I am working on writing an ethanol determination lab protocol for my biochemistry class. I have uploaded the instructions. I am confused on how to determine the necessary amounts of ethanol. If the most concentrated ethanol standard is 5x less than the NAD+ concentration of 1mM, then the highest ethanol concentration should be 0.2mM. The standards are supposed to cover a range of 500 fold, so the lowest ethanol concentration should be 0.0004mM. If the given standard ethanol solution has a concentration of 2mM and the reaction solutions are prepared in quantities of 250microliters, that would require an amount far smaller than we can pipette. What am I missing? I could also use help calculating what concentrations of the unknown solutions I should be using (beer or wine). Thank you!
Last lab: Ethanol Determination
The purpose of this lab is to allow students to develop a protocol to determine the ethanol
content in several beverage samples, using the enzyme alcohol dehydrogenase (ADH). The
reaction catalyzed by ADH is shown below.
H O O H H
NH2 NH2 OH H
N NAD+ NADH The formation of NADH is the basis for estimating the quantity of ethanol in the unknown
samples. In this reaction, ethanol is oxidized to acetaldehyde with concomitant reduction of
NAD+ to NADH. The formation of NADH can be monitored by measuring the change in
absorbance at 340 nm. The reduced form of the coenzyme absorbs quite strongly at this
wavelength (?340 = 6.2 ? 103 M-1cm-1), but the oxidized form does not absorb significantly.
A major problem associated with using this reaction to quantitatively determine ethanol content
relates to the equilibrium constant for the reaction. The equilibrium for the process lies far in the
direction of ethanol production, that is far to the left as written above. To function as a valid
quantitative assay for ethanol, essentially all of the substrate (ethanol in this instance) must be
converted to product. We can manipulate the conditions in several ways to make this happen.
1. The pH of the buffer will be set at 8.8. This will give a relatively low concentration of H+,
one of the products of the reaction.
2. The concentration of ethanol will be at least 5 fold less than the concentration of NAD+, thus
even after complete ethanol oxidation, there will be an NAD+ excess to push the equilibrium
in the desired direction. Additionally, despite the production of NADH, the ratio of
NAD+/NADH will have a value around 5 or greater even with all of the ethanol oxidized.
3. Semicarbazide will be included in the reaction mixture. It will react spontaneously with
acetaldehyde, as seen in the following reaction. The concentration of free acetaldehyde will
be reduced to very low levels, further driving the ethanol oxidation towards products. The
general principle of coupling an exergonic reaction to an endergonic reaction to shift the
equilibrium is used extensively in biochemical systems.
O O O N H 2N
H NH2 NH2 H 2O H Department of Chemistry & Biochemistry 1 Arizona State University Methods Development
You must first generate a detailed experimental protocol for the ethanol determination, based on
the instructions and reagents described below. The experimental design should include: a)
assays for standard solutions and unknown solutions, b) appropriate blanks and reagent controls.
The protocols should have sufficient detail to allow any of your classmates to use without
encountering difficulties. You may be called upon during the recitation to present part or all of
your experimental protocol.
Reagents available to you
Solution 1: Buffer: 22mM glycine, pH 8.8 containing 0.15 M sodium pyrophosphate and
0.16 M semicarbazide Solution 2: NAD+ 3 mM Solution 3: Standard ethanol solution: 2 mM Solution 4: Yeast alcohol dehydrogenase, 15 mg/ml in 0.05 M glycine, pH 8.0, prepared on
the day of the experiment. Assume this solution contains 5000 units/ml. Solution 5: Unknown solutions: Dilute your unknowns with distilled water to concentrations
that are similar to the standard ethanol solution (see info below). Add in a manner
similar to the ethanol standards. Instrumentation and Equipment
The platereader, adjustable volume pipettes (5-50 ?l and 50-300?l), and microplates are
When designing experiments, it is important to keep costs in mind. This means some attention
should be given to using the lowest amounts of materials, consistent with the laboratory
equipment and instrumentation that are available for the experiments. Absorbance
measurements can be made easily from 250 ?l of reaction mixture.
Complete reaction mixtures should contain:
a) enough buffer solution to give 75 mM sodium pyrophosphate
b) 1 mM NAD+
c) 25-50 units of enzyme
d) ethanol (amounts determined by volume of standard solution and diluted unknown)
e) water to make final volume of 250 ?l
It is usually best to perform the absorbance measurements of the standard ethanol solutions in
duplicate and you should have a minimum of 9 ethanol standards. These standards should cover
a range of 500 fold. To provide an example (keep in mind that these will not be your
concentrations): if your most concentrated ethanol standard had an ethanol concentration of 500
?M, then the most dilute ethanol standard would have an ethanol concentration of 1?M. Keep in
mind that you want the ethanol to be the limiting reagent with the NAD+ in a 5-fold excess over
the ethanol for the standard with the highest ethanol concentration.
For your unknown solutions, you should plan on doing several different dilutions of the samples,
with the absorbance of each of these solutions read in duplicate. You will need to have an idea
Department of Chemistry & Biochemistry 2 Arizona State University of the concentrations of your unknowns. Beer with a 5% alcohol content has an ethanol
concentration of ~ 1M while 12% alcohol content wine has an ethanol concentration of ~ 2.5M.
The volume of standard ethanol solution and diluted unknown solution should be varied to cover
an appropriate concentration range of ethanol.
You might also need to check your samples to insure that they do not absorb any light at 340 nm
at the concentrations used in your reaction mixtures. You should include a method for doing this
in your protocol. All standards and samples should be incubated for at least 30 minutes before
making final absorbance readings.
This lab has many similarities to the Bradford assay that you used in the Protein Spectroscopy
lab during the fourth week of lab. You will be making a standard curve and wanting to include
unknowns that have concentrations similar to your standards. Look at the protocol for the
Bradford assay to help you with the preparation of the current lab. It would probably be ideal to
make a table for reagent addition, much like you had for the Bradford assay.
Appropriate unknowns: you are allowed to bring unknowns in Eppendorf tubes (you will not
need more than 1.5 mls). Suggested unknowns are beer or wine (generally anything under ~15%
alcohol is fine). Be sure to obtain Eppendorf tubes from your TA the week prior to this lab.
Make a standard curve of absorbance vs. [ethanol] and use this to determine the ethanol
concentrations of your unknowns. Compare this concentration to the concentration claimed by
the maker of your beverage.
N. O. Kaplan and M. M. Ciotti, ?Methods in Enzymology III,? 253-256 (1957).
H. U. Bergmeyer, ?Methods in Enzymatic Analysis,? 2nd Edition, 3, 1499 (1974), 3rd Edition, 6,
598 (1986). Department of Chemistry & Biochemistry 3 Arizona State University
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