An S-alkylating reagent with positive charges as an efficient solubilizer of denatured disulfide-containing proteins

Hidenori Yamada, Masaharu Seno, Ayumi Kobayashi, Takeshi Moriyama, Megumi Kosaka, Yuji Ito, Taiji Imoto

Research output: Contribution to journalArticlepeer-review

37 Citations (Scopus)


A novel S-alkylating reagent, N-(3-bromopropyl)-N, N, N', N', N'-pentamethyl-1, 3-pro-panedi(ammonium bromide) (TAP2-Br) which carries two positive charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteinecontaining denatured proteins (or peptides). S-Alkylation with TAP2-Br introduces two positive charges per cysteine residue, which will effectively shift the net charge of a protein in the positive direction. Disulfide-containing proteins, 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-alkylating reagents such as monoiodoacetic acid, bromosuccinic acid and (3-bromopropyl)trimethylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP2-Br effectively solubilized not only basic proteins (lysozyme and RNase) but also an acidic protein containing a fairly large number of cysteine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were greatly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-containing denatured proteins and to decrease the hydrophobicity of peptides containing cysteine residue(s).

Original languageEnglish
Pages (from-to)852-857
Number of pages6
JournalJournal of biochemistry
Issue number4
Publication statusPublished - Oct 1994


  • Disulfide-containing protein
  • Double cationic reagent
  • Peptide mapping
  • S-alkylation
  • Solubilization of denatured protein.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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