Cathodic stripping voltammetry: Part II. Study of the release of inorganic sulfide from proteins during denaturation in alkaline media

The concentration of inorganic sulfide ion liberated from a wide range of proteins denatured in 0.2 M NaOH at 25°C was measured using direct cathodic stripping voltammetry (c.s.v.), as well as by ion selective electrode potentiometry and c.s.v. after separation of the H₂S by an isothermal microdiffusion technique. The sulfide produced in 0.2 M NaOH was equivalent to the number of protein disulfide bridges broken, and using several model proteins it was shown that only surface, or solvent-accessible disulfide bonds are attacked. The reaction obeyed first-order kinetics, and the rate was proportional to hydroxide ion concentration. Some simple disulfide compounds were also studied, and possible reaction mechanisms for the formation of sulfide ion are discussed. Some iron proteins were denatured in 0.2 M NaOH and the dissociation of the heme-protein bond was monitored by measuring the iron redox wave at the mercury electrode. Normal and cancerous blood serum samples were analysed by c.s.v. measurement of the sulfide released in alkali, both before and after separation of the albumin and globulin by precipitation and gel permeation chromatography.