,
> >University College Dublin,
> >Dublin 4, Ireland.
> >E-Mail:- McGuirk at ollamh.ucd.ie> >-------------
> You might also want to check out the methods described by J.D. Fox and
> J.F. Robyt "Miniaturization of three carbohydrate analyses using a
> microplate reader", which appeared in Analytical Biochemistry 195(1991)
> 93-96. I don't know if it will suit your needs, but the three assays are
> reducing sugars (via the copper-bicinchoninate method), total
> carbohydrate (phenol-sulfuric acid) and glucose (via glucose oxidase).
> Application to measuring saccharide hydrolysis is also illustratred.
>> G. Cote