Chemiluminescent picture of diphenyleneiodonium‐inhibited NADPH oxidase: a bimodal process and its logistic–exponential model‐based description

Abstract

A chemiluminescence (CL) study of diphenyleneiodonium-inhibited NADPH oxidase was performed on a cellular system containing neutrophils stimulated by phorbol myristate acetate, indicating a complex bimodal structure of CL processes corresponding to different stages of the inhibition. The complex structure of these processes was described by a superposition of two logistic-exponential (LE) models, characterizing these processes as bimodal ones. To determine the mechanistic foundation of the LE model-described processes, a generalized form of the second-order dynamic system of CL reactions, the solution to which corresponds to the LE model, was constructed. The diphenyleneiodonium effects on neutrophil NADPH oxidase were separated from the total bimodal CL of the whole measurement system by the use of difference CL processes. These difference processes were also found to be bimodal; thus, inhibitor-induced reduction of CL could be described by a second-order dynamic system. The rate constants and initial concentrations in this dynamic system were determined by the least squares method applied to numerical solutions approximating the difference processes. Using interrelations between the parameters of the dynamic system, cooperative effects in the inhibitor reactions with NADPH oxidase were found and described quantitatively. Other evidences of cooperativity were obtained from integral characteristics of the CL reduction process, i.e. dose-response and progress curves, determined by numerical integration of the LE models constituting the superposition. On this basis, it was also possible to detect a specific binding of the inhibitor to the enzyme. Finally, putative reaction mechanisms suggested by the model obtained were considered and compared with those known at present.