THE MECHANISM OF THE INHIBITION OF HEMOLYSIS : V. INHIBITORY PROCESSES OCCURRING IN THE COURSE OF SIMPLE HEMOLYTIC REACTIONS

This paper is concerned with hemolytic systems containing sodium taurocholate, sodium glycocholate, or saponin, and either human red cells or the ghosts (stromata) of human red cells. The lysins are allowed to react with the cells for a short time (1 minute or less), and with the ghosts for a long time (4 hours), and the quantity of lysin remaining in the bulk phase, after the removal of the cells or of the ghosts, is found by (a) colorimetric methods, and (b) methods which measure its hemolytic activity. In the experiments in which the lysins react with the cells for a time so short that none of them is hemolyzed, it is found: (1) that the suspension medium of a cell suspension contains inhibitory substances which render a small amount of the lysin non-lytic, (2) that on the addition of the lysin to the cell suspension, a further and much larger amount of lysin is rendered non-lytic, and (3) that the quantity of chromogenic material in the bulk phase, after the lysin has been in contact with the cells and the latter have been removed, is substantially the same as that initially present. No appreciable quantity of lysin, accordingly, accumulates in increased concentration at the cell surfaces. The results of the colorimetric determinations show that the apparent disappearance of lysin from the bulk phase, once thought to be due to an accumulation of lysin at the cell surfaces, is the result either of an inhibitory process or of a sudden utilization of lysin unrecognized by existing theory. In the experiments in which the lysins react with stromata for 4 hours, it is found: (1) that the suspension medium of a stroma suspension contains inhibitory substances which render some of the lysin non-lytic, (2) that when the lysin reacts with the stromata over a period of time, a much greater quantity of lysin is rendered non-lytic, and (3) that the concentration of chromogenic material in the bulk phase of the lysin-stroma system, after 4 hours of reaction, is substantially the same as it was initially. The observations can be accounted for by supposing that the lysin molecules are as chromogenic after reacting with the cell components as before, and by their not being bound to the cell or to the ghost, but diffusing back, combined with the components with which they have reacted, into the bulk phase. Such a process would have similarities to the process of penetration and breakdown of mixed protein-lipoid films. Because it is not possible at present to decide how much of the lysin is rendered inert because of utilization in a reaction with cell components, and how much because of the effect of inhibitory substances, difficulties and uncertainties arise in connection with the expressions which have been used to describe the kinetics of hemolysis.