A novel method for detection of nitric oxide binding sites by using a chemiluminescence-headspace gas technique

Nitric oxide (NO) is important in many physiological, pharmacological, and pathological processes. According to current concepts, guanylyl cyclase is considered to be a receptor for NO in vascular and nonvascular smooth muscle and other tissues. Since there are no suitable radioisotopes of oxygen and nitrogen available for conventional radioligand-receptor binding studies for NO, a novel method was developed to identify NO binding site(s). A chemiluminescence-headspace gas assay was utilized to measure the sequestration of NO in biological systems, and this was used as an index of NO binding. In the present report, myoglobin (a hemoprotein, Mb) was used as a prototype macromolecule to develop the binding assay for subsequent application to studies of putative NO receptors. Solutions containing various concentrations of Mb were incubated with NO in sealed micro-Fernbach flasks at 37 degrees C in an argon atmosphere for 30 min; NO remaining in the headspace gas was analyzed by means of the chemiluminescence assay. The magnitude of NO sequestration was dependent on Mb concentration, and 5 nM Mb was the lowest Mb concentration for which NO sequestration was measurable. Application of the method to the measurement of NO sequestration by bovine serum albumin (BSA) and pulmonary artery medial layer homogenate (BPA-M) revealed that the lowest BSA concentration at which NO sequestration was measurable was 1.6 microM, which was 320 times greater than that for Mb. Applicability of the method to address the question of putative NO receptors was indicated by significant NO sequestration after incubation with 20% (w/v) homogenate of BPA-M, which is responsive to NO and putative NO prodrugs.