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100 mg of TTC was reacted with 5 ml of 10% NaOH to produce the formazon. The

formazon was dried weighed and a known concentration ranging from 1 to 25microgram
were prepared by dilution of the formazon in toluene. These were then subjected to a

spectrophotometer for the preparation of the standard curve.

In the case of enzyme studies, to the known amount of the buffered extract (Buffer

beings specific for each enzyme study). 1.0ml of 0.01% TTC (freshly prepared) and1 ml of
o
the specific substrate was added and incubated at 40 C for 30 minutes. The red coloured
formazon developed due to reduction of TTC (indicate of enzyme activity) was extracted
with 6ml toluene and the intensity of colour was read at spectrophotometer.

Preparation of Enzyme powder

The fresh root and shoot systems were separated and ground with chilled Butanol,
Benzene, Acetone mixture (In the proportion of 2:1:1) and kept in the fridge overnight. Then

the extract was dried at room temperature and made into fine powder. This enzyme powder

was used for various enzymatic activities. The method of Kannan (1968 b) was employed for
estimating the activities of the enzyme.

Alcohol dehydrogenase :( ADH)

50mg of dried material was ground with 5ml of 0.02M sodium pyrophosphate buffer

(pH 8.6). To the entire solution, 1 ml of 0.1 M acetone and 1 ml of freshly prepared 0.1%
o
TTC were added and the mixture was inoculated at 45 C for 30 minutes. The developed red
colour with 6 ml of toluene and was read in a spectrophotometer at 454 nm. A tube
containing 6 ml of toluene alone was used as blank.

Glucose Dehydrogenase: (GDH)

50mg of dried material was ground with 5ml of citrate phosphate buffer (pH 5.0). To
the entire mixture 1 ml of 0.1% glucose and 1 ml of 0.1% freshly prepared TTC added and

o
incubated at 45 C for 30 minutes. The red colour developed in the solution and with it was
mixed with 6ml of tolueneanditwasreadinspectrophotometerat454 nm along with a blank.
Statistical Analysis

Result obtained in the present studies was subjected to various statistical analysis of

Standard deviation, Student’s t-test, ANOVA and Critical Differences.

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