Protein Protocols Protein Protocols

596 Aitken and Learmonth
6. Finally, add 100 µL 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 (A final).
7. The concentration of thiols present may be calculated from the molar absorbance of the
TNB anion. (See Note 3.)
∆A 412 = E 412TNB 2– [RSH] (1)
Where ∆A 412 = A final – (3.1/3.2) (A DTNB – A buffer)
and E 412 TNB 2– = 1.415 × 10 4 cm –1 M –1 .
If using denaturing buffer, use the value E 412 TNB 2– = 1.415 × 10 4 cm –1 M –1 .
3.2. Analysis of Disulfide Thiols
1. Sample should be carboxymethylated (see Chapter 59) or pyridethylated (see Chapter 62)
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 µL, is usually required) should be dissolved
in 6 M guanidinium HCl, 0.1 M Tris-HCl, 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 (see Subheading 3.1.).
Alternatively, gel filtration into the same buffer may be carried out.
5. Analysis of newly exposed disulfide thiols can thus be carried out as described in Subheading
3.1.
4. Notes
1. It is not recommended to use urea in place of guanidinium HCl, since this can readily
degrade to form cyanates, which will react with thiol groups.
2. Unless newly purchased, it is usually recommended to recrystallize DTNB from aqueous
ethanol.
3. Standard protocols for use of Ellman’s reagent often give E412 TNB2– = 1.36 × 104 cm –1M –1 .
A more recent examination of the chemistry of the reagent indicates that these are more
suitable values (4), and these have been used in this chapter.
References
1. Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77.
2. Zahler, W. L. and Cleland, W. W. (1968) A specific and sensitive assay for disulfides.
J. Biol. Chem. 243, 716–719.
3. Anderson, W. L. and Wetlaufer, D. B. (1975) A new method for disulfide analysis of
peptides. Analyt. Biochem. 67, 493–502.
4. Riddles P. W., Blakeley, R. L., and Zerner, B. (1983) Reassessment of Ellman’s reagent.
Meth. Enzymol. 91, 49–60.