4.11. Ellman’s Test for Determination of Protein-Bound Sulfhydryl (PB-SH) Groups

To calculate the sulfhydryl (-SH) group bound to a protein in the reaction mix, ZnJ6 was added to bovine insulin in an equimolar ratio; ZnJ6 alone served as control. One mM DTT was added to the solution to reduce the insulin, as described above. The amount of protein bound-SH (PB-SH) in the reaction mix was calculated before and after incubation for 2 h. To calculate the PB-SH, DTNB was first added to the mixture, and total -SH groups (T-SH) were measured by taking absorbance at 412 nm [before precipitation with trichloroacetic acid (TCA)]. Next, a parallel mixture was TCA precipitated, centrifuged to remove all the proteins, and the DTNB was added to the protein-free supernatant. This step eliminated the effect of DTT on binding to DTNB. The value obtained was nonprotein bound-SH (NP-SH). The difference between the T-SH and the NP-SH values gave the PB-SH groups [45]. Total-SH groups were quantified by adding 50 µL of reaction sample in 950 µL DTNB reagent (0.1 mM DTNB, 2.5 mM sodium acetate, and 100 mM Tris, pH 8). For each measurement of DTNB binding before and after TCA precipitation was measured. The mixture was incubated for 5 min at RT before monitoring the absorbance at 412 nm.