Enzymes 147 



While it may prove impossible to prevent artifacts com- 

 pletely and in every case, every effort must be made to re- 

 duce their occurrence. It is imperative to insure the prompt- 

 est possible precipitation of reaction products formed. This 

 can be done mainly by increasing the insolubility of the pre- 

 cipitate, e.g., by lov^ ionic strength, an appropriate pH, and 

 a high concentration of the precipitating ion. In addition, 

 incubation should not be prolonged beyond the optimum.^^ 

 The details of these measures will be given with the individ- 

 ual techniques. Presaturation of the incubating medium with 

 the precipitate to be formed is also practiced, but this pro- 

 cedure is not very reliable. If saturation is not complete, it is 

 of little help; if it is, the shghtest evaporation of the medium 

 or change in its temperature may lead to supersaturation, 

 with a resulting indiscriminate precipitation. A high rate of 

 enzymatic activity is also important because of the critical 

 nature of the difference between rates of hydrolysis and of 

 diffusion. Enzymatic activity can be enhanced by a high 

 substrate concentration, the use of activators and incubation 

 at the optimal pH. Inhibitors should not be employed ex- 

 cept for specific purposes. 



It is obvious that slow reactions for the precipitation of the 

 primary product of enzymatic activity cannot localize cor- 

 rectly, no matter how insoluble the final precipitate may be. 

 For instance, indoxyl liberated enzymatically can be visual- 

 ized by oxidation into insoluble indigo. The picture obtained, 

 however, can serve only as a rough indication of the true 

 distribution of activity, since the process of oxidation is a 

 relatively slow one. 



The precipitate obtained in the course of enzymatic reac- 

 tions is often colorless and invisible under the microscope. 

 In such cases it must be transformed into a colored substance. 

 The choice of the appropriate chemical procedure to ac- 

 complish this will vary with the nature of the primary pre- 

 cipitate. If the steps are well planned, there is Httle danger 

 of producing spurious secondary localizations, because the 



