Conversion of the reactive sulfhydryl groups of proteins to the S-trifluoroethyl derivatives : Application to human hemoglobin

Abstract

The S-2,2,2-trifluoroethyl residue (-SCH 2CF 3) has been incorporated into human hemoglobin, Hb 4(SH) 2, as an extrinsic probe at Cys-β93 through formation of a disulfide bond. The thiol group was activated by reaction with 5,5′-dithiobis(2-nitrobenzoic acid), Hb 4(SSCH 2CF 3) 2 then being obtained by reaction with 2,2,2-trifluoroethanethiol. Both disulfide interchange reactions proceed to completion with a modest excess of reagents using dilute solutions of hemoglobin (0.005 m heme). The stoichiometry of each disulfide interchange reaction is readily determined by measurement of 5-thio-2-nitrobenzoic acid, a product of each reaction. The functional properties of Hb 4 (SSCH 2CF 3) 2 were found to be similar to those of Hb 4(SH) 2. At pH 7.0 and 20°C the partial pressure of oxygen required for half saturation was 0.45 mm Hg in 0.050 m 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilo-triethanol, 4.1 mm Hg in 0.050 m potassium phosphate, and 16.5 mm Hg in 0.010 m inositol hexaphosphate. The n values of the Hill plots were 1.45, 1.80, and 2.3, respectively. The equilibrium constant for the tetramer-dimer dissociation reaction, K 4,2, of the carbon monoxide derivative was 2.1 × 10 −7 m. The time course for combination with carbon monoxide was homogeneous at 432 nm. In the presence of inositol hexaphosphate the time course of combination with carbon monoxide was wavelength dependent.