Assay of [3H]dihydrorotenone binding to complex I in intact human platelets

Abstract

We have developed an assay for the binding of [3H]-dihydrorotenone ([3H]DHR), an analogue of the pesticide rotenone, to the mitochondrial enzyme, complex I, in intact human platelets. The highly hydrophobic nature of dihydrorotenone, which diffuses easily through biological membranes, rendered the isolation of mitochondrial fractions unnecessary. This allowed us to reduce the amount of blood required and to shorten the processing of samples considerably. [3H]-DHR binding was saturable, specific, and highly reproducible. We also found that MPP+ (1-methyl-4-phenyl-pyridinium species), which is accumulated actively by platelets, inhibited [3H]DHR specific binding in a concentration-dependent manner. This method could provide a simple tool for the study of complex I in those disorders, such as Parkinson's disease (PD), in which a defect of this enzyme has been suggested.