Specificity of NEM toward cysteine residues

Mikhail Alexeyev malexe at biost1.thi.tmc.edu
Tue Aug 13 11:53:15 EST 1996


In article <dodel-1308961627590001 at chokotoff.biocell.fundp.ac.be>,
dodel at biocell.fundp.ac.be (Dominique DELFORGE) wrote:

> In our experiments, we recently used two different cysteine-directed
> chemical reagents i.e. 5,5'-dithiobis(2-nitrobenzoic)acid (DTNB) and
> N-ethylmaleimide (NEM) in order to determine the possible presence of a
> cysteine residue in the active site of L-alanine dehydrogenase. The
> results obtained show that the enzyme is not inactivated by DTNB even when
> used in large excess relative to the enzyme with long reaction times. On
> the other hand, the enzyme is rapidely inactivated by NEM when used in
> large excess.
> 
> My questions are: Does someone know something about the chemical
> specificity of N-ethylmaleimide ? Is this reagent completely specific of
> cysteine residues ? When used in large excess, does the reagent react with
> other residues than cysteine ? With which residues does it then also react
> ? Is DTNB more specific (or exclusively specific) of cysteine residues ?
> Do you have litterature references about the chemical specificity of NEM ?
> 
> 

Sorry, my references are home, but my feeling is that both reagents are
quite specific. Also, you do not give much information on assay conditions
for L-alanine dehydrogenase which led me to a speculation that can
potentially explain your results without resorting to search for
alternative specificities.

As far as I know, DTNB formes mixed DISULFIDES with SH-groups of proteins
which (at least, theoretically) can be reduced if your enzyme
storage/assay buffer containes reducing reagents like DTT, 2ME, DTE or
other. To the contrary, modification with NEM is irreversible.

Another explanation lies in steric or electrostatic effects. DTNB is more
bulky and more charged than NEM is. Large excess of NEM required to
inactivate enzyme may indicate some steric/electrostatic problems and DTNB
molecule could be just too charged/bulky to do the job. 

My interest is far from protein chemistry, but I wouldn't use DTNB to
block sulfhydrils. How common is it to use DTNB for this purpose?

Regards,
M.A.



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