BBL433

Experiment 2:
Assay of Alcohol Dehydrogenase (ADH)

DATE OF EXPT : Monday, 29/01/2024 (Grp.4)

UTKARSH ANAND 2021BB10322

MEENAKSHI MINA 2021BB10012
Background

Alcohol dehydrogenase (ADH) is a group of enzymes found in many organisms. These

enzymes facilitate the conversion between alcohols and aldehydes or ketones, while
reducing nicotinamide adenine dinucleotide (NAD+) to NADH. They play a crucial role in
breaking down alcohols that can be toxic to the body and participate in generating useful
aldehyde, ketone, or alcohol groups during the biosynthesis of various metabolites. ADHs are
natural redox catalysts capable of both oxidizing alcohols and reducing carbonyl compounds.
They are expressed at highest levels in the liver but occur in other tissues as well .

When NAD+ is reduced to NADH, it strongly absorbs ultraviolet light at 340 nm, while the
oxidized form shows virtually no absorption at this wavelength. In a reaction mixture containing
ethanol, NAD+, and an enzyme in buffer, the reaction proceeds until equilibrium is reached.
The increase in absorbance at 340 nm can be measured as NADH is formed.
Materials And Equipment Required :-

 1. UV-Visible Spectrophotometer
 2. Cuvettes
 3. Pipettes and Tips
 4. Micro-centrifuge Tubes (MCTs)

Reagents Required :-

 1. 0.05M Phosphate Buffer (pH = 8.8)

 2. 50 mM NAD+
 3. 2M Ethanol
 4. Bradford Reagent
 5. BSA Standard
Procedure
1. Test Solution:
1. Combine 0.5 ml of Phosphate buffer, 0.3 ml of NAD+, and 0.1 ml of Enzyme (Final
Reaction Volume = 0.9 ml).
2. This mixture will serve as the test solution.
2. Blank Solution:
1. Mix 0.65 ml of Phosphate buffer, 0.3 ml of NAD+, and 0.1 ml of Enzyme (Final Reaction
Volume = 1.05 ml).
2. This mixture will serve as the blank solution.
3. Calibration:
1. Transfer 1 ml of the Blank solution to a cuvette.
2. Calibrate the blank in a UV spectrophotometer at 340 nm.
Procedure Cont.
4. Reaction Start:
1. Begin the reaction by adding 0.15 ml of alcohol to the test solution.
2. Mix well and immediately transfer 1 ml of the reaction mixture to the
cuvette.
3. Measure the absorbance at 340 nm promptly to capture initial product
formation.
5. Readings:
1. Place the cuvette inside the UV spectrophotometer.
2. Take readings at every 30 seconds for 2-3 minutes.
6. Enzyme Quantification:
1. Quantitate the moles of enzyme provided using Bradford-based protein
estimation.
2. Follow this protocol:
a) Prepare Test: Mix 0.1 ml of enzyme with 0.9 ml of Bradford reagent.
b) Prepare Blank: Mix 0.1 ml of phosphate buffer with 0.9 ml of Bradford
reagent.
c) Incubate both mixtures for 5-10 minutes.
Observations :

Absorbance of BSA : 0.601, 0.596, 0.594.

Average = 0.597
 Graphical Analysis
Abs.-time plot for original conc. Of Ethanol

Eqn. of st. line = 0.0009x + 0.0857

dA/dT = Slope = 0.0009
We know,
A = E*C*L
 dC/dT = (1/EL)*[dA/dT]
 dC/dT = (1/6220)*0.0009
 dC/dT = 0.144 μM/sec.
 dC/dT = 8.64 μM/min
 Graphical Analysis
Abs.-time plot for halved conc. Of Ethanol

Eqn. of st. line = 0.0005x + 0.0329

dA/dT = Slope = 0.0005
We know,
A = E*C*L
 dC/dT = (1/EL)*[dA/dT]
 dC/dT = (1/6220)*0.0038
 dC/dT = 0.08 μM/sec.
 dC/dT = 4.8 μM/min
As expected, the activity has roughly halved
from 8.64 μM/min (for E/20) to 4.8 μM/min
(for E/40) as the Ethanol conc. has been
halved.
Calculations :
 A=E*C*L
 C=A/E*L=0.0034/30=1.134*10^-4
 Moles of Enzyme = [(0.529-0.057)/0.006] *(1/147)=5.348 μmol/L
 Kcat = (0.000134)/5.348 sec^-1 = 2.506*10^-5 Sec^-1

Result :
Kcat = 2.506*10^-5 Sec^-1
Enzyme Activity = 0.036U

Sources of Error :
1)Cuvette might not be cleaned properly.
2)Amount of enzyme added might not be accurate.
3)Error in the measurement by spectrophotometer.
4) Absorbance values might have been measured much later after reaction have occurred.
Precautions :
 The buffer should be added first in the reaction system then NAD+ and then
Enzyme in exact concentration.
 After adding alcohol to the MCT labelled "test" it should be mixed well and
immediately transferred it to the cuvette and start measuring the
absorbance.
 Make sure the reagents are not contaminated. Cuvettes should be clean.
 It should be ensured that alcohol is added in the last after adding all other
reagents.
 Readings should be carefully noted down after each interval of 30 seconds for
2 - 3 minutes.

Thank You!