Describes the methods used to isolate the oxygen-sensitive iron-sulfur protein FNR in the laboratory.
Authors: Burgess et al., Translator: Chen Wei, this experiment is from "Protein Purification Guide".
Operation method
Separation techniques of oxygen-sensitive proteins: [4Fe-4S]-FNR as an example Move I. Anaerobic separation of 4Fe-FNR 2.1 Principle and procedure for purification of O2 unstable 4Fe-FNR 2.1.1 General Preparations for Achieving and Maintaining an Oxygen-Free Environment Prior to protein purification, common procedures must be adapted to establish and maintain an anaerobic environment for all chemicals, solutions, and equipment used in the protein purification process (Sutton and Kiley, 2003). This can often be accomplished by setting up a sparging station and using an anaerobic glove box ( C o y Laboratory Products, Inc., M o d e l A ) . A sparging station typically consists of an argon or nitrogen tank, equipment to provide copper-scrubbed (Sargent-Welch furnace) gas, and a customized manifold (Fig. 42.1). The venting system removes trace O2 from chemicals, or dissolved O2 from solutions used for protein isolation and identification, by dispersing inert gas into butyl rubber-sealed glass vials containing O2-sensitive solid reagents [e.g., sodium dithionite (D T H )] For this purpose, the manifold is equipped with a plurality of tubing lines, each of which is connected to a gas dispersion tube ( U p c h u r c h Scientific) or syringes (Fig. 42. 1). Standard syringes can also be used for aeration. The glove box provides sufficient space for storage and handling during protein purification. The anaerobic chamber (vinyl c h a m b e r ) is filled with a gas mixture consisting of 80 % N 2 , 1 0 % 0 ) 2 , and 1 0 % H 2 , which allows any O2 entering the box to react with the H2 via a palladium catalyst to produce water that is absorbed by the desiccant. In anaerobic environments, F P L C or H P L C purification systems for purifying proteins are advantageous because the seals resist oxygen diffusion and the newer designs are tight enough ;iption (small body size) to be operated in anaerobic chambers (assuming minimal heat production). In addition, most of them are automated designs and are easier to operate in an anaerobic chamber. However, if an older style F P L C is used, most of the components, including the pump, column, and detection system, need to be placed outside the anaerobic chamber and connected to the buffer and component collector in the anaerobic chamber by means of oxygen-impermeable P E E K tubing (U pchurch Scientific) (Fig. 42. 2), so that the interior of the entire system is maintained anaerobically. The solution flows through the path of the F P L C , from the liquid container located inside the anaerobic chamber to the pump and/or column outside the anaerobic chamber, and then back to the component collector inside the anaerobic chamber (Fig. 42. 2). Most solutions (e.g., buffers) are first vented with argon for at least 20 m in before entering the anaerobic chamber through the air valve and then equilibrated in the anaerobic chamber for at least 12 h before use. All glassware and plastic items should be equilibrated in the anaerobic chamber for at least 24 h before use to ensure that any inadvertent introduction of oxygen into the chamber is minimized. Among the large accumulations of F N R auxin-free proteins during the human induction phase, these cultures were decanted into a 9 L large-mouth glass vial and passed through argon gas at 4°C overnight. We were fortunate to find that the above process was expressed more efficiently than in a stable anaerobic environment, and our recent finding that both iron-sulfur cluster biosynthetic pathways are attenuated under anaerobic conditions compared with aerobic conditions (M e t t e r t e t a L , 2008) may provide an intrinsic mechanism for this phenomenon. The large-mouthed glass vials used to ventilate the cultures with argon were supplied with two-hole stoppers inserted into two glass tubes: the first connects the line from the argon tank to the gas dispersal tube, which touches close to the bottom of the glass vials; the second, which is shorter (3-4 inches), allows the gas to be released from the aeration vessel. After the overnight aeration step, the organisms are quickly emptied into a container (4 L beaker) sized to fit into the air valve and immediately transferred to the anaerobic chamber, where the organisms are dispensed into 500 mL centrifuge flasks that have been equilibrated in the anaerobic chamber for more than 24 h. The flasks are then removed from the anaerobic chamber. Before removing the centrifuge flasks from the anaerobic chamber, the caps with O-ring seals (Beakman) on the centrifuge flasks were tightly sealed. The organisms were centrifuged at 8000 r/m i n at 4°C to settle for 15 m i n ( B e c k m a n Avanti J-25 centrifuge, J L A 10. 