European Biophysics Journal

, 38:407

Enzyme solid-state support assays: a surface plasmon resonance and mass spectrometry coupled study of immobilized insulin degrading enzyme

  • Giuseppe Grasso
  • Ashley I. Bush
  • Roberta D’Agata
  • Enrico Rizzarelli
  • Giuseppe Spoto
Original Paper

DOI: 10.1007/s00249-008-0384-y

Cite this article as:
Grasso, G., Bush, A.I., D’Agata, R. et al. Eur Biophys J (2009) 38: 407. doi:10.1007/s00249-008-0384-y

Abstract

Solid-support based assays offer several advantages that are not normally available in solution. Enzymes that are anchored on gold surfaces can interact with several different molecules, opening the way to high throughput array format based assays. In this scenario, surface plasmon resonance (SPR) and mass spectrometry (MS) investigations have often been applied to analyze the interaction between immobilized enzyme and its substrate molecules in a tag-free environment. Here, we propose a SPR-MS combined experimental approach aimed at studying insulin degrading enzyme (IDE) immobilized onto gold surfaces and its ability to interact with insulin. The latter is delivered by a microfluidic system to the IDE functionalized surface and the activity of the immobilized enzyme is verified by atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI) MS analysis. The SPR experiments allow the calculation of the kinetic constants involved for the interaction between immobilized IDE and insulin molecules and evidence of IDE conformational change upon insulin binding is also obtained.

Keywords

Solid-state assay Surface plasmon resonance Mass spectrometry Insulin degrading enzyme Conformational change 

Copyright information

© European Biophysical Societies' Association 2008

Authors and Affiliations

  • Giuseppe Grasso
    • 1
  • Ashley I. Bush
    • 2
    • 3
  • Roberta D’Agata
    • 1
  • Enrico Rizzarelli
    • 1
  • Giuseppe Spoto
    • 1
    • 4
  1. 1.Dipartimento Scienze ChimicheUniversità di CataniaCataniaItaly
  2. 2.Department of PsychiatryMassachusetts General HospitalCharlestoneUSA
  3. 3.Department of Pathology, Mental Health Research Institute of VictoriaThe University of MelbourneParkvilleAustralia
  4. 4.Istituto di Biostrutture e Bioimmagini, CNRCataniaItaly

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