M. N. SWARTZ, X. O. KAPLAN AND M. E. FRECH 



67 



had been boiled in demineralized water. This finding was quite fortuitous, 

 since subsequently it has been shown that the boiling of crude extracts in 

 buffers such as tris (hydroxymethyl) aminomethane, (tris) , has led to in- 

 activation of the enzyme. Further work has revealed several interesting 

 points on the heat stability of this enzyme. The ability of the enzyme to 



25 



10 20 30 



TIME (Min.) 



5 10 15 20 

 TIME (Min.) 



Fig. 4. Effect of prolonged boiling on DPN pyrophosphatase. The enzyme prepara- 

 tion was a 1-6 sonicate of Proteus vulgaris cells in water, and assays were carried out 

 in usual fashion. 



Fig. 5. Stabilization of enzyme to heat by pyrophosphate. The reaction mixture 

 contained 0.5 ml of crude enzyme preparation that was boiled for 2 minutes, 2 /x moles 

 of DPN, .08 M tris buffer at pH 7.5, and 1 /j. mole of C0CI2 in a final volume of 1.5 ml. 

 Curve 1, undialyzed enzyme. Curve 2, enzyme dialyzed for 18 hours against distilled 

 water, then 2 /x moles of inorganic pyrophosphate added to 0.5 ml of enzyme and 

 boiled for 2 minutes. Curve 3, same as curve 2 except that the pyrophosphate was 

 added to the reaction after the enzyme had been boiled. (Science 123: 50, 1956.) 



Table 1. Activity of Mycobacterium butyricum and of 

 proteus vulgaris at various temperatures 



TEMP., °C units/ml. 



A. Mycobacterium butyricum* 



20 543 



30 915 



40 1140 



50 882 



60 111 



TEMP., "C 



units/ml. 



B. Proteus vulgaris 



30 200 



40 350 



50 430 



60 321 



65 104 



70 



* Data from M. Kern. In press. 



