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Team:UC Davis/Protein Engineering Test
From 2014.igem.org
Assay Components
Enzyme, Substrates, and Coenzymes
The main components of our assay are aldehyde dehydrogenase (ALDH), the NAD+ coenzyme, and the aldehyde substrate. Since NADH is a product of oxidation by aldehyde dehydrogenases, we were able to use a simple spectrophotometric approach to quantify the reaction rate of these enzymes on different aldehyde substrates. Using a plate reader, we tracked the change in absorbance at 340nm and measured the accumulation of NADH over time as aldehydes were converted into carboxylic acids. In addition to these basic components, there were several necessary additions made to our assay.
Isopropyl Alcohol
We were faced with two options when it came to introducing the aldehydes from an olive oil sample to our enzymes: solubilization or extraction. We determined that the extraction of aldehydes from a sample of olive oil would be ideal for our application; this would avoid the use of emulsifiers and other compounds which could potentially interfere with the enzymes and electrode. We used isopropyl alcohol as a representative solvent to extract the aldehydes from a sample of olive oil. Isopropyl alcohol was present at a concentration of 10% during all of our assays.
Dithiothreitol (DTT)
Aldehyde dehydrogenases possess a catalytic cysteine group which attacks carbonyl carbon of an aldehyde to create an intermediate thiol ester. Since several cysteine groups exist around the catalytic center of the aldehyde dehydrogenases we investigated, it was imperative that these residues did not oxidize to produce disulfide bridges. Thus, 1mM dithiothreitol (DTT) was used in all assays to prevent oxidation of the enzyme.
Tween 20 Aliphatic, straight-chain aldehydes are inherently hydrophobic and had a difficult time solubilizing in our initial assay conditions. Even in 10% isopropyl alcohol, longer chain aldehydes (e.g. nonanal, decanal) started to crash out of solution at concentrations greater than 100uM. To alleviate these issues, we conducted our assays in a final concentration of 1% Tween 20. While we found that the electrode could not operate in the presence of Tween 20, it was necessary in our enzyme characterization assays to collect data for long chain aldehydes.
The main components of our assay are aldehyde dehydrogenase (ALDH), the NAD+ coenzyme, and the aldehyde substrate. Since NADH is a product of oxidation by aldehyde dehydrogenases, we were able to use a simple spectrophotometric approach to quantify the reaction rate of these enzymes on different aldehyde substrates. Using a plate reader, we tracked the change in absorbance at 340nm and measured the accumulation of NADH over time as aldehydes were converted into carboxylic acids. In addition to these basic components, there were several necessary additions made to our assay.
| Standard NAD(H) Assay Components and Concentrations | |||
|---|---|---|---|
| Order of Addition | Volume | Components | Concentration |
| 1 | 130uL | Tween 20 | 1.54% (v/v) |
| DTT | 1.11 mM | ||
| 2 | 20uL | Aldehydes | 10x Final Concentration in Isopropyl Alcohol |
| 3 | 50uL | ALDH | 0.004 mg/mL |
| NAD+ | 4mM | ||
| DTT | 1.11mM | ||
| Final Volume | 200uL | ||
| Unless stated, all solutions were prepared in 11.1mM Potassium Phosphate, 111.1mM Potassium Chloride solution | |||
| Assays were conducted in a Costar 96 well flat bottom plate. Biotek Epoch/Synergy H1 plate readers were used to measure NADH accumulation at A340 over the course of 30 minutes. | |||
We were faced with two options when it came to introducing the aldehydes from an olive oil sample to our enzymes: solubilization or extraction. We determined that the extraction of aldehydes from a sample of olive oil would be ideal for our application; this would avoid the use of emulsifiers and other compounds which could potentially interfere with the enzymes and electrode. We used isopropyl alcohol as a representative solvent to extract the aldehydes from a sample of olive oil. Isopropyl alcohol was present at a concentration of 10% during all of our assays.
Dithiothreitol (DTT)
Aldehyde dehydrogenases possess a catalytic cysteine group which attacks carbonyl carbon of an aldehyde to create an intermediate thiol ester. Since several cysteine groups exist around the catalytic center of the aldehyde dehydrogenases we investigated, it was imperative that these residues did not oxidize to produce disulfide bridges. Thus, 1mM dithiothreitol (DTT) was used in all assays to prevent oxidation of the enzyme.
Tween 20 Aliphatic, straight-chain aldehydes are inherently hydrophobic and had a difficult time solubilizing in our initial assay conditions. Even in 10% isopropyl alcohol, longer chain aldehydes (e.g. nonanal, decanal) started to crash out of solution at concentrations greater than 100uM. To alleviate these issues, we conducted our assays in a final concentration of 1% Tween 20. While we found that the electrode could not operate in the presence of Tween 20, it was necessary in our enzyme characterization assays to collect data for long chain aldehydes.
