Krawkov’s method of preparing diastase from saliva (Green, p. 46) may also be mentioned, as it is of general application. It consists in salting out the enzymes, by saturating the clear solution (in this case saliva diluted with an equal volume of water) with neutral ammonium sulphate. The precipitate which is caused by the saturation is collected on a filter, and washed for a short time with strong alcohol. It is then allowed to stand under absolute alcohol for one or two days, and finally dried at 30° C. On extraction with water it yields a solution which is strongly diastatic, and which gives no proteid reactions.
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.
2Mn++ + 2H+ + 0 = 2Mn+++ + H2O
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+++ + H2O + 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.
Victor Henri has shown that the power of metals in the colloidal state to bring about these catalytic actions varies with the metal employed, and is in inverse ratio to the size of the particle. There is a very interesting and wide field of research open here in order to determine the conditions under which the various metals act. To this end the ashes from the purest enzyme preparations might be studied, and methods devised for producing these metals in the colloidal state, for it seems evident that it is the state of the body acting which gives it the properties observed, and not its chemical properties in the usual sense.