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<DIV><SPAN class=308034711-31082005><FONT size=3><STRONG>Ellman's
Reagent</STRONG> </FONT></DIV>
<DIV>
<P>In order to quantitative available sulphydryl groups, the classic reagent is
</SPAN> Ellman's reagent<SPAN class=308034711-31082005>, </SPAN><SPAN
class=308034711-31082005>(</SPAN>DTNB<SPAN class=308034711-31082005>),
which is used<SPAN class=308034711-31082005> </SPAN>for the
modification of free thiols in proteins and peptides[1]</SPAN>.
It <SPAN class=308034711-31082005>typically reacts with the thiol
containing molecule to </SPAN>releasing the 5-thio-2-nitrobenzoate (TNB) anion,
which absorbs<SPAN class=308034711-31082005> </SPAN>at 412 nm <SPAN
class=308034711-31082005>(</SPAN>absorption coefficient of 13600 M−<SPAN
class=308034711-31082005>1</SPAN>cm−<SPAN class=308034711-31082005>1</SPAN>
[<SPAN class=308034711-31082005>2</SPAN>]<SPAN
class=308034711-31082005>)</SPAN>.<SPAN class=308034711-31082005> It's
presence can be plotted against a standard curve to determine the total amount
of free thiols in proteins or peptide. </SPAN><SPAN
class=308034711-31082005>Typically reactions arte conducted </SPAN> in
phosphate buffer, <SPAN class=308034711-31082005>(0.1 M<SPAN
class=308034711-31082005> </SPAN></SPAN>pH 7.2<SPAN class=308034711-31082005>)
</SPAN>at 25 °C under <SPAN class=308034711-31082005>strictly
</SPAN>anaerobic conditions<SPAN class=308034711-31082005>. Around </SPAN>3.6 x
10-5 M of free thiol <SPAN class=308034711-31082005>is relatively easy to
detect. Ten fold further dilution appears to be the limit of thee assay
using 1 cm pathlength cells. </SPAN>A <SPAN class=308034711-31082005>double
vacuum manifold </SPAN>system <SPAN class=308034711-31082005>(under Argon)
</SPAN>and<SPAN class=308034711-31082005> a </SPAN>stoppered sidearm
cuvette <SPAN class=308034711-31082005>are routinely used in our laboratory
</SPAN>to maintain <SPAN class=308034711-31082005>strictly
</SPAN>anaerobic<SPAN class=308034711-31082005> </SPAN>conditions.<SPAN
class=308034711-31082005> Otherwise, oxidation to disulphides may occur
preventing accurate quantification of the thiol groups present.</SPAN></P>
<P><SPAN class=308034711-31082005><STRONG>References</STRONG></SPAN></P>
<P><SPAN class=308034711-31082005></SPAN><SPAN class=308034711-31082005>[1]
Riddles, P. W.; Blakely, R.; Zerner, B. Reassessment of Ellman's
reagent.<STRONG> </STRONG><SPAN title="Methods in enzymology.">Methods
Enzymol.</SPAN> 1983;91:49-60.</SPAN></P></DIV>
<DIV><SPAN class=308034711-31082005>[2] Li, T.-Y.; Minkel, D. T.; Shaw III, C.
F.; Petering, D. H. Biochem. J. (1981) 193, 441-446.</SPAN></DIV>
<DIV><SPAN class=308034711-31082005></SPAN> </DIV>
<DIV><SPAN class=308034711-31082005>Dr. Fyaz M. D. Ismail</SPAN></DIV>
<DIV><SPAN class=308034711-31082005>Laboratory of Medicinal
Chemistry</SPAN></DIV>
<DIV><SPAN class=308034711-31082005>Liverpool John Moores
University,</SPAN></DIV>
<DIV><SPAN class=308034711-31082005>Byrom Street,</SPAN></DIV>
<DIV><SPAN class=308034711-31082005>Liverpool L3 3AF</SPAN><SPAN
class=308034711-31082005></DIV>
<DIV><BR></DIV></SPAN>
<BLOCKQUOTE
style="PADDING-LEFT: 5px; MARGIN-LEFT: 5px; BORDER-LEFT: #0000ff 2px solid">
<DIV class=OutlookMessageHeader dir=ltr align=left><FONT face=Tahoma
size=2>-----Original Message-----<BR><B>From:</B>
everybody-bounces@orglist.net [mailto:everybody-bounces@orglist.net]<B>On
Behalf Of </B>Anil Saini<BR><B>Sent:</B> 31 August 2005 10:59<BR><B>To:</B>
everybody@orglist.net<BR><B>Subject:</B> ORGLIST: Quantification of SH
group<BR><BR></FONT></DIV>
<P>Helo to every one <BR>I want to know how to quantitate the sulfahydrl group
(-SH) in a linear peptide chain spectrophotometrically. and if there is any
other method to quantitate it what will be the strategy for
that.<BR><BR>another thing i want to know is that how to precipitate
urea from a solution of denatured protiens.<BR>I thanks in
advance<BR><BR><BR>Anil </P><BR><BR><A
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