Thursday, October 8, 2009

Estimation of Disulfide Bonds Using Ellman’s Reagent

Ellman’s reagent has been widely used for the quantitation of thiols in peptides and proteins. It has also been used to assay disulfides present after blocking any free thiols (e.g., by carboxymethylation) and reducing the disulfides prior to reaction with the reagent. It is also commonly used to check the efficiency of conjugation of sulfhydryl- containing peptides to carrier proteins in the production of antibodies.

Ellman’s reagent or DNTB (5,5'-dithiobis(2-nitrobenzoic acid)) was first introduced in 1959 for the estimation of free thiol groups. The procedure is based on the reaction of the thiol with DTNB to give the mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by the absorbance of the anion (TNB 2-) at 412 nm.

Materials :
  1. Reaction buffer: 0.1M phosphate buffer, pH 8.0.
  2. Denaturing buffer: 6M guanidinium chloride, 0.1M Na2HPO4, pH 8.0.

    It is not recommended to use urea in place of guanidinium HC1, since this can readily degrade to form cyanates, which will react with thiol groups

  3. Ellman's solution: 10 mM (4 mg/mL) DTNB in 0.1M phosphate buffer, pH 8.0.

    Unless newly purchased, it is usually recommended to recrystallize DTNB from aqueous ethanol

  4. Dithiothreitol (DTT) solution: 200 mM in distilled water.
Methods :

A. Analysis of Free Thiols
  1. It may be necessary to expose thiol groups, which may be buried in the interior of the protein. The sample may therefore be dissolved in reaction buffer or denaturing buffer. A solution of known concentration should be prepared with a reference mixture without protein. Sufficient protein should be used to ensure at least one thiol per protein molecule can be detected; in practice, at least 2 nmol of protein (in 100 micro-liter) are usually required.
  2. Sample and reference cuvets containing 3 mL of the reaction buffer or denaturing buffer should be prepared and should be read at 412 nm. The absorbance should be adjusted to zero (Abuffer).
  3. Add 100 micro-liter of buffer to the reference cuvet.
  4. Add 100 micro-liter of Ellman's solution to the sample cuvet. Record the absorbance (ADTNB).
  5. Add 100 micro-liter of protein solution to the reference cuvet.
  6. Finally, add 100 micro-liter protein solution to the sample cuvet, and after thorough mixing, record the absorbance until there is no further increase. This may take a few minutes. Record the final reading (Afinal).
  7. The concentration of thiols present may be calculated from the molar absorbance of the TNB anion.
    U-A412 = E412TNB2 - [RSH] (1)
    Where U-A412 = Afinal- (3.1/3.2) (ADTNB – Abuffer)
    and E412TNB2- = 1.415 x 104 cm-1 M -1.

    If using denaturing buffer, use the value E412TNB2- = 1.37 x 104 cm-1 M -1.

    Standard protocols for use of Ellman's reagent often give E412TNB2- = 1.37 x 104 cm-1 M -1.

B. Analysis of Disulfide Thiols
  1. Sample should be carboxymethylated or pyridethylated without prior reduction. This will derivatize any free thiols in the sample, but will leave intact any disulfide bonds.
  2. The sample (at least 2 nmol of protein in 100 micro-liter is usually required) should be dissolved in 6M guanidinium HC1, 0.1M Tris-HC1 pH 8.0 or denaturing buffer, under a nitrogen atmosphere.
  3. Add freshly prepared DTT solution to give a final concentration of 10-100 mM. Carry out reduction for 1-2 h at room temperature.
  4. Remove sample from excess DTT by dialysis for a few hours each time, with two changes of a few hundred mL of the reaction buffer or denaturing buffer. Alternatively, gel filtration into the same buffer may be carried out.

Reference: Number 21 at References