On the use of the experimentally determined enzyme inhibition constant as a measure of absolute binding affinity

Fouad H. Darras, Yuan-Ping Pang

Research output: Contribution to journalArticle

7 Scopus citations


Defined as a state function representing an inhibitor's absolute affinity for its target enzyme, the experimentally determined enzyme inhibition constant (Ki) is widely used to rank order binding affinities of different inhibitors for a common enzyme or different enzymes for a common inhibitor and to benchmark computational approaches to predicting binding affinity. Herein, we report that adsorption of bis(7)-tacrine to the glass container surface increased its Ki against Electrophorus electricus acetylcholinesterase (eeAChE) to 3.2 ± 0.1 nM (n = 5) compared to 2.9 ± 0.4 pM (n = 5) that was determined using plastic containers with other assay conditions kept the same. We also report that, due to binding or “adsorption” of bis(7)-tacrine to the inactive eeAChE, the bis(7)-tacrine Ki increased from 2.9 ± 0.4 pM (n = 5) to 734 ± 70 pM (n = 5) as the specific eeAChE activity decreased from 342 U/mg to 26 U/mg while other assay conditions were kept the same. These results caution against using Kis to rank order binding potencies, define selectivity, or benchmark computational methods without knowing detailed assay conditions.

Original languageEnglish (US)
Pages (from-to)451-454
Number of pages4
JournalBiochemical and Biophysical Research Communications
Issue number4
StatePublished - Aug 5 2017



  • Adsorption
  • Affinity
  • Dissociation constant
  • Inhibition constant
  • Nonspecific binding
  • Nontarget site binding

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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