Thiol groups exist in living organisms as proteins such as albumin, glutathione, free thiol groups, etc. Thiols have a strong reducing ability and respond quickly to reactive oxygen species (ROS), eliminating them and forming disulfides. Protein thiol groups are more susceptible to oxidative modification than other amino acid side chains, and several proteins and enzymes are regulated by the redox state of thiol groups.

Thiol concentration in serum is one of the indicators of the redox environment in vivo, and is also attracting attention as an oxidative stress marker. It has been reported that serum thiol concentrations are decreased in various diseases such as cancer and liver diseases, suggesting that serum thiol concentration may be an indicator related to diseases and aging.

This method determines the total amount of SH groups by observing the color development in the visible region by the reaction of Ellman's reagent (DTNB: 5,5′-Dithiobis(2-nitrobenzoic acid)) with thiol (SH) groups. DTNB reacts with SH groups to produce TNB (5-Mercapto-2-nitrobenzoic acid). The concentration of thiol groups in the sample is determined by measuring the absorbance at a wavelength of 412 nm.

◯ Samples that can be directly quantified with this kit

Examples: serum, plasma

※Dissolve the standard sample (acetylcysteine powder) in the amount of water indicated on the label immediately prior to use.

※Store the dissolved standard solution frozen.

(1) Add 20 µl of DIW, standard solution, and assay sample to wells of a 96-well plate.

(2) Apply 200 µl of RA solution to each well.

(3) Incubate the plate at room temperature for 5 minutes.

(4) Measure absorbance OD1 at the wavelength of 412 nm.

(5) Add 30 µl of RB solution to each well.

(6) Incubate the plate at room temperature for 5 minutes.

(7) Measure absorbance OD2 at the wavelength of 412 nm.

(8) Calculate the total antioxidant capacity using the following formula:

(1) Linearity

Figure 1: Dilution linearity of standard sample

• “Tissue sulfhydryl groups.” Arch. Biochem. Biophys. 82 (1): 70–7. (1959).

• Einaga H., Yoshihara E., Matsuo Y. and Yodoi J. Oxidative stress and redox regulation －protein oxidative modification and activation －. Journal of Analytical Bio-Science, Vol.32, No 4. 2009

• “Reduced Level of Serum Thiols in Patients with a Diagnosis of Active Disease. “Journal of Anti-Aging Medicine.Vol.6, Number 4, 2003

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