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Kinetic Studies on Immune Hemolysis

IV. Rate Determination of the Mg⁺⁺ and Terminal Reaction Steps¹

From the Department of Microbiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland

Abstract

1. The hemolytic action of complement is arrested much more promptly by ethylene diamine tetra acetate as well as by an unrelated specific precipitate when hemolytic antibody rather than complement is the limiting factor.

2. This suggests that the rate of reaction in the limited antibody system is controlled by the Ca⁺⁺ step while in the limited complement system the Mg⁺⁺ step is the pace-making reaction.

3. The Mg⁺⁺ step may be blocked promptly and effectively by dilution and chilling. On this basis a sampling procedure for kinetic analysis has been devised by which the rate of the Mg⁺⁺ step may be measured.

4. Kinetic reaction curves are presented which show that the velocity of the Mg⁺⁺ step and of the terminal transformation reaction are identical. The average time required for a cell to go through the terminal reaction is about 2 minutes.

5. Sensitized erythrocytes may be taken through the Ca⁺⁺ step in the cold. Upon addition of Mg⁺⁺ and an appropriate fraction of complement at 37°C the Mg⁺⁺ step proceeds with little or no lag.

Footnotes

¹ The work reported in this communication was supported in part by grants from the National Science Foundation, The American Cancer Society, Sidney M. Cone Foundation, as well as by a contract with the Office of Naval Research (NONR-248-17).