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Spectrophotometric titration with N-ethylmaleimide

Addition of thiols to activated double bonds 3.8.9.1. Spectrophotometric titration with N-ethylmaleimide (NEM) CHjSH CH-CO  [Pg.110]

The spectrophotometric procedure requires freshly prepared stock solutions of NEM (1.25 x 10 M) in either 5 M guanidine hydro- [Pg.110]

0 ml NEM solution -f 0.5 ml protein solution D 2.0 ml solvent + 0.5 ml protein solution The absorbancy (Aj) of solution A (sample cell) versus solution B (reference cell), and the absorbancy (A2) of solution C versus solution D, are then determined in 1 cm pathlength spectrophotometer cells. The reaction with denatured proteins is generally rapid and complete within 10 min under the conditions specified here. Lack of further change in absorbance over an additional period of 10 min indicates that the reaction is indeed complete. [Pg.111]

The at 305 nm for NEM is 620 whereas S-(ethylsuccinimido)-cysteine does not absorb at this wavelength. Hence, the sulfhydryl titer is given by the following relationship. [Pg.111]

Parenthetically, it should be noted that the reaction of maleic anhydride with sulfhydryl-containing proteins also leads to the formation of S-succinylcysteine derivatives, by the reaction sequence [Pg.112]




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