The purification of F1-ATPase is based on a classical biochemical methods which proved to be crucial for our understanding of the mechanism of ATP production by other ATP synthases. The main advantage of this technique is that it produces a highly pure enzyme suitable for structure determination by X-ray crystallography or cryo-electron microscopy. Although this protocol is optimized for the isolation of F1-ATPase from trypanosomes, it can be adapted to other sources, such as tissue or culture cells, and to various pathogenic protists.
This technique may lead to the identification of new drugs to treat severe human diseases. For example, Mycobacterium tuberculosis F-ATPase is an authenticated drug target for the treatment of tuberculosis. To begin the protocol, thaw and gently resuspend mitochondrial vesicles, also known as mitoplasts, previously isolated by hypotonic lysis from one times 10 to the 11 to two times 10 to the 11 cells of procyclic Trypanosoma brucei in five milliliters of ice-cold Buffer A.Keep the sample chilled.
Next, determine the protein concentration in the suspension by the bicinchoninic acid, or BCA, protein assay according to the manufacturer's instructions. Perform a BSA dilution series in ultra pure water to construct the standard curve. Dilute a small amount of the sample 20 to 100 times with ultra pure water to fit into the range of the BSA standards.
After calculating the total protein amount in the sample, bring the protein concentration to 16 milligrams per milliliter by diluting it with additional Buffer A.Then fragment the mitoplasts into inverted vesicles and membrane pieces by sonication seven times for 15 seconds each, with a total energy of 70 to 100 joules per impulse with a microtip with a diameter of 3.9 millimeters. While fragmenting, incubate the sample on ice for 30 seconds between impulses. After the sonication, the suspension might appear slightly darker.
Sediment the membrane fragments by ultracentrifugation at 54, 000 g for 16 hours or at 98, 000 g for five hours at four degrees Celsius. Decant the supernatant and proceed with the chloroform extraction. Calculate the volume of Buffer B based on the total amount of Buffer A used.
Transfer the frozen sediment from the centrifuge tube into the homogenizer. Resuspend the pellet of mitochondrial membranes in Buffer B with the aid of a small Dounce homogenizer. Transfer the suspension to a 50 milliliter conical tube.
Remove the sample from ice, and keep the sample and all the solutions at room temperature for the remaining steps. Then add chloroform saturated with two molar tris adjusted with HCl to pH 8.5, one to one. Close the cap tightly, and shake the sample vigorously for exactly 20 seconds.
Immediately centrifuge the mixture at 8, 400 g for five minutes at room temperature. Next, transfer the upper, cloudy aqueous phase to 1.6 milliliter microtubes. Add protease inhibitors to the sample to replace the inhibitors removed by the chloroform treatment.
Centrifuge the samples at 13, 000 g for 30 minutes at room temperature. After the spin, transfer the supernatant to fresh microtubes, and repeat the centrifugation to remove any remaining insoluble material. Equilibrate the five milliliter anion exchange Q column attached to a fast protein liquid chromatography system with 50 milliliters of Q column buffer at a flow rate of five milliliters per minute until the absorbance at 280 nanometers and the conductivity stabilize.
Load the supernatant on the equilibrated column at a flow rate of one milliliter per minute. Wait until the absorbance at 280 nanometers stabilizes at the background. Apply a 25 milliliter linear gradient of the Q column elution buffer from 0%to 100%at a flow rate of 0.5 milliliters per minute and collect one milliliter fractions.
Assay 10 microliters of each individual fraction corresponding to the major elution peak for ATP hydrolytic activity per on milliliter of reaction mixture by the Pullman ATPase assay at pH 8.0. Pool the fractions that exhibit ATPase activity. Concentrate the pooled sample by membrane ultrafiltration using a spin column with a 100, 000 molecular weight cutoff PES filter to 200 to 500 microliters.
Then proceed to size exclusion chromatography. Equilibrate the Superose 6 Increase column attached to a liquid chromatography system with at least 48 milliliters of SEC buffer at a flow rate of 0.5 milliliters per minute. Apply the sample to the column.
Run the chromatography at a flow rate of 0.25 milliliters per minute, while collecting 0.25 milliliter fractions. Next, run 10 microliters of the fractions that correspond to the peaks of the UV 280 nanometer absorbance trace on SDS-PAGE. Then stain the samples with Coomassie Blue.
Assay the fractions corresponding to the first major peak containing the F1 peak for the ATP hydrolytic activity and azide sensitivity by the Pullman ATPase assay. Then perform a BCA assay to determine the protein concentration. Finally, keep the purified F1-ATPase at room temperature, and use it within three days after purification for downstream applications.
This elution profile of an anion exchange chromatography displays the UV absorbance at 280 nanometers and concentration of sodium chloride in the elution buffer. The selected fractions were separated on the SDS-PAGE gel and stained with Coomassie Blue dye. The fractions that correspond to the major elution peak from anion exchange chromatography and contain the F1-ATPase were pooled, concentrated, and used as the input for size-exclusion chromatography.
The major contaminant is dihydrolipoyl dehydrogenase, which elutes from the size-exclusion chromatography column as a discrete peak. The F1-ATPase elutes in the first dominant, largely symmetric peak. The Coomassie Blue staining of a typical band pattern after the separation of the purified F1-ATPase via SDS-PAGE shows sporadic weak bands visible above the beta subunit, which represent subcomplexes of the alpha three beta three headpiece, dimers and oligomers of the alpha and beta subunits, and are devoid of any contaminants detectable by mass spectrometry.
While performing the chloroform extraction, the critical step of the protocol, it is essential to shake the sample vigorously and proceed to the centrifugal separation of the organic and aqueous phases immediately. Following this procedure, the isolated F1-ATPase can be characterized in detail by mass spectrometry. Further, it can be used in activity assays or for structural studies, either on its own or in the presence of various inhibitors or substrate analogs.
