Thiols, spectrophotometric titration

In Section V we will discuss acid-base equilibria. It is, of course, quite possible and often very useful to study other reactions of proteins by spectrophotometric titration methods. The reaction of thiol groups with heavy metals and the binding of a coenzyme or an inhibitor by an enzyme represent familiar examples. However, it will be most convenient to limit our present discussion to hydrogen ion equilibria. 

It may seem odd that the technique of spectrophotometric titration, so extensively applied to the tyrosyl residues of proteins, has not been used in the study of protein sulfhydryl groups. The only study of this sort known to the author is the determination of the ionization constant of —SH grouj of thiolated gelatin (introduced by reaction with A -acetylhomocysteine thiolactone). By measuring the change in absorptivity as a function of pH at 2380 A, Benesch and Benesch (1958) found the apparent acidity of the thiol groups of the modified gelatin (pK = 9.8) to be nearly the same as that of an appropriate model compound, A -acetylhomocysteine (pK = 10.0). 

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

The overexpression and purification of the dimeric, 144 amino acid TnSOl MerR protein in large quantities has made detailed chemical studies possible (143, 145, 171). A variety of studies including gel filtration, equilibrium dialysis, nonequilibrium dialysis, and spectrophotometric titration have indicated that the MerR proteins isolated to date bind a single mercuric ion per protein dimer (82, 145, 171, 202). All of these studies have been performed in the presence of 10-10,(XX)-fold excess buffer thiol competitors, indicating that Hg(II) binds tightly to the protein. 

As the four microscopic constants cannot be determined by a titration curve, spectrophotometric analysis (UV absorption of R-S ) was necessary. The pX (8.65) of cysteine betaine (ionization of a thiol in the presence of a positive nitrogen) and the pK (8.75) of S-methyl cysteine (ionization of an amino group in the presence of neutral sulfur) closely mimic the and /c2 dissociation pathways and suggest that these values should be close to each other... 

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