Capillary electrophoretic reactor for estimation of spontaneous dissociation rate of Trypsin–Aprotinin complex

Yumiko Sasaki, Yosuke Sato, Toru Takahashi, Mitsuo Umetsu, Nobuhiko Iki

Research output: Contribution to journalArticle

Abstract

A capillary electrophoretic reactor was used to analyze the dissociation kinetics of an enzyme–inhibitor complex in a homogeneous solution without immobilization. The complex consisting of trypsin (Try) and aprotinin (Apr) was used as the model. Capillary electrophoresis provided a reaction field for Try–Apr complex to dissociate through the steady removal of free Try and Apr from the Try–Apr zone. By analyzing the dependence of peak height of Try–Apr on separation time, the dissociation rate kd H was obtained as 2.73 × 10−4 s−1 (298 K) at pH 2.46. The dependence of kd H on the proton concentration (pH = 2.09–3.12) revealed a first-order dependence of kd H on [H+]; kd H = kd + k1[H+], where kd is the spontaneous dissociation rate and was 5.65 × 10−5 s−1, and k1 is the second-order rate constant and was 5.07 × 10−2 M−1 s−1. From the kd value, the half-life of the Try–Apr complex at physiological pH was determined as 3.4 h. The presence of the proton-assisted dissociation can be explained by the protonation of -COO of the Asp residue in Try, which breaks the salt bridge with the –NH3 + group of Lys in Apr.

Original languageEnglish
Article number113406
JournalAnalytical Biochemistry
Volume585
DOIs
Publication statusPublished - 2019 Nov 15

Keywords

  • Aprotinin
  • Biomolecular complex
  • Capillary electrophoresis
  • Dissociation kinetics
  • Enzyme
  • Inhibitor
  • Trypsin

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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