Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in many metabolic pathways in living cells.  The fraction of free and enzyme-bound NADH has been shown to be sensitive to mitochondrial activities of brain tissues and cancer cells.  Here, we have investigated the effects of molecular crowding on the reaction kinetics of NADH and lactate dehydrogenase (LDH) using two-photon time-resolved anisotropy.  Synthetic (Ficoll-70) and proteins (bovine serum albumin, BSA, ovalbumin) were used to induce biomimetic crowding.  Computer simulations and kinetic analysis were used as predictive approach and a guide to our experimental design and data interpretation.  The observed associate anisotropy of NADH-LDH mixture indicates a dependence on both the type and concentration of crowding agents.  Complementary studies on intracellular NADH in cultured cells were also carried out and the results will enhance the potential of NADH as a natural biomarker for a myriad of biochemical reactions in living cells.

Experimental and simulation analysis of NADH-enzymes binding in a crowded environment

245th National Meeting (2013)

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