However, more recent investigations clearly indicate that there is an actual combination between the substrate material and the enzyme, which combination then breaks down with a resultant change in the substrate material and a freeing of the enzyme for repeated recombination with additional substrate, with the net result that the chemical change in the substrate material is enormously accelerated. That such a combination between substrate and enzyme actually exists has been demonstrated in two different ways: (_a_) experimentally, by mixing together solutions of an enzyme and of its substrate, each of which is filterable through paper or through a porous clay filter, with the result that the active material in the combined solutions will not pass through these same filters; and (_b_) mathematically, by a study of the curves representing the reaction velocities of typical reactions which are proceeding under the influence of an enzyme, which show that so long as there is a large excess of substrate material present, the accelerating influence of the catalyst is uniform over given successive periods of time, but that when the quantity of substrate material becomes smaller than that which permits the maximum combining power of the enzyme to be exercised, the reaction velocity immediately slows up. Again, the fact that the specificity of the action of an enzyme, i.e., the limitation of the action of that enzyme to a specific single compound or group of similar compounds, is definitely related to the molecular configuration of the molecule of the substrate, as has been found to be true in all those cases where the molecular configuration of the substrate material has been established (see pages 56 to 58), is an added indication that there is some kind of a union between the enzyme and the substrate as a first step in the catalytic process. As to the nature of this supposed combination of substrate and enzyme, two theories are held. The first is that this union is in the form of an actual molecular combination, or chemical compound, and the other is that it is a purely physical, or colloidal complex. The latter view has by far the greater weight of theoretical and experimental evidence in its support. The relation of electrolytes to the catalytic effect of enzymes, the appearance of the reacting masses under the ultramicroscope, and the effect of heat upon the reacting mixtures, all point to the conclusion that the phenomenon is colloidal rather than molecular in character. This view also makes the remarkable catalytic effects which take place in living protoplasm, which undoubtedly exists in the colloidal condition, much more easily understood. This phase of the matter will be much more apparent after the chapter dealing with the physical chemistry of the protoplasm has been studied.