To 1 g human plasma, 5 ml 0.1 M 2-mercaptoethanol (SigmaAldrich) with 0.5 % sodium ascorbate (Sigma-Aldrich) was added and clarified the supernatant by centrifugation.
To 20 μl sample, 1 μl of the above enzyme preparation was added and incubated at 37 °C for 4 h in dark. Samples were diluted and 10 μl of it was used for the assay