Analysis and Characterization of

Alcohol Dehydrogenase
For: CH-533-01
A Little Background on Alcohol
Dehydrogenase (ADH)
ADH is the enzyme which catalyzes the
reduction of acetaldehyde to ethanol by
NADH. This fermentation occurs in yeast
during anaerobic conditions.

Acetaldehyde + ADH---->
 In The Beginning...
We Had Two Research Questions:
1. How does the pH change the activity of the
enzyme?
2. How does the stereospecificity of the enzyme
for the alcohol effect the catalysis of the
oxidation?
 Our General Procedure...
1. Variation of pH:
- Used Sodium Pyrophosphate Buffers at pH of
6.0, 7.0, 8.0, 8.8, 9.79, 10.8
- Each trial solution had 1.300 mL of the 50mM of
buffer. (Buffers were at varying pHs)
- The buffer was combined with 0.100 mL of 95%
ethanol, and 1.500 mL of 15mM -NAD.
- Each starting solution was observed for 3
minutes at A340 by a Spectrophotometer 20.
 Game Plan Continued...
- After 3 minutes, 0.100 mL of 0.08 unit/ mL of
yeast Alcohol Dehydrogenase (ADH) in the
phosphate buffer was added to the cuvette for an
additional 6 minutes.
- This process was continued for each pH.
2. Analyzation of stereospecificity of ADH
- Essentially the same as the pH runs only ethanol
is replaced by (R)-2-hexanol and (S)-2-hexanol.
 Continued again...
-Both specificities were performed at a pH of
8.8.
- We also wanted to perform the stereospecific
experiment with -NAD, but it proved too
costly.
 Calculations...
Units/ mL enzyme =
(A340nm/min.Test -Anm/min. Blank)(3)(df)
(6.22)(0.1)
3 = Total volume (mL) of assay
df = Dilution Factor
6.22 = Millimolar extinction coefficient of b-NADH at
340 nm
0.1 = Volume (mL) of enzyme used
Enzyme Activity vs. pH
 Change in Absorbance for Chiral 2-
Hexanol

0.04
0.03
Absorbance/min

(S)-(-)-2-hexanol
Chang in

0.02
0.01 (R)-(+)-2-hexanol
0
-0.01 DA DA Activity
blank blank
Conclusions from pH Experiment
Our results show that reactivity of alcohol
dehydrogenase does change with pH.
Below a pH of 8, reactivity began to drop off.
And above a pH of 10, reactivity dropped
off.
Therefore, our optimum pH for the reaction
was broader than the literature pH of 8.6 to
9.0 (Vallee and Hoch).
 pH (Cont.)
This difference probably results because the
final pH of the solution was not measured.
More kinetic runs should have been run,
especially in the pH regions with a rapid
change in activity.
 Conclusions from
Stereospecificity Experiment
Data do not show a significant activity of
the alcohol dehydrogenase.
2-hexanol was not soluble in our reaction
cuvette, even after addition of dioxane.
2-hexanol is somewhat sterically hindered.
No literature data on 2-hexanol.
Wanted to use chiral 2-butanols or
glyceraldehydes.
Either unavailable or very expensive.
 Stereospecificity (cont.)
The data is completely unreliable because
no activity of the enzyme could be seen.
More dioxane in the cuvette could have
helped dissolve the 2-hexanol, but the 2-
hexanol probably would not be able to be
oxidized by the alcohol dehydrogenase.