Labs
ONPG Lab
How Temperature Impacts Hydrolysis
- Class Alignment: Biology, chemistry
- Lab/home availability: Lab only
- Conduction Difficulty: Easy
- Time Required: ~1.5-2 hours
Introduction
In this lab, you will investigate how changes in temperature impact the rate of hydrolysis by measuring the breakdown of a substrate called ONPG by the enzyme lactase. By using color change to track the reaction, you'll be able to determine the optimal temperature for the enzyme to function.
(hover-over definitions:)
Hydrolysis: A chemical process in which a substrate reacts with water and separates into two fragments.
ONPG (Ortho-nitrophenyl-β-galactoside): A synthetic substrate that is used in replacement of Lactose due to its similar structure. It is consumed by the enzyme lactase, and upon consumption, it produces a yellow liquid. This color change is used to track the reaction in this experiment.
Optimal temperature: A particular range of temperature that allows a particular process to occur. In the context of this experiment, the temperature where enzyme activity peaks (because enzyme activity increases as temperature increases, and eventually drops once temperature gets too high).
Safety Notes
If chemicals come into direct contact with eyes or skin, immediately wash hands thoroughly and flush eyes with running water for more than fifteen minutes.
Do not pour ortho-nitrophenol down the sink, as it is toxic to aquatic life. Dispose of all waste in a chemical waste container.
Ensure you have access to a laboratory coat, safety goggles, and disposable gloves. Caution is required with the following chemicals:
ONPG
Can irritate skin and eyes. Avoid direct contact and inhalation.
Hazard Level: Low
Ortho-nitrophenol (Product)
Can irritate skin, eyes, and respiratory. Avoid direct contact and inhalation.
Hazard Level: Moderate
Lactase enzyme
Can irritate skin or eyes. Avoid direct contact.
Hazard Level: Low
Phosphate Buffer (Buffer Solution)
Can irritate eyes. Avoid direct contact.
Hazard Level: Low
Background Information
Lactase is an enzyme that typically breaks lactose, a substrate, down into glucose and galactose. Due to the colorless nature of the products from that reaction, this lab uses a structurally similar substrate called ONPG, to allow for the physical observation of the reaction. When ONPG is broken down, it produces galactose and ortho-nitrophenol, which is a yellow liquid that strongly absorbs blue light at 420 nanometers, making it easier to quantitatively measure.
A spectrophotometer can precisely quantify the color change through change in blue light absorbance at 420 nanometers, so the wavelength of 420 nanometers should be used to measure the production of ortho-nitrophenol. The rate of ortho-nitrophenol production will be used as a measurement of overall lactase activity throughout this experiment.
Enzyme activity is affected by temperature, as increasing temperature increases reaction rate by causing molecules to collide more frequently until the optimal temperature is reached. Beyond the optimal temperature, the enzyme may denature, decreasing the production of galactose and ortho-nitrophenol.
(Denature: The process in which an enzyme loses its natural structure and function, usually due to high heat. This process results in a permanent impairment of an enzyme’s ability to break down substrates.)
(Quantitatively measure: To measure something using exact numbers, rather than just describing it in general terms. In this lab, you will measure an exact absorbance value instead of just describing the color of the product.)
(Spectrophotometer: A device used to measure the amount of light that gets absorbed versus the amount that passes through a sample at a specific wavelength. This gives a precise, numerical value for how much of a colored substance is present.)
Variables
Independent Variable
Temperature of the reaction mixture (°C). Increments of temperature (10, 20, 30, 40, 50 °C) were selected to observe how temperature affects enzyme (lactase) activity. The temperature will be manipulated using water baths and measured by a temperature probe.
Dependent Variable
Rate of ONPG hydrolysis by lactase, measured as the change in absorbance at 420 nm over time (5 minutes) using a spectrophotometer. Used to determine ortho-nitrophenol production.
Controlled Variables
Figure 1. The Significance Controlled Variables, and the Method of Control.
Controlled Variable | Significance | Method of Control |
|---|---|---|
Concentration of ONPG | Concentration of ONPG impacts the frequency of enzyme–substrate collisions, altering reaction rate. | Use the same ONPG concentration and volume for all trials. |
Concentration of lactase | Concentration of lactase increases the availability of active sites and increases the reaction rate. | Use the same lactase solution and volume for all trials. |
pH of the reaction mixture | pH of the reaction mixture impacts the reaction site, altering reaction rate. | Use the same buffer solution and pH for all trials. |
Wavelength | Using different wavelengths would change measured absorbance values. | Measure absorbance of 420 nm for all samples to track ortho-nitrophenol production. |
Sampling time intervals | Inconsistency can alter the calculated reaction rate. | Record absorbance at fixed time intervals for all trials. |
Duration of the reaction | Duration of the reaction must be consistent or the substrate may begin to deplete. | Measure absorbance over the same total time for all trials. |
Materials
Figure 2. List of Apparatus and Chemicals Required for Investigation.
Apparatus | Chemical |
|---|---|
· Spectrophotometer · Water bath · Temperature probe · Test tube rack · Stopwatch · Micropipettes (0–1000 µL) · Graduated cylinder (50 mL) · 15 * Cuvettes · 15 * Test tubes | · ONPG (ortho-nitrophenyl-β-galactoside) solution 20 mL · Lactase (β-galactosidase) enzyme solution 10 mL · Buffer solution at 7.0 pH 50 mL · Distilled water 50 mL |
Experiment Preparation
· Prepare 50 mL of pH 7.0 buffer solution to maintain constant pH during all reactions.
· Prepare 20 mL of 2–5 mM ONPG solution as the substrate.
· Prepare 10 mL of lactase (β-galactosidase) enzyme solution.
· Preheat the water bath to 10°C for the first trial, adjust temperature for each trial. Confirm temperature with probe.
· Fill a cuvette with a buffer and insert a blank cuvette into the spectrophotometer to calibrate.
Experimental Procedure
Label test tubes for each temperature and replicate (3 replicates per temperature), plus one control tube per temperature.
For each trial, prepare two separate tubes per replicate: an "ONPG tube" (2 mL ONPG + 1 mL buffer) and a "lactase tube" (1 mL lactase solution). Label control tubes with 2 mL ONPG + 2 mL buffer (no lactase).
Set the water bath to the target temperature for that trial (start with 10°C).
Place the ONPG tubes, lactase tubes, and control tubes in the water bath and allow them to equilibrate for 5 minutes before mixing, confirming the target temperature with the probe.
Once equilibrated, pour the lactase tube into its matching ONPG tube and invert 2–3 times to mix. Start the stopwatch immediately upon mixing.
Keep the reaction tube in the water bath between readings to hold it at the target temperature throughout the reaction.
At 30-second intervals, transfer a 1 mL aliquot of the reaction mixture into a clean cuvette containing the stop solution to halt the reaction immediately.
Measure the absorbance of each stopped aliquot at 420 nm using the spectrophotometer. Record readings every 30 seconds for a total of 5 minutes.
Repeat steps 4–8 for the control tube at the same temperature, using the same time intervals.
Repeat steps 3–9 for the remaining temperatures (20, 30, 40, and 50°C), resetting the water bath temperature each time.
Repeat the entire process for all three replicates per temperature. Subtract the control absorbance from the corresponding reaction absorbance at each timepoint, then average the replicate values when calculating reaction rates.