178 



MANUAL OF MICROBIOLOGICAL METHODS 



the sonic oscillator required for cell disruption will vary for different 

 species. In addition, some enzymes are more labile than others under 

 the conditions of sonic disruption. Although maximum protein concen- 

 tration in the extract may be obtained after 50 min exposure, for example, 

 maximum enzyme yield may be achieved after only 20 min and the total 

 activity of the particular enzyme may decrease after that period (see 

 Table 17). The nature of the suspending fluid markedly influences the 



Table 17. Effect of Disintegration Time on Enzyme Yield 



Time, min 



Enzyme, units per ml 



Protein, mg per ml 



Specific activity, units per mg protein 



10 



2.0 

 61.7 



0.03 



20 



3.2 

 96.3 



0.03 



30 



2.0 

 111.6 

 0.02 



40 



1.8 

 145.8 

 0.01 



50 



1.0 

 154.8 

 0.003 



composition of the extract obtained by sonic disruption; alkaline buffers 

 (pH 8-9) often give better results. Thus, the conditions used for the 

 preparation of cell-free extracts must be carefully investigated in order to 

 obtain adequate results, reflecting the particular biochemical reaction of 

 interest to the investigator. 



Combination of methods may be employed: extraction of dried cells 

 at low temperatures, oscillation of suspensions prepared from dried cells 

 or frozen ceil pastes, etc. 



Protein Fractionation 



The cell-free extracts of bacteria, prepared according to the foregoing 

 methods, contain many of the bacterial enzymes. It is often the case 

 that a given biochemical reaction cannot be easily measured in such an 

 extract owing to the presence of interfering reactions catalyzed by other 

 enzyme systems which are also present. Therefore, it often becomes 

 necessary to separate the desired enzyme system from some or all of the 

 other enzyme systems present. This result may be achieved either by 

 isolation of enzyme protein or by isolation of enzyme activity, using 

 enzymatic activity as a measure of the process. Since bacterial enzymes 

 are protein in nature, most of the problems encountered in enzyme protein 

 isolation are similar to those of animal protein fractionation. The isola- 

 tion of enzyme activity is often accomplished by utilizing fortuitous 

 differences in physical properties (e.g., sensitivity to heat or pH) of the 

 desired active protein. A useful example is the purification of the 

 enzyme myokinase from rabbit-muscle extract (Colowick and Kalckar, 

 1943). In contrast to other enzymes, the enzymatic activity of myo- 

 kinase is relatively stable to heat and acids. The protein is thus easily 

 separated from interfering reactions by heating the extract at 90°C for 



