Procedure 7.3 Spectrophotometric Titration of PC2 and Glutathione

Procedure 7.3 Spectrophotometric Titration of PC2 and Glutathione with Cadmium(ll) and Copper(ll) 

Prepare a 5 ml aqueous solution of 5 mM CdCl2 and a 5 ml aqueous solution of 5 mM CuCl2. Use acid-washed volumetric glassware. 

Prepare 25 ml of 0.15 mM (as SH) peptide [MM(PC2) = 539.58 g/mol not including triflate or acetate anion MM(glutathione, reduced) = 307.33] in your Tris-HC1 buffer. Prepare each just prior to analysis to minimize SH oxidation. 

If necessary, the SH content of your peptide can be determined by a DTNB test.15 

To 5 ml of your PC2 solution, add 5 pi Cd2+ solution. Allow the reaction to equilibrate with occasional stirring for 2-5 min. Obtain the absorbance at 250 nm (Cd2+-mercaptide). 

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