Validity of the `Bioassay' for Thioredoxin-Reductase Activity-Reply

In Reply.—  It is our belief that a critical review of the literature, including your references 3 and 4, on the thioredoxin reductase (TR) bioassay, provides sufficient information to confirm our results. A most important feature of our spin-labeled substrate is its inability to penetrate the plasma membranes of keratinocytes and melanocytes. Therefore, the nitroxide radical reduction that we measure is catalyzed by an enzyme acting at the outer membrane surface.¹ We understand your criticism that nitroxide radicals are easily reduced to the electron resonance spectroscopy-silent hydroxylamine product by many biological redox systems (your reference 3), but in our bioassay we have shown that the nitroxide radical is reduced at the cell surface to a secondary amine.² The inability of our spin-labeled substrate to penetrate the plasma membrane rules out nonspecific reactions that would undoubtedly occur in the cytosol or on mitochondrial membranes. Indeed, if substrate transport occurred,