Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

This procedure uses a stopped flow instrument to detect the different spectral forms of SID A and measure rates. The reaction catalyzed by SID A can be divided into reductive and oxidative half reactions of FAD. The oxidized flavin binds to N-A-D-P-H and reacts to form reduced flavin and NADP plus reaction of molecular oxygen and binding of ornithine results in formation of C four.

A hydroxy flavin after hydroxylation of ornithine, the hydroxy flavin dehydrates to form the oxidized enzyme. NA DP plus remains bound throughout the catalytic cycle and is the last substrate to be released. The stopped flow instrument facilitates rapid mixing of small volumes of reactants and records.

The spectral changes over time Stuff loss spectrophotometer allows one to measure the rate of formation and of a enzyme catalyzed reaction specifically of transient intermediates. Two members of my lab, Rita Robinson and Enviro Romero will demonstrate these experiments using set A at flavor mono oxygenase as a model system. Pour 250 milliliters of 100 millimolar potassium phosphate buffer into a 500 milliliter buchner flask containing a stir bar.

Seal the flask with a rubber stopper, then place it on a stir plate and connect the short tube of the flask to a sch flank line. Degas the buffer under vacuum for an hour at room temperature with agitation, followed by flushing with argonne. Repeat five times.

Finally flush the flask with Argonne for 10 seconds. Disconnect the flask from the vacuum manifold and place it inside the glove box. Continue to stir vigorously with the flask open.

Fill the reservoir syringes with 18.13 units per milliliter glucose oxidase, and 100 millimolar glucose solutions. Now set the drive valves to the load position. Fill the drive syringes.

Next, turn the F valve to the drive position. Using the pro data control software, click empty the stop syringe, then push the buffer from the drive syringe F through the flow circuit by manually raising the corresponding drive ram. Continue with the other three drive syringes equilibrate for an hour.

Now refill the reservoir syringes and repeat flushing of the stopped flow system. To prepare oxygen saturated buffer. Bubble the solution with 100%oxygen for one hour with agitation.

Then remove the short needle from the vial and wait 10 seconds. Proceed to remove the long needle and place the close vial in the glove box. For the N-A-D-P-H solution.

Place a 1.5 milliliter einor tube containing one milligram of N-A-D-P-H into the glove box. Dissolve in 300 microliters of anaerobic, 100 millimolar potassium phosphate buffer. pH 7.5.

Remove 30 microliters to determine the N-A-D-P-H concentration. Now to remove oxygen from the enzyme stock, transfer the solution into a 25 milliliter vial containing a stir bar cap with a wheat and stopper and aluminum seal. Place on ice.

Connect to a schlink line, then degas by vacuum for 20 minutes, followed by flushing with argonne. Finally, flush the vial with Argonne for 10 seconds and disconnect it from the vacuum manifold. Place the vial on ice inside the glove box.

In the control panel window of the pro data control software. Select photo diode array, external trigger and logarithmic scale. Also launch the pro data viewer software and define the directory for data file storage.

Now replace the solution in the reservoir syringes C and F with anaerobic 100 millimolar potassium phosphate buffer pH 7.5. Turn the C and F valves to the drive position. Click empty the stop syringe in the pro data control software.

Then push the buffer from both drive syringes through the flow circuit by manually raising the corresponding drive ram. Repeat flushing with reservoir syringes C and F three more times. To ensure complete removal of glucose oxidase from the flow circuit, proceed to click on baseline in the pro data control software.

Replace the solution of the reservoir syringe F with the enzyme solution. Turn the F valve to the drive position. Click empty the stop syringe in the pro data control software.

Push the enzyme solution from drive syringe F through the flow circuit by manually raising the corresponding drive ram. Return to the pro data control software to set the acquisition time to 60 seconds. Verify that both drive syringes are filled with their corresponding solutions and that the drive rams are in contact with the drive syringe.Pistons.

Turn both valves to the drive position and click on acquire in the pro data control software to perform the drive. Acquire spectral data of the oxidized form of the enzyme in triplicate using the A 4 52 nanometer value obtained from the drives. Determine the enzyme concentration before mixing and prepare four milliliters of N-A-D-P-H solution.

1.5 fold more concentrated fill reservoir syringe C with the N-A-D-P-H solution and empty and refill the stop syringe three times as shown earlier. Now, acquire spectra during the reduction of the enzyme flush contents from the flow circuit with anaerobic 100 millimolar potassium phosphate buffer pH 7.5. Now click on baseline in the pro data control software.

Then mix 200 microliters of enzyme stock solution with 1000 microliters of anaerobic, 100 millimolar potassium phosphate buffer. pH 7.5. Replace the solution of the reservoir syringe A with the enzyme solution.

Turn the A valve to the drive position in the pro data control software. Click the stop syringe empty. Push the enzyme solution from drive syringe A through the flow circuit by manually raising the corresponding drive ram.

Set the delay time to 0.5 seconds and acquisition time to 60 seconds. Verify that all drive syringes are filled with their corresponding solutions and the drive rams are in contact with the drive syringe pistons. Then turn all drive valves to the drive position and click on acquire in the pro data control software.

Obtain spectral data of the oxidized form of the enzyme in triplicate. Next, replace the solution of the reservoir syringe B with an N-A-D-P-H solution. 1.5 fold more concentrated than the enzyme solution in syringe A.Turn the B valve to the drive position and empty and refill the stop syringe three times as shown earlier in the control panel window.

Set the delay time at 0.5 seconds. An acquisition at 20 seconds, replace the solution of the reservoir syringe sea with the oxygenated buffer. Turn the C valve to the drive position.

Flush three times in the pro data control software. Set the delay time at 15 seconds and acquisition time at 900 seconds. Continue to acquire spectra during the re oxidation of the fully reduced enzyme in the stopped flow instrument.

The anaerobic reduction of SID A with N-A-D-P-H can be monitored by measuring changes in the redox state of the flavin using absorbent at 452 here. Spectral changes are recorded over time from oxidized SID A to the last spectrum of fully reduced SID A.The rate of this enzymatic step can be determined by fitting the data to the appropriate equation. The rate of oxidation and the reaction intermediates can also be determined using the double mixing mode of the stopped flow in this reaction by AF Sid A.The C four A Hydroxi flavin intermediate is clearly detected with a lambda max of three 80 nanometers and the oxidized enzyme has a lambda max of 450 nanometers.

Once mastered, this technique can be completed in four to six hours if it is performed properly. Remember to ensure the solutions are anaerobic. After watching this video, you should have a good understanding of how to investigate both the relative and the ulcerative ion of FLA independent mono further downstream experiment like rapid chemical.

Quentin can help to elucidate the sad nature of the intermediates.