Ellman\'s reagent (5,5\'-dithiobis-(2-nitrobenzoic acid)) is an important reagen

Question

Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid)) is an important reagent for quantifying thiols by liberating a yellow chromophore (5-mercapto-2-nitrobenzoic acid), which absorbs light at 410 nm. The reaction is shown below. Ellman's reagent has been used for many years to quantify the number of free cysteine residues that are found in a protein. You have purified protein USF and want to determine the number of cysteine residues found in this protein. Treatment of a 0.4 mg/mL solution of protein USF with 1.0 mM Ellman's reagent at pH 10.0 yields an increase in absorbance at 410 nm of 0.75. Assuming the reaction went to completion, calculate the number of free cysteine residues in protein USF using the following data. Molecular weight of protein USF: 50,000 g/mol Extinction coefficient for the yellow chromophore at 410 nm (epsilon_410): 13,600 M^1 cm^-1 Path length of cuvette using to measure increase in A_410: 1 cm

Explanation / Answer

concentration of the protein = 0.4 mg/mL

= (0.0004g/mL * 1000)/(50000 g/mol)

= 8*10-6 M

As concentration of Ellman's reagent is very high with respect to the concentration of protein, so it can be assumed that concentration of the sample solution do not change after the reaction.

A = cl

where, A = absorbance, c = concentration of the chromophore , l = length of the cell

A = 0.75 = 13600 M-1cm-1 * c * 1 c,

or , c = 5.515*10-5 M

According to the reaction 1 mol R-SH react with 1 mole of the reagent to produce 1 mole of the yellow chromophore.

But concentration increases (5.515*10-5 M/8*10-6 M) 7 fold after the reaction, which means there are 7 cysteine residues in the protein molecule.

Related Questions

Navigate

• Browse (All)
• Browse E
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.