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 journalArticle

36 Citations (Scopus)

Abstract

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
Volume116
Issue number4
Publication statusPublished - Oct 1994

Fingerprint

Disulfides
Charge
Proteins
Protein
Cysteine
Solubility
Alkylation
Muramidase
Peptides
Hydrophobicity
Enzymes
Hydrophobic and Hydrophilic Interactions
Kunitz Soybean Trypsin Inhibitor
Iodoacetic Acid
Egg White
Pancreatic Ribonuclease
Decrease
Soybean
Acids
High-performance Liquid Chromatography

Keywords

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

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Radiology Nuclear Medicine and imaging
  • Molecular Biology
  • Biochemistry

Cite this

An S-alkylating reagent with positive charges as an efficient solubilizer of denatured disulfide-containing proteins. / Yamada, Hidenori; Seno, Masaharu; Kobayashi, Ayumi; Moriyama, Takeshi; Kosaka, Megumi; Ito, Yuji; Imoto, Taiji.

In: Journal of Biochemistry, Vol. 116, No. 4, 10.1994, p. 852-857.

Research output: Contribution to journalArticle

Yamada, Hidenori ; Seno, Masaharu ; Kobayashi, Ayumi ; Moriyama, Takeshi ; Kosaka, Megumi ; Ito, Yuji ; Imoto, Taiji. / An S-alkylating reagent with positive charges as an efficient solubilizer of denatured disulfide-containing proteins. In: Journal of Biochemistry. 1994 ; Vol. 116, No. 4. pp. 852-857.
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