FW: ORGLIST: Quantification of SH group

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From: Ismail, Fyaz (F.M.Ismail$##$livjm.ac.uk)
Date: Wed Aug 31 2005 - 08:15:58 EDT

Ellman's Reagent

In order to quantitative available sulphydryl groups, the classic =
reagent is Ellman's reagent, (DTNB), which is used for the =
modification of free thiols in proteins and peptides[1]. It typically =
reacts with the thiol containing molecule to releasing the =
5-thio-2-nitrobenzoate (TNB) anion, which absorbs at 412 nm (absorption =
coefficient of 13600 M?1cm?1 [2]). It's presence can be plotted against =
a standard curve to determine the total amount of free thiols in =
proteins or peptide. Typically reactions arte conducted in phosphate =
buffer, (0.1 M pH 7.2) at 25 C under strictly anaerobic conditions. =
Around 3.6 x 10-5 M of free thiol is relatively easy to detect. Ten fold =
further dilution appears to be the limit of thee assay using 1 cm =
pathlength cells. A double vacuum manifold system (under Argon) and a =
stoppered sidearm cuvette are routinely used in our laboratory to =
maintain strictly anaerobic conditions. Otherwise, oxidation to =
disulphides may occur preventing accurate quantification of the thiol =
groups present.


[1] Riddles, P. W.; Blakely, R.; Zerner, B. Reassessment of Ellman's =
reagent. Methods Enzymol. 1983;91:49-60.

[2] Li, T.-Y.; Minkel, D. T.; Shaw III, C. F.; Petering, D. H. Biochem. =
J. (1981) 193, 441-446.
Dr. Fyaz M. D. Ismail
Laboratory of Medicinal Chemistry
Liverpool John Moores University,
Byrom Street,
Liverpool L3 3AF

-----Original Message-----
From: everybody-bounces$##$orglist.net =
[mailto:everybody-bounces$##$orglist.net]On Behalf Of Anil Saini
Sent: 31 August 2005 10:59
To: everybody$##$orglist.net
Subject: ORGLIST: Quantification of SH group

Helo to every one
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.

another thing i want to know is that how to precipitate urea from a =
solution of denatured protiens.
I thanks in advance



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