FLIM quantification of temperature-dependent enzyme-NADH binding dynamics

Metabolic enzyme-coenzyme interactions are temperature-dependent; yet, conventional biochemical assays lack the temporal and spatial resolution to resolve rapid thermal effects on cellular metabolism and cellular binding equilibria. Here, fluorescence lifetime imaging microscopy (FLIM) of the metabolic coenzyme nicotinamide adenine dinucleotide (NADH) was used to quantify steady-state and rapid temperature-dependent changes in enzyme-coenzyme binding dynamics through estimation of dissociation constants ( K D ) and corresponding fractions of free NADH ( α 1 ). A computational model describing the temperature dependence of enzyme-coenzyme binding affinity was constructed to relate changes in K D to temperature-dependent shifts in α 1 . This model was validated in LDH-NADH solutions, MCF-7 cells, and primary murine hippocampal neurons using FLIM. Both steady-state and rapid temperature increases induced increases in free NAD(P)H across all samples, consistent with a reduction in binding affinity (increased K D ). Rapid thermal gradients induced by short pulses of infrared light produced greater increases in free NADH in LDH-NADH solutions than in cells. These results establish FLIM of NADH as a non-contact, quantitative method to evaluate the temperature-dependence of enzyme-coenzyme binding at millisecond timescales. Additionally, integration of temperature-dependent dissociation constant modeling with FLIM provides a generalizable framework for studying dynamic enzymatic binding processes within living cells that are otherwise inaccessible using conventional assays.