Low-affinity binding determined by titration calorimetry using a high- affinity coupling ligand: A thermodynamic study of ligand binding to protein tyrosine phosphatase 1B

Yan Ling Zhang, Zhong Yin Zhang

Research output: Contribution to journalArticle

123 Scopus citations

Abstract

A competition-based method is used for the determination of the thermodynamic parameters for a low-affinity ligand binding reaction by isothermal titration calorimetry. This method is based on the coupling of a high-affinity ligand to the binding of the low-affinity ligand. Results are presented for the binding of a nonhydrolyzable phosphotyrosine analog phosphonodifluoromethyl phenylalanine (F2Pmp)-containing peptide (Ac-Asp- Ala-Asp-Glu-F2Pmp-Leu-NH2), arsenate, and inorganic phosphate to the intracellular human protein tyrosine phosphatase 1B (PTP1B). The binding constants are 3.3 x 106, 4.3 x 103, and 48 M-1 for the F2Pmp-containing peptide, arsenate, and inorganic phosphate, respectively. The binding of arsenate and inorganic phosphate to PTP1B is enthalpy driven. This is in contrast to the binding of the F2Pmp-containing peptide which is mainly driven by entropy. The calorimetrically determined binding constants are in agreement with the K(i) values determined by enzyme inhibition studies. This demonstrates that isothermal titration calorimetry can be used to quantitatively determine the thermodynamic parameters for the interactions between proteins and low-affinity ligands if a proper coupling ligand can be identified.

Original languageEnglish (US)
Pages (from-to)139-148
Number of pages10
JournalAnalytical biochemistry
Volume261
Issue number2
DOIs
StatePublished - Aug 1 1998

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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