98 MASS. EXPERIMENT STATION BULLETIN 175. 



In all the experiments fresUy collected material was used, and the 

 determinations made almost immediately after collection. The usual pro- 

 cedure was as follows : — 



A weighed amount of leaf was ground thoroughly with a weighed 

 amount of acid-washed sand and a certain volume of double distilled water, 

 and the whole washed into the apparatus with sufficient double distilled 

 water to bring the volume up to the standard volume used in the particu- 

 lar series in question. This, of course, gave to each flask a standard con- 

 stant dilution value. To this mixture was then added a like volume of 

 1 per cent, solution of Merck's perhydrol, thus making the H2O2 concen- 

 tration of the total mixture .5 per cent. The amount of oxygen liberated 

 in ten minutes was arbitrarily taken as the measure of enzyme activity. 

 Several different forms of apparatus were used, but for large amounts of 

 leaf any ordinary water displacement method was found to be very sat- 

 isfactory. (Care should be exercised where this mode of analysis is used, 

 to take into accoxint the absorption of oxygen by the water.) In making 

 determinations where the amount of material was very small, the ap- 

 paratus designed by Lohnis for use in milk examinations was found to be 

 more convenient. Practically all determinations were made at tempera- 

 tures ranging from 17° to 23° C. The action of the catalase is much 

 accelerated by shaking, as pointed out by Loew, and each test was shaken 

 imder exactly similar conditions in all the determinations made. It was 

 found necessary to use this method for the determination of the catalase 

 activity, as any method involving titration, such as the permanganate 

 method, was unsatisfactory, due to the reaction of certain constituents 

 in the tissue with the reagents. 



Table VI. shows the relative amounts of oxygen developed in normal 

 tobacco leaves, and it is to be noted that the catalase of the dark green 

 leaves was much more active than that of the light green leaves. This 

 was found to hold true, to a certain extent, for light and dark green leaves 

 even on the same plant. The basal leaves of older plants, which in some 

 cases were almost mature, and of a Ughter color than the middle and upper 

 leaves, developed in every case relatively less oxygen. This was partic- 

 ularly true in the case of Havana tobacco. Broadleaf did not show such 

 a wide divergence, but it should also be stated that in the Broadleaf plants 

 employed in the determinations the basal leaves did not show any great 

 color difference. 



As will be noted, some of these experiments were made with plants 

 grown under field conditions, but a greater number were made with 

 plants grown in the greenhouse, under control conditions. 



