Ellman's Reagent/DTNB (PROTOCOL)

[Shaun D. Black]

Hi All, 

I had a couple of folks ask for my opinion and method for use of Ellmen's
reagent (DTNB) in the detemination of protein thiols (Cys-SH).  Below, I've
included a protocol, references, and general information that should be
helpful to all who anticipate using this method.  Best Regards,  Shaun



     =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 
     =  Use of Ellman's Reagent for Determination of Protein Thiols  =
     =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

=-=-=-=-=-=-=-=
=  Background =
=-=-=-=-=-=-=-=

Ellman's reagent or 5,5'-dithiobis(2-nitrobenzoate) (DTNB) is a symmetrical
aryl disulfide which readily undergoes the thiol-disulfide interchange
reaction in the presence of a free thiol:

        -                -                           -
     COO              COO                         COO
      \___        ___/                             \___
      /   \      /   \           |    -            /   \     |
 O N-   O   -S-S-  O   -N O   +  Cys-S  ---->  O N-  O  -S-S-Cys  (Mixed
  2   \___/      \___/   2       |              2  \___/     |   disulfide)
        
          (Excess)                                        +
                                                     -
                                                  COO
                                                   \___
                                                   /   \  -    (2-nitro-5-
                                               O N-  O  -S    thiobenzoate)
                                                   \___/          (TNB)

The TNB dianion has a relatively intense absorbance at 412nm compared to
both disulfides.  Because the stoichiometry of protein thiol to TNB formed
is 1:1, TNB formation can be used to assess the number of thiols present.
In the absence of denaturants, only accessible thiols will react, whereas
in the presence of chaotropic agents the total number of reduced Cys residues
present can be measured.  Reduction of the protein followed by treatment
with chaotropes and DTNB can yield the total number of cysteines (Cys-SH
plus Cys-S-S-Cys).
                                               -                     -
     The reaction is sensitive to alkaline pH ( OH) competes with R-S ),
acidic pH (disulfides can be broken), oxygen (reoxidation of R-SH), and
temperature (thermochromism).  Consequently, the reaction is usually 
carried out with an excess of DTNB to protein, at neutral pH, fixed temp-
erature, and sometimes under anaerobic conditions.  Furthermore, TNB is
sensitive to various buffer ions, so the extinction coefficient used to
calculate number of thiols must be properly matched to the reaction
conditions.  This is also true of reactions carried out in the presence of
the denaturants urea or guanidine (GuHCl).  The following table summarizes
some useful data on DTNB and TNB under various conditions:

--------------------------------------------------------------------------
Compound   lambda max (pH~7)   Extinction: (in PO4)  (in Tris)  (in GuHCl)
---------  -----------------   --------------------  ---------- ----------
DTNB            324 nm                17,780           16,600      ----
TNB             412 nm                14,150            ----      13,700
--------------------------------------------------------------------------
Extinction coefficients are in units of per Molar per cm path length, 30degC

Decreased TNB extinction in the presence of GuHCl is due to a shift in the
lambda max from 412 to 422 nm.  The extinction of DTNB at 412 nm is 212/M/cm,
small but measurable.

=-=-=-=-=-=-=-=
=  Protocol   =
=-=-=-=-=-=-=-=

Stock Solutions:

     Protein solution (native or previously reduced and desalted)
                      (~5-40uM in 0.1M potassium phosphate buffer, pH 7.4)
     DTNB stock  (20 mM in 0.1M KPO4, pH 7.4; MW 396.35; 7.93 mg/ml)
                 (DTNB is very slow to go into solutions; vortex periodically
                  over the period of 2-3 hours to achieve complete solution,
                  which will be very light yellow in color)
     Urea or Guanidine-HCl  (high purity, crystalline solids)


Major Equipment:

     Dual beam spectrophotometer set in kinetic mode at 412nm
     Thermostated cells, 30degreesC

Procedure:

     Sample cell 1    = 1.0 mL protein solution + 50 uL 20mM DTNB
     Reference cell 1 = 1.0 mL buffer + 50 uL DTNB

     Record the absorbance before addition of the DTNB as "zero".  Follow
the reaction at 412 nm at 30degC continuously.  Record past the time when
the reaction reaches a maximum.  If any significant down slope is seen
after the maximum absorbance is reached, this will need to be corrected for
in order to get the correct number of accessible thiols.

     In the second experiment, add crystalline Guanidine-HCl to your
protein solution to achieve a final concentration of about 4M.  Note the
volume change and the resulting significant dilution of your protein 
solution!

     Sample cell 2    = 1.0 mL protein solution (+4M GuHCl) + 50 uL 20 mM DTNB
     Reference cell 2 = 1.0 mL buffer (+4M GuHCl) + 50 uL 20mM DTNB

     Again, record OD 412 nm at 30 degC up to and beyond peak absorbance.
This experiment will yield the total number of reduced thiols.

     Sample cell 3    =  1.0 mL pre-denatured,reduced, and dialyzed protein
                          + 50 uL 20mM DTNB
     Reference cell 3 =  1.0 mL buffer (+4M GuHCl) + 50 uL 20 mM DTNB

     Again, record OD 412 nm at 30degC vs time to peak absorbance and beyond.
This experiment will yield the total number of Cys residues in the protein.

=-=-=-=-=-=-=-=-=-=
=  Data Analysis  =
=-=-=-=-=-=-=-=-=-=

Calculate the number of thiols modified (or concentration thereof) using
the maximum absorbance at 412 nm measured (or the corrected value thereof),
Beer's Law, and the appropriate extinction coefficient; divide this value
by the accurately known molar protein concentration in the reaction cuvette
for each of the three experiments.  This will give you accessible thiols,
total Cys-SH, and total Cys-SH plus Cys-SS-Cys.  Do each experiment in
duplicate or triplicate and average the respective replicates.  If your
numbers are statistically integers, you're done.  If not, consider repeating
the protocol, but using anaerobic (degassed) buffers for all reactions.

=-=-=-=-=-=-=-=-=-=-=
=  Some References  =
=-=-=-=-=-=-=-=-=-=-=

"A colorimetric method for determining low concentrations of mercaptans"
  Ellman, G.L. (1958) Arch. Biochem. Biophys. 74, 443-450.

"Reassessment of Ellman's reagent" Riddles, P.W., Blakeley, R.L., and Zerner,
  B. (1983) Methods Enzymol. 91, 49-60.

"Studies on the identity of the heme-binding cysteinyl residue in rabbit
  liver microsomal cytochrome P-450 isozyme 2"  Black, S.D. and Coon, M.J.
  (1985) Biochem, Biophys. Res. Commun. 128, 82-89.


  =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 
  = Shaun D. Black, PhD   | Internet address:     shaun at jason.uthct.edu = 
  = Dept. of Biochemistry | University of Texas Health Center, at Tyler = 
  =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=