Molecular state of cytochrome c oxidase on polyacrylamide gel electrophoresis

Abstract

The molecular state of cytochrome oxidase, complex IV (EC 1.9.3.1), was studied by polyacrylamide gel electrophoresis. Cytochrome oxidase in the presence of non-ionic detergent Emasol 1130 ran as a single band under conditions where there is a small sieving effect as in a 2.5% polyacrylamide gel. This polymeric form is an association of different molecular species that can be dissociated on a preparative electrophoresis system. In such a system, five species of different molecular size with enzymic activity are obtained, though the specific activity is lower in the first running fractions. After treatment in highly dissociating conditions (phenol-acetic acid-water (2:1:1, w/v/v) the polymeric form and the different species present two main fractions on analytical polyacrylamide electrophoresis (7.5% in acrylamide, 35% acetic acid, and 5 m urea). The original form after treatment with 2.5% sodium dodecylsulfate (SDS) also presents two fractions on analytical electrophoresis in the presence of 2.5% sodium dodecylsulfate. These results suggest that the basic subunit structure of the various forms of the cytochrome oxidase consists of two closely related polypeptides. The highest activity was found with the polymeric form of cytochrome oxidase, a fact which may have physiological significance in relation to the natural state of this enzyme in the mitochondrion.