Determination of S-nitrosohemoglobin using a solid-state amperometric sensor

Nitric oxide (NO, nitrogen monoxide), generated in biological systems, play s important roles as a regulatory molecule. Its ability to bind to hemoglob in (Hb) iron is well known. Moreover, it may lose an electron, forming the nitrosonium ion, involved in the synthesis of nitrosothiols (RSNO). It has been suggested that S-nitrosohemoglobin (SNO-Hb) may act as a reservoir of NO. The S-nitrosylation of Hb can be detected after the incubation of CysNO and Hb for 60 mim with a molecular ratio (CysNO/hem) of 1:1. Upon increasi ng the ratio to 10:1, about 50% of total Hb (100% of beta -chain -SH 93) wa s derivatized in 60 min. In this paper, we describe a new method for the qu antitative assay of SNO-Hb, after the liberation of NO by Cu2+/Cu+ and the simultaneous assessment of NO by solid-state amperometric sensor. The assay described by us is sensitive, rapid, easy to perform, and inexpensive. For this reason, we believe that it may represent an important analytical impr ovement for the study of the S-transnitrosylation reactions between RSNO an d the Hb Cys-beta 93 and SNO-Hb and glutathione. (C) 2000 Academic Press.