Reversible denaturation of the soybean Kunitz trypsin inhibitor

Robin Roychaudhuri, Gautam Sarath, Michael Zeece, John Markwell

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70°C, changes in UV difference spectra showed increased absorbance at 292 and 297nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65°C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70°C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90°C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered β-sheet proteins.

Original languageEnglish (US)
Pages (from-to)20-26
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume412
Issue number1
DOIs
StatePublished - Apr 1 2003

Fingerprint

Kunitz Soybean Trypsin Inhibitor
Denaturation
Hot Temperature
Cooling
Heating
Protein folding
Proteins
Fluorophores
Dithiothreitol
Protein Folding
Tryptophan
Trypsin
Restoration
Tyrosine
Conformations
Molten materials
Fluorescence

Keywords

  • ANS fluorescence
  • Antiparallel β-sheet
  • Circular dichroism
  • Disulfide bonds
  • Folding
  • Protein structure
  • Soybean Kunitz trypsin inhibitor
  • Thermal denaturation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Reversible denaturation of the soybean Kunitz trypsin inhibitor. / Roychaudhuri, Robin; Sarath, Gautam; Zeece, Michael; Markwell, John.

In: Archives of Biochemistry and Biophysics, Vol. 412, No. 1, 01.04.2003, p. 20-26.

Research output: Contribution to journalArticle

Roychaudhuri, Robin ; Sarath, Gautam ; Zeece, Michael ; Markwell, John. / Reversible denaturation of the soybean Kunitz trypsin inhibitor. In: Archives of Biochemistry and Biophysics. 2003 ; Vol. 412, No. 1. pp. 20-26.
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