METCALF 



.25 .5 1.0 



3.0 4.0 



TIME (Houri) 



Figure 2. The effect of neuraminidase concentration upon the release of neuraminic 

 acid from mixtures of influenza virus (A2/Jap 30 5/57) and neuramin mucoid at 37^ C. 



Using neuraminmucoid and enzyme mixtures incubated at 37° C, 

 free neuraminic acid was linearly split off during the first 20 min- 

 utes. When the same mixtures were combined and incubated at 20° C 

 a slow linear increase in free neuraminic acid was observed during 

 the 30 minute test interval. No reaction took place within 30 minutes 

 at 4° C. The use of the IR method for measurement of enzyme action 

 confirmed the findings of the TB A analyses. Differences observed in 

 the results obtained by the two methods were indicative either of a 

 different order of sensitivity, or a difference in the substance being 

 measured. For example the IR titration gave a yield of approxi- 

 mately 90 per cent split product compared to about 53 per cent for 

 the TBA method. Enzymic action at 20° C demonstrated by TEA 

 analysis was not shown by the IR method. 



The concentration of enzyme affected the release of neuraminic 

 acid as shown in Figure 2, Not only was the rate of release propor- 

 tional to enzymic concentration, but also to the total amount re- 

 leased. The same result using other substrate materials at 37° C 



346 



