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Sensitization with increasing concentrations of homologous immune serum shifts the isoelectric point of the antigens studied progressively to the alkaline side. Antigens maximally sensitized with rabbit sera have shown isoelectric points of pH 5.6 to 5.8. The globulins precipitated or salted out of the same immune sera have been isoelectric at pH 5.1 to 5.2. The combination of antigen with antibody depends of course upon specific affinities; the surface properties of the sensitized antigen, agglutination and phagocytosis depend primarily upon the properties of the sensitizing serum substances combined with and deposited on the antigen surface.

As a further test of the theory of tropin action proposed in the preceding paper artificial surfaces have been prepared, and have been found to be phagocytized according to prediction from the theory. Protein was adsorbed on collodion particles according to the technique of F. S. Jones. These particles were then agglutinated and prepared for phagocytosis by the corresponding protein precipitin sera. The precipitating, agglutinating, surface and tropin effects for each serum or serum globulin fraction have been found to be in satisfactory quantitative correspondence. All of these effects were serologically specific; all remained almost unaffected by inactivation of the immune sera for 30 minutes at 56 degrees C. or by washing of the particles after sensitization. The surfaces of particles maximally sensitized by homologous rabbit immune serum or one of its globulin fractions have shown certain characteristic properties, i.e., they were cohesive, had wetting properties characteristic for protein, and were isoelectric at pH. 5.5 to 5.8. The same set of properties were found for immune precipitate in the zone of maximal precipitation. The same properties have also been found for maximally sensitized acid-fast bacteria, and for maximally sensitized sheep erythrocytes. These results indicate, we believe, that precipitation, agglutination, the surface changes and increased phagocytosis are all consequences of one underlying phenomenon. This phenomenon is the specific chemcal combination with, and deposit on the surface of the antigen of antibody protein. The several serological reactions then follow as consequences of the properties of the sensitized surface and of the special environing conditions. The antibody is contained in the globulin fractions of immune serum, and appears to be a globulin with physico-chemical differences from normal serum globulin.

1. Using the unfertilized egg of the sea urchin, Arbacia, as osmometer, it was found that the rate with which water enters or leaves the cell depends on the osmotic pressure of the medium: the velocity constant of the diffusion process is higher when the cell is in concentrated sea water, and lower when the sea water medium is diluted with distilled water. Differences of more than tenfold in the value of the velocity constant were obtained in this way. When velocity constants are plotted against concentration of medium, a sigmoid curve is obtained. 2. These results are believed to indicate that cells are more permeable to water when the osmotic pressure of the medium is high than when it is low. This relation would be accounted for if water should diffuse through pores in a partially hydrated gel, constituting the cell membrane. In a medium of high osmotic pressure, the gel is conceived to give up water, to shrink, and therefore to allow widening of its pores with more ready diffusion of water through them. Conversely, in solutions of lower osmotic pressure, the gel would take up water and its pores become narrow.

The work reported in this and in previous papers (1, 7) demonstrates the following relations for acid-fast bacteria and rabbit polymorphonudear leucocytes: 1. The combination of a substance or substances present in fresh immune rabbit serum, heated or unheated, or in fresh unheated normal rabbit serum, with a substance or substances in the bacterial surface causes an increase in cohesiveness, decrease in surface potential difference and characteristic alteration in wetting properties of the bacteria, and prepares the bacteria for phagocytosis. 2. (a) The effective substance or substances in the serum may become so altered as the result of heating or aging that combination with the bacterial surface, while causing changes in bacterial surface properties indistinguishable by the present physical-chemical tests from these just mentioned, may not lead to phagocytosis, or may lead to phagocytosis with a prezone not paralleled by a prezone in the changes in surface properties. (b) Sensitization of bacteria with human sera causes changes in surface properties similar to those caused by rabbit sera, but does not lead to phagocytosis by rabbit leucocytes. The spreading requirements of rabbit polymorphonuclear leucocytes are evidently highly selective.

