Titles: enzyme kinetic
Author: Praopan Yodrabum
Lab: Virtual lab by Labster for BIO 1201L-03
Introduction: To investigate the alcohol, dehydrogenase CADH). enzyme, using
spectrophotometer try to determine it is kinet
...
Titles: enzyme kinetic
Author: Praopan Yodrabum
Lab: Virtual lab by Labster for BIO 1201L-03
Introduction: To investigate the alcohol, dehydrogenase CADH). enzyme, using
spectrophotometer try to determine it is kinetic parameter. This enzyme is important in the
manifestation of Alcohol flush syndrome.
Mission # 1:
Hypothesis: Enzymes are biological catalyst, so by lowering the activation energy, enzyme
accelerate the reaction rate by up to a million times faster.
Procedure:
Prepare the master mix in 15 ml tube which is include: Ethanol, NAD * and buffer. Do
not add the enzyme into the master mix because the reaction will start immediately and our
measurement will be inaccurate.
Ethanol is added because it's substrate the enzyme
NAD+ is function as co - factor.
Buffer is 1 it's kept pH of the master mix stable
Prepare the empty cuvette to set the measurement baseline to determine the
concentration of an unknow sample, it's important to prepare a reference blank each time.
a measurement is performed using the spectrophotometer. The reference blank is a solution that
contain no analyte of interest.
Prepare the reference blank: The lab assistant has already prepared a cuvette for us. Then
just simply open the spectrophotometer, put the cuvette that contain reference blank " into the
spectrophotometer and close it. Then press start to begin the measurement. Add the master mix
and " ADH18 * 1 '. enzyme into the new cuvette. so, the cuvette contain 0.16 ml of
10 MM ethanol, 0.2 ml of 10. PM NAD, 0.54 ml of buffer and 0.1 ml of 1 mg/ ml ADH so the
total volume will be 1 mL. So, the substrate concentration in this matter is " 0.0016 IM "
Take the cuvette the contain reference blank " out of the spectrophotometer then place the
cuvette that contain " master mix and ADH ' instead then begin the measurement by pressing
close and start.
(NAD+ Co - factor function as an electron transporter or electron acceptor)
Given ethanol with a stock concentration of 50 MM. then calculate the ethanol volume
for two master mixes with final substrate concentration of om and 0.0053 MM in a final volume
of 1 ml. We will have the ethanol volume of " 0 mL and 0.106 mL. "
The formula is C1 V1 = C2 V2
where “C” is the concentration and “V” is the volume.
Result: We can investigate ADH enzyme kinetic measuring. NADH at 340. nm
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