AN S-ALKYLATING REAGENT WITH POSITIVE CHARGES AS AN EFFICIENT SOLUBILIZER OF DENATURED DISULFIDE-CONTAINING PROTEINS

A novel S-alkylating reagent, -(3-bromopropyl)-N,N,N',N',N'-pentamethy l-1,3-pro- panedi(ammonium bromide) (TAP2-Br) which carries two positi ve charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteine-containing denatured prote ins (or peptides). S-Alkylation with TAP2-Br introduces two positive c harges per cysteine residue, which will effectively shift the net char ge of a protein in the positive direction. Disulfide-containing protei ns, such as hen egg-white lysozyme, RNase A, BSA, and soybean trypsin inhibitor (Kunitz type), were reduced and S-alkylated with TAP2-Br to evaluate the potential of this reagent compared with other S-alkylatin g reagents such as monoiodoacetic acid, bromosuccinic acid and (3-brom opropyl)trimethylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP 2-Br effectively solubilized not only basic proteins (lysozyme and RNa se) but also an acidic protein containing a fairly large number of cys teine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were gre atly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-co ntaining denatured proteins and to decrease the hydrophobicity of pept ides containing cysteine residue(s).