A spectrophotometric method for kinetic studies with quinone-dependent oxidoreductases. Application to detection in membranes of nitrate reductase activity with menadione and duroquinone as electron donors

A simple method for estimating the activity of membrane-bound quinone-dependent oxidoreductases is described. Nitrate reductase from membranes of Escherichia coli is used as a model with menadione and duroquinone, commercially available analogues of the physiological substrates, menaquinone and ubiquinone, as electron donors. These analogues are reduced by KBH4 (which is specific for aldehydes and ketones) using molar ratios of [KBH4]/[menadione] = 20 and [KBH4]/[duroquinone] = 10. The appearance of the oxidized state can be monitored with a classical spectrophotometer and does riot require a dual wavelength or diffusing-medium apparatus. To avoid turbidity in the cuvette, small amounts of membrane containing overexpressed enzyme ni-e used. (C) 1998 Elsevier Science Inc.