The calculation of serum protein thiols is described in the following article:

http://www.sciencedirect.com/science/article/pii/S0076687994330441

(Hu ML. Measurement of protein thiol groups and glutathione in plasma.Methods Enzymol. 1994;233:380-5.)

Briefly, 0.20 ml of plasma sample is mixed with 0.6 ml of the Tris-EDTA buffer with the following addition of 40ul of DTNB (10 mM) and 3.16 ml of methanol. The tube is incubated for 20 minutes, following by centrifugation at 3000 g for 10 min at room temperature. Afterwards the absorbance of the supernatant is measured at 412 nm.

Total SH groups are calculated using the following formula and the molar extinction coefficient (13,600 cm-1 M-1):

(A - B - 0.03) × (4.0/0.2)/13.6 = (A - B - 0.03) × 1.47 mM,

where A is the absorbance of supernatant, B is DTNB blank absorbance and 0.03 is the sample blank (or it is to be estimated experimentally).

Dear Alexey

thanks for the reply but since i have this paper and i have followed the Method by Hu but i m unable to get the protein thiols reading.

Also i have followed method by sedlak and lindsay ,1976,Motchnik 1994

and ellman method though i m getting the free- SH reading more than the total -SH

i wil mail you the protocol which i m following

Try this

http://www.ncbi.nlm.nih.gov/pubmed/18620047

@Roberto

thanks for the reply..

serum protein-SH was measured by spectrophotometric method using 5'-5'-dithio-bis-2-nitrobenzoic acid according to Motchink et al., 1994.