It has been shown previously that a neuron-specific protein kinase C (PKC) substrate, neurogranin (Ng), is a redox-sensitive protein with four cysteine (Cys) residues readily oxidizable by nitric oxide (NO) donors (Sheu et al., 1996). The NO oxidation of Ng results in a conformational change detectable by increased electrophoresis mobility when resolved in non-reducing sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). In this study, we directly examine the redox reaction between dissolved NO gas and cysteine (Cys) as well as NO and bacterial expressed Ng in its reduced form, at concentrations approximate to physiological levels in phosphate buffer solution. The reaction kinetics are measured directly by a newly developed, chemically-modified electrode (CME) with nan...[ Read more ]

It has been shown previously that a neuron-specific protein kinase C (PKC) substrate, neurogranin (Ng), is a redox-sensitive protein with four cysteine (Cys) residues readily oxidizable by nitric oxide (NO) donors (Sheu et al., 1996). The NO oxidation of Ng results in a conformational change detectable by increased electrophoresis mobility when resolved in non-reducing sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). In this study, we directly examine the redox reaction between dissolved NO gas and cysteine (Cys) as well as NO and bacterial expressed Ng in its reduced form, at concentrations approximate to physiological levels in phosphate buffer solution. The reaction kinetics are measured directly by a newly developed, chemically-modified electrode (CME) with nanoparticles of transitional metal palladium loaded as catalytic centers. Our CME detects the oxidation of Cys and Ng in a linear range from submicro to micromolar concentration at +450 mV, versus a saturated calomel reference electrode (SCE). The oxidation of NO does not occur at this potential but can be optimally detected at +700 mV (vs. SCE) by our CME with a linear current-to-concentration range of nM to μM. It thus provides a selective control to examine oxidation of Cys or Ng by NO at +450 mV without interference current generated by NO itself. With this CME as a detector, we found that (1) the oxidation of either Cys or Ng by NO is a fast reaction and proceeds to near completion within l-2 min at its physiological concentration; and (2) with the completion of the oxidation reaction, the amount of NO in the final reaction mixture remains almost the same as in the initial stage before the oxidation reaction. The vigorous reaction kinetics of both NO to Cys and NO to Ng implies that NO can achieve better local action on cellular proteins than following concentration gradient to diffuse out to exert its effect on targets located in neighboring cells. In addition, our data are consistent with a reaction mechanism for NO to Ng that involves formation of S-nitrosothiol as intermediate during the conversion of thiol groups into disulfide bond.