Determination of interactive thiol ionizations in bovine serum albumin, glutathione, and other thiols by potentiometric difference titration.
A potentiometric difference titration (PDT) method is used to study the ionization behavior of the thiol group in bovine serum albumin and in the following less complex compounds: glutathione, cysteine, 2-mercaptoethanol, 3-mercaptopropionic acid, 2-mercaptoethylamine, cis-2-mercaptocyclobutylamine, 2-aminothiophenol, and 5-mercapto-2-nitrobenzoic acid. In the PDT method the pH dependence of the amount of protons released in the reaction RSH + CH3SO2SCH3 leads to RSSCH3 + CH3SO2- + H+ is measured in order to obtain the pH dependence of the molar proton content of the thiol (hu) relative to the molar proton content of its methylthio derivative (hm). The pH dependence of hu--hm reflects the ionization behavior of the thiol group and of other groups whose ionization is thermodynamically linked to that of the thiol group. Data presented here indicate that the ionization behavior of the single thiol group in albumin is strikingly different in the native and the urea-denatured proteins. Three ionizable groups appear to affect ionization of the thiol in the native protein whereas only one group appears to affect ionization of the thiol in the urea-denatured protein. Furthermore, the measured PDT curves are consistent with an abnormally high acidity (pK less than 5) for the thiol in native albumin and a normal acidity for the thiol in the urea-denatured protein. Comparisons of microscopic ionization constants determined for cysteine by using the PDT method with those determined by other methods indicate that the PDT method should be useful in characterizing the ionization behavior of thiol groups in proteins and other polyprotic substances.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D005978:Glutathione / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007201:Indicator Dilution Techniques; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives; D011199:Potentiometry; D012710:Serum Albumin, Bovine / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000032:analysis*
D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D005978:Glutathione