A new cycling assay for NAD that uses thiazolyl blue as a terminal electron acceptor has been found to offer significant advantages over the more established procedure that employs 2,6dichlorophenolindophenol. With thiazolyl blue, the cycling assay is linear with NAD at picomole levels, and with time for at least 120 min. In contrast, with 2,6-dichlorophenolindophenol as terminal acceptor, the cycling assay deviates considerably from linearity at picomole levels of NAD, and the reaction rates become linear for shorter periods of time as the level of NAD increases. Data are given which provide a basis for choosing optimal assay conditions using the new thiazolyl blue cycling technique.
Recent studies of pyridine nucleotides in muscle extracts have involved the isolation of the bound forms of NAD+ and NADH by means of ultrafiltration (1). Because the NADH content of rabbit muscle is small [40-50 nmoles/g fresh tissue (1) ] and only limited amounts of extract can be ultrafiltered, a highly sensitive assay technique is required. A study of the suitability of the cycling procedure of Slater et al. (2) revealed that it was insufficiently sensitive as well as nonlinear in the required range.
Nisselbaum and Green (3) have recently proposed the use of thiazolyl blue as a terminal electron acceptor in the cycling assay for pyridine nucleotides. We have found that a modified version of their method possesses both the required sensitivity and the linearity for the analysis of ultramicro levels of bound NAD+ and NADH. The present report describes this modified thiazolyl blue technique. We also discuss certain important features of the assay that have not been previously noted.