Question

Using Ellman’s reagent, find the number of cysteines in a protein that has a concentration of 30 mg/ml solution with a molecular weight of 15,000 Da. Data-- 0.1 ml of the protein was reacted with the reagent and diluted to 10 ml and the absorbance at 412 nm was 1.4 A. Hint, first find the moles of protein used for the experiment, then calculate the moles of Ellman’s reagent that reacted

Answer 1

For Protein molecular weight [kDa]:  15000

At Concentration of 30 mg/ml

The moles of protein is 0.000002

Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB) is a chemical used to quantify the number or concentration of thiol groups in a sample.

This reaction is rapid and stoichiometric, with the addition of one mole of thiol releasing one mole of TNB. The TNB²⁻ is quantified in a spectrophotometer by measuring the absorbance of visible light at 412 nm, using an extinction coefficient of 14,150 M⁻¹ cm⁻¹ for dilute buffer solutions and a coefficient of 13,700 M⁻¹ cm⁻¹ for high salt concentrations, such as 6 M guanidinium hydrochloride or 8 M urea. Unfortunately the extinction coefficient for dilute solutions was underestimated in the original 1959 publication, as 13,600 M⁻¹ cm⁻¹, and as noted in a recent article, this mistake has persisted in the literature.Commercial DTNB may not be completely pure, so may require recrystallization to obtain completely accurate and reproducible results.