Protein sulfenic acids (SOHs) are the principal oxidation products formed when redox active proteins interact with peroxide molecules. We have developed a new antibody reagent that detects protein SOHs derivatized with dimedone. Using this new antibody, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is the predominant protein sulfenate present in isolated rat ventricular myocytes under basal conditions. During oxidative stress with hydrogen peroxide (H(2)O(2)), GAPDH SOH labeling is lost, but a number of secondary dimedone-reactive protein sulfenates then appear. As the sulfenate labeling is lost, the Cys-149 sulfinic/sulfonic acid oxidation states of GAPDH appear. This hyperoxidized GAPDH is associated with both the inhibition of glycolysis and its ability to reduce H(2)O(2). We examined whether inactivation of GAPDH was causative in the generation of secondary protein sulfenates that coincide with its hyperoxidation. The selective GAPDH inhibitor koningic acid (which functions by forming a covalent adduct at Cys-149) fully prevented basal SOH labeling, as well as subsequent peroxide-induced hyperoxidation. However, koningic acid-mediated inhibition of GAPDH alone did not induce the formation of intracellular H(2)O(2) or secondary protein sulfenates and also failed to potentiate their peroxide-induced formation. Overall, GAPDH appears to have peroxidase-like properties, but its inhibition failed to impact on downstream oxidant signaling involving secondary protein sulfenation.