This method is a useful tool to introduce students to the concepts governing enzyme reactions. The main advantage of this technique is that it is suitable for students with very little wet lab experience, providing robust results for the students to observe and to interpret. To begin the protocol, label a 15 milliliter tube suspension.
Crush one lactase tablet containing 200 milligrams of enzyme into an even powder using a mortar and pestle. Place the resulting powder into the labeled tube and re-suspend it in 10 milliliters of 100 millimolar phosphate buffered saline, or PBS. Vortex the solution for one minute to maximize the enzyme extraction.
Next, transfer one milliliter from the suspension to a 1.5 milliliter tube and spin the sample for one minute at 10, 000 times G to sediment the solid particles. Transfer 500 microliters of the supernatant to a clean 1.5 milliliter tube labeled lactase extract. Place 390 microliters of 100 millimolar PBS into a 1.5 milliliter tube labeled reaction A.Then add 100 microliters of five millimolar ONPG solution and mix well by vortexing.
Add 10 microliters of extract into the reaction A tube and vortex the contents. Observe the reaction mixture often for five minutes and note any colorimetric changes of the solution. Set up two 1.5 milliliter tubes, one labeled reaction B, one labeled control.
Add 390 microliters of 100 millimolar PBS to the reaction B tube, 400 microliters of 100 millimolar PBS to the control tube, and then add 100 microliters of five millimolar ONPG to each tube. Mix the contents by vortexing. Add 10 microliters of lactase extract to the reaction B tube, mix by vortexing, and allow the reaction to proceed for one minute at room temperature.
Once one minute has elapsed, add 500 microliters of one molar sodium carbonate to both tubes to inhibit the lactase enzyme by increasing the pH, stopping the reaction. Transfer 500 microliters from each tube into clean spectrophotometer cuvettes and measure the absorbance at 420 nanometers. Set up three control tubes and three reaction tubes and label them four degrees Celsius, room temp, and 37 degrees Celsius.
Following the addition of the enzyme extract, incubate one tube on ice, one at room temperature, and one at 37 degrees Celsius in a preheated water bath for one minute. Then stop the reaction by adding 500 microliters of one molar sodium carbonate to each tube. Measure the absorbance at 420 nanometers for each tube.
Record the values, subtracting the control value from the reaction value in each case. Using the protocol, the control remains clear while the reaction solution containing extract from the lactase tablet turns yellow as ONPG is hydrolyzed, releasing ortho nitrophenol. Following analysis of samples using the spectrophotometer, production of ortho nitrophenol is lowest when the reaction is incubated on ice and highest when the reaction is carried out at 37 degrees Celsius.
While attempting this procedure, it's important to remember to have the reactions at the appropriate temperature prior to adding the enzyme extract.
