As a highly reactive substance produced in biological systems by the one-electron reduction of oxygen, superoxide (O(2) (-)) seemed a likely candidate as a bactericidal agent in leukocytes. The reduction of cytochrome c, a process in which O(2) (-) may serve as an electron donor, was found to occur when the cytochrome was incubated with leukocytes. O(2) (-) was identified as the agent responsible for the leukocyte-mediated reduction of cytochrome c by the demonstration that the reaction was abolished by superoxide dismutase, an enzyme that destroys O(2) (-), but not by boiled dismutase, albumin, or catalase. Leukocyte O(2) (-) production doubled in the presence of latex particles. The average rate of formation of O(2) (-) in the presence of these particles was 1.03 nmol/10(7) cells per 15 min. This rate, however, is only a lower limit of the true rate of O(2) (-) production, since any O(2) (-) which reacted with constituents other than cytochrome c would have gone undetected. Thus. O(2) (-) is made by leukocytes under circumstances which suggest that it may be involved in bacterial killing.

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Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent.

Hepatic Triphosphopyridine Nucleotide-Cytochrome c Reductase: Isolation, Characterization, and Kinetic Studies

Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra

Ferricytochrome c: I. GENERAL FEATURES OF THE HORSE AND BONITO PROTEINS AT 2.8 A RESOLUTION

The microestimation of succinate and the extinction coefficient of cytochrome c.

Appendix—Spectrum of horse-heart cytochrome c

The chromatographic behaviour of cytochrome c on cation exchangers.

A study of the relationship of the structure of the horse-heart cytochrome c haemochromogen to the enzymic activity of the protein could be undertaken in view of: (a) the firm thio ether bonds which link the protein to the haem (Theorell, 1938; Theorell, 1939; Paul, 1951), enabling the isolation, after proteolytic digestion, of a relatively small portion of the protein adjacent to the haem (Tsou, 1951a, b), and (b) the isolation, by column chromatography on cation exchangers, of a graded series of denatured forms of the protein, showing a gradation of concomitant changes in enzymic activity, on the one hand, and of various typical properties of the cytochrome c haemochromogen, such as the pH range of stability of the reduced spectrum and the percentage combination with carbon monoxide, etc., on the other. While this work was in progress a series ofpapers by Tuppy & Bodo (1954a-c) and Tuppy & Paleus (1955) established the following amino acid sequence for that part of the cytochrome c protein next to the haem:

E. Margoliash

Papers

Appendix—Spectrum of horse-heart cytochrome c

The chromatographic behaviour of cytochrome c on cation exchangers.

A study of the cytochrome c haemochromogen

The use of ion exchangers in the preparation and purification of cytochrome c.