The Reduced Pyridine Nucleotide of Human Erythrocytes Dehydrogenases

Kiese (1) reported the presence of an enzyme in erythrocytes that would catalyze the reduction of methemoglobin by reduced triphosphopyridine nucleotide in the presence of methylene blue. This enzyme was purified further by Huennekens et al. (2) and reported to be active both with TPNH and, to a lesser extent, with DPNH. It was postulated that this enzyme was responsible for the physiological maintenance of hemoglobin in the red cell in the reduced state, on the assumption that some unknown electron carrier was present in red cells that could act in the same manner as methylene blue does in the assay system. Shrago and Falcone (3) purified this TPNH dehydrogenase further and reported it to be essentially free of heme protein. A DPNH dehydrogenase with methemoglobin reductase activity has been isolated from normal human red cells (4). This enzyme is lacking in red cells of persons with hereditary methemoglobinemia (5). A second DPNH dehydrogenase, prepared from methemoglobinemic red cells, has quite different properties from the DPNH dehydrogenase of normal cells, as well as being present in lesser amount (6). Evidence will be presented here that there are four reduced pyridine nucleotide dehydrogenases in human red cells: TPNH dehydrogenase A, TPNH dehydrogenase B, DPNH dehydrogenase I, and DPNH dehydrogenase II. The two TPNH dehydrogenases resemble each other closely, but are separable by chromatography. DPNH dehydrogenase I is the major “diaphorase” of human red cells previously described (4), while DPNH dehydrogenase II is a minor component previously purified from methemoglobinemic cells (6). Described here are the preparation of two TPNH dehydrogenases from normal human red cells. Certain samples of these preparations contained considerable DPNH dehydrogenase II activit,y. The properties of these four dehydrogenases were compared and their possible role in methemoglobin reduction was estimated.