There are several other methods for the preparation and purification of enzymes, but up to the present it may safely be stated that no one has succeeded in preparing an enzyme in a state of purity. In considering the mechanism by which enzymes act, it must be remembered in the first place that they are colloids, and, as such, will form absorption compounds with the substrate, or body, upon which they are acting. It is difficult to understand how an enzyme can exert an action on the substrate, unless it enters into some kind of combination with it, although this may be only a temporary one. The action of some enzymes has been found to be due to extremely small amounts of certain metals, e.g. in the case of the oxidizing enzyme laccase, the metal is manganese. In the purest samples of this enzyme prepared, 0·16 per cent. Mn was found, and it has been supposed by some observers that the whole of the action of the enzyme may be attributed to the _physical state_ of the manganese which it contains. According to this hypothesis,[101] the active part of the enzyme is the ion Mn. This ion may exist in the solution in two conditions, differing by the electric charge which they carry. One of them Mn_{++} carries two positive charges, the other Mn_{+++} carries three. In the first phase, Mn_{++} is transformed into Mn_{+++} by absorbing the charge of one ion of hydrogen (H_{+}), and two hydrogen ions thus discharged, in the _nascent state_, unite with the oxygen dissolved in the liquid to form water. [101] Duclaux. See Bibliography. 2Mn_{++} + 2H_{+} + 0 = 2Mn_{+++} + H_{2}O In the second phase the ion Mn_{+++} with three charges will be transformed into the ion with two charges, by decomposing a molecule of water, of which the nascent oxygen will attach itself to the oxidizable body R yielding the oxide RO. 2Mn_{+++} + H_{2}O + R = 2Mn_{++} + 2H_{+} + RO and the same cycle of operations will begin again, and continue indefinitely. In the above illustration the enzyme action was an oxidizing one. In the case of puering it is a hydrolytic action, in other words a molecule of water is added to the skin substance. The fibre or some portion of it is converted first into proteoses, and finally into peptones, and simpler bodies. The active metal in these cases appears to be calcium, but by what mechanism it brings about the hydrolysis is at present unknown. Pozerski[102] found that the pancreatic juice, which is secreted after injections of certain sera (anti-pancreatic action), and as a consequence has no pancreatic action, contains no calcium, but pancreatic juice secreted under the influence of pilo-carpin is more or less rich in calcium, and its proteolytic action increases about equally with the amount of calcium contained in it. The same probably holds good for the intestinal juice. [102] Koch’s Jahresbericht über Gärhungs Organismen, 1908, 635.