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Related Concept Videos

Temperature Dependence on Reaction Rate02:55

Temperature Dependence on Reaction Rate

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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
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FLIM quantification of temperature-dependent enzyme-NADH binding dynamics.

Jocelyn Martinez1, Anna Theodossiou1, Linghao Hu1

  • 1Department of Biomedical Engineering, Texas A&M University, College Station, TX, 77840, USA.

Biomedical Optics Express
|July 16, 2026
PubMed
Summary

This study uses fluorescence lifetime imaging microscopy (FLIM) to measure temperature effects on enzyme-coenzyme binding. FLIM quantifies changes in metabolic coenzyme nicotinamide adenine dinucleotide (NADH) binding dynamics, revealing temperature-dependent shifts in cellular metabolism.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Microscopy

Background:

  • Enzyme-coenzyme interactions are temperature-sensitive.
  • Conventional assays lack the resolution to study rapid thermal effects on cellular metabolism and binding.
  • Understanding these dynamics is crucial for cellular function.

Purpose of the Study:

  • To develop and validate a method to quantify rapid temperature-dependent changes in enzyme-coenzyme binding dynamics.
  • To investigate the effects of steady-state and rapid temperature increases on metabolic coenzyme binding.
  • To establish fluorescence lifetime imaging microscopy (FLIM) as a tool for studying these processes.

Main Methods:

  • Utilized FLIM of nicotinamide adenine dinucleotide (NADH) to measure binding dynamics.
  • Estimated dissociation constants (KD) and fractions of free NADH (α1).
  • Developed a computational model to describe temperature dependence of binding affinity and validated it in solutions and cells.

Main Results:

  • FLIM successfully quantified temperature-dependent changes in enzyme-coenzyme binding.
  • Increased temperatures led to increased free NAD(P)H, indicating reduced binding affinity.
  • Rapid thermal gradients caused greater increases in free NADH in solutions compared to cells.

Conclusions:

  • FLIM of NADH is a non-contact, quantitative method for evaluating enzyme-coenzyme binding temperature dependence at millisecond timescales.
  • Integrating FLIM with dissociation constant modeling offers a generalizable framework for studying dynamic enzymatic binding in living cells.
  • This approach overcomes limitations of conventional assays for studying rapid cellular thermal effects.