The rate of swelling of unfertilized sea urchin eggs in hypotonic sea water was investigated. Analysis of curves leads to the following conclusions. 1. The rate of swelling follows the equation, See PDF for Equation where V(eq)., V(0), and V(t) stand for volume at equilibrium, at first instant, and at time t, respectively, the other symbols having their usual significance. This equation is found to hold over a wide range of temperatures and osmotic pressures. This relation is the one expected in a diffusion process. 2. The rate of swelling is found to have a high temperature coefficient (Q(10) = 2 to 3, or micro = 13,000 to 19,000). This deviation from the usual effect of temperature on diffusion processes is thought to be associated with changes in cell permeability to water. The possible influence of changes in viscosity is discussed. 3. The lower the osmotic pressure of the solution, the longer it takes for swelling of the cell. Thus at 15 degrees in 80 per cent sea water, the velocity constant has a value of 0.072, in 20 per cent sea water, of 0.006.

1. Solutions containing NH(4)OH and NaOH, and CO(2) and HCl may be used to produce various combinations of extracellular and intracellular reactions in starfish eggs, Gonionemus, and Nitella cells. 2. Staining by basic dyes is, with a constant intracellular reaction, favored by increased extracellular alkalinity. With a constant extracellular reaction, staining is hindered by increased intracellular alkalinity. 3. These facts are in opposition to the view that staining of cells by basic dyes is chiefly governed by a combination of the dyes with cell proteins. It is more in harmony with the view that the combination is with a substance or substances of acid nature.

Antisera against several strains of acid-fast bacteria have been separated into their euglobulin, pseudoglobulin and albumin fractions. The globulin fractions have been found to possess the essential properties of bacteriotropic sera: thus they alter the bacterial surface properties, and, in quantitative correspondence, cause agglutination and phagocytosis; these several effects withstand washing of the sensitized bacteria; the effects are little if at all affected by inactivation of the antisera before fractionation; the combination of antibody and antigen is serologically specific. The conclusion is drawn that the contact of antigen with fresh homologous immune serum results in the deposit on the antigen surface of a substance or substances contained in the globulin fractions of the antiserum; as a consequence of this surface deposit leucocytes can spread upon and engulf the antigen.

Using unfertilized eggs of Arbacia punctulata as natural osmometers an attempt has been made to account for the course of swelling and shrinking of these cells in anisotonic solutions by means of the laws governing osmosis and diffusion. The method employed has been to compute permeability of the cell to water, as measured by the rate of volume change per unit of cell surface per unit of osmotic pressure outstanding between the cell and its medium. Permeability to water as here defined and as somewhat differently defined by Northrop is approximately constant during swelling and shrinking, at least for the first several minutes of these processes. Permeability is found to be independent of the osmotic pressure of the solution in which cells are swelling. Water is found to leave cells more readily than it enters, that is, permeability is greater during exosmosis than during endosmosis.

1. The effect of HCl, NaOH, CO(2), and NH(3) on the volume of unfertilized Arbacia eggs was tested over a wide range of pH values. 2. No swelling occurred, except in HCl solutions, and there not until after injury or death had occurred. 3. Whereas the volume of erythrocytes and of proteins such as gelatin is known to be dependent on the pH of the solution, such a relation does not exist in the case of living and uninjured cells, at least of the type tested.

1. Permeability to water in unfertilized eggs of the sea urchin, Arbacia punctulata, is found to be greater in hypotonic solutions of dextrose, saccharose and glycocoll than in sea water of the same osmotic pressure. 2. The addition to dextrose solution of small amounts of CaCl(2) or MgCl(2) restores the permeability approximately to the value obtained in sea water. 3. This effect of CaCl(2) and MgCl(2) is antagonized by the further addition of NaCl or KCl. 4. It is concluded that the NaCl and KCl tend to increase the permeability of the cell to water, CaCl(2) and MgCl(2) to decrease it. 5. The method here employed can be used for quantitative study of salt antagonism.