66 INFLUENCE OF TEMPERATURE ON BIOLOGICAL SYSTEMS 



pear to be no cross reactions of the antibodies. For example, the anti- 

 body to the DPN pyrophosphatase from Proteus vulgaris will not inhibit 

 the DPN pyrophosphatase from Staphylococcus aureus. Further work is 

 currently in progress in Dr. Osier's laboratory in correlating the properties 

 of the various protein inhibitors and the related antibodies. 



Role of Inhibitor-Enzyme Complex. One cjuestion that may be raised 

 concerning this inhibitor and the inhibitor-enzyme complex relates to its 

 role in the cell. Does this enzyme occur naturally in the cell in the inactive 

 form or is this an artifact produced by sonic disruption of the cell, allowing 

 the inhibitor and enzyme to interact? In the case of Mycobacterium butyri- 

 cum, no DPNase activity is evident in either whole cells or sonic extracts 

 unless they are boiled. The situation with regard to the DPN pyrophos- 

 l)hatase from Proteus is much less clear. Whole cells exhibit pyrophos- 

 phatase activity. Sonic extracts are inactive. Heating these extracts pro- 

 duces demonstrable enzymatic activity, approximately 25% greater than 

 that of a comparable whole cell preparation. There is some evidence to 

 suggest that the enzyme in the whole cell is on the cell surface and is dif- 

 ferent from the enzyme isolated from sonicates. However, at the moment 

 this must remain a moot point. 



HEAT-STABLE ENZYMES 



Heat-Stability of Crude Enzyme Preparations. Although the DPN 

 pyrophosphatase from Proteus is surprisingly heat-stable for an enzyme, 

 it is nevertheless inactivated by rigorous and prolonged heating. In figure 

 4 it can be seen that the activity of the crude extract is maximal after two 

 minutes of boiling, and that after 15 minutes the activity is reduced by 

 75%. The remarkable stability to heat of this enzyme resembles the stabil- 

 ity of trypsin and myokinase. 



Reversible Denaturation of Purified Enzymes. The effect of tempera- 

 ture on the activity of the enzymes from Mycobacterium and Proteus 

 may be seen in table lA and B. In each instance the enzymes have been 

 purified about 40-fold. The first step in this purification was boiling, in 

 order to destroy the inhibitor. The enzymatic reactions were then run at 

 the temperatures indicated. The temperature optimum for the DPN pyro- 

 phosphatase of Proteus is about 50°C, while that of the DPNase of Myco- 

 bacterium is about 40°C. In both instances the purified enzymes are heat- 

 labile and most of the activity is lost when the reaction is run at 60° or 

 70°C. However, this inactivation is reversible; for upon cooling the reac- 

 tion mixtures and incubating them at 37°C, the expected increase in ac- 

 tivity is observed. 



Factors Affecting Heat Stability of Enzymes. Originally, the 'heat- 

 activated' pyrophosphatase of Proteus was demonstrated in extracts that 



