A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD(+)/NADH in Streptococcus mutans.

Nicotinamide adenine dinucleotide is a coenzyme present in all kingdoms of life and exists in two forms: oxidized (NAD(+)) and reduced (NADH). NAD(H) is involved in a multitude of essential metabolic redox reactions, providing oxidizing or reducing equivalents. The ratio of free intracellular NAD(+)/NADH is fundamentally important in the maintenance of cellular redox homeostasis (Ying, 2008). Various chromogenic cycling assays have been used to determine the ratio of NAD(+)/NADH in both bacterial and mammalian cells for more than forty years (Bernofsky and Swan, 1973; Nisselbaum and Green, 1969). Here, we describe in detail an assay to determine the ratio of free intracellular NAD(+) to NADH in Streptococcus mutans. This cycling assay is a modified version of the protocol first described by Bernofsky and Swan (Bernofsky and Swan, 1973), using the extraction buffer described by Frezza et al. (2011), followed by the reduced MTT precipitation described by Gibbon and Larher (Gibon and Larher, 1997). As depicted in Figure 1, alcohol dehydrogenase is used to drive a series of redox reactions utilizing exogenously added ethanol and NAD(+) from sample extracts as initial substrates, phenazine ethosulfate (PES) as an electron carrier, and thiazolyl blue tetrazolium bromide (MTT) as a terminal electron acceptor. 6 M NaCl is used to stop the reaction. The reduced MTT (formazan dye) is purple in color and can be quantified by measuring absorbance at 570 nm. This protocol is divided into three steps: A. Preparation of cell pellets of S. mutans; B. Preparation of deproteinated cell extracts containing NADtotal or NADH; C. NAD(+)/NADH cycling assay. This method has proven robust in measuring the NAD(+)/NADH ratio in S. mutans under a variety of conditions, and should be applicable to other Gram-positive bacteria.