500 rotary head). The bottle is then returned to the anaerobic chamber, the supernatant is poured off, and the procedure is repeated until all organisms have been collected. At this point, the anaerobic bacteriophage precipitate can be stored at -80°C or used immediately for lysis. 2.1.3 Preparation of bacterial lysates Bacteriophage lysates were prepared basically as described by S u t t o n and Kiley (2003) by passing an anaerobic bacteriophage suspension containing a reducing agent through a French press at a pressure of 20,000 psi with a continuous injection of argon gas in a collection flask. The greatest technical challenge in this step was maintaining anoxic conditions. To minimize bacterial contact with O 2 , a pre-cooled French press vessel and the bottle containing the anoxic bacterial precipitate were first placed into the anaerobic chamber through two cycles in the air valve. The flange press vessel is immediately rinsed twice with an anaerobic buffer = Normally, the bacterial deposits are resuspended (to ∼0.5% of the original volume) in anaerobic buffer A supplemented with sodium dithionite (D T H ) and dithiothreitol (D T T ) (Table 42.1), and are immediately transferred with a pipette to the rinsed, pre-cooled flange press vessel. Seal the flannel press container with the bacterial suspension and connect the sample outlet tube to a capped 18-gauge needle and remove the entire container from the anaerobic chamber. Once the vessel was assembled into the French Press, the needle was inserted into a glass vial with a butyl rubber cap that was pre-ventilated with argon gas for collection of bacterial lysates. After a single pass through the French Press at 20,000 psi, the anaerobic lysates are collected in sealed glass vials, which are then immediately taken back to the anaerobic chamber, transferred to ultracentrifuge tubes, and sealed with gas-tight lids before being removed from the anaerobic chamber. Ultracentrifugation was performed at 4°C, 45,000 r/m i n for 60 min (Beck-m a n O p t i m a L E -80K , 70. I T i rotor) to remove fragments of the organism. The samples were then taken back to the anaerobic chamber and the supernatant used as a source of 4F e-F N R protein isolation. 2.1.4 Protein purification [4Fe-4S]-FNR was routinely purified using an F P L C ( P h a r m a c i a L C C -501 Plus system) equipped with a 5 m L BioR e x -70 cation exchange column (BioRad Laboratories). The buffers were placed in an anaerobic chamber and pumped through the port of the anaerobic chamber via PEEK tubing to the external FPC. Prior to isolation of the bacterial lysate, the BioRex-70 column was washed for I h with anaerobic buffers B and A containing DTT and DTH and equilibrated in buffer A. The column was then washed with anaerobic buffers B and A containing FNR and DTH. The FNR-containing bacterial lysate was first transferred to a super ring (s u p e r b o p ), sealed, and connected to the up-sampling ring before it was taken outside the anaerobic chamber. The 4F e - F N R was eluted through a gradient of 0. 1 to 0. 55 m o l / L K C l at a flow rate of 10 m L / h and then pumped back into the sample chamber through a P E E K tube, which was connected to the component collector in the anaerobic chamber. Based on conductivity meter measurements, F N R was eluted with approximately 0. 4 m o l /L potassium chloride. The [4Fe-4S] 2+ clusters exhibit a distinctive green color due to a maximum absorption peak for visible light at 420 nm. The colored fraction is then collected and concentrated by flowing through a gravity flow column equipped with Iml BioRex-70 cation exchange resin. The column is stored in an anaerobic chamber and equilibrated with Anaerobic Buffer A. In addition, since the presence of the reducing agents D T T and D T H can interfere with the subsequent biochemical characterization of 4P V F N R , this step can also be used to remove these reducing agents from the protein solution. The 4 F e - F N R obtained by this method had a peculiar dark green color and contained about I.0 m m o l / L of protein with a cluster retention of about 80 %. 2.2 Procedure The operating procedure for stepwise separation of 4F e -F N R is summarized as follows® . Day 1 (1) In the early afternoon, inoculate strain P K 827 in 100 mL of LB medium containing I mL of 20 % glucose, 50 ul of 200 m g/m L benzylpenicillin (A p ) and incubate overnight at 37°C. The culture is then incubated at 37°C. The culture is then incubated at 37°C. (2) Prepare and autoclave four 2L flasks, each containing l l l X M 9 (Mer, 1972) basic medium. The next day (1) To each I L of M 9 medium add:10 m L 2 0 % glucose, 10 m L 2 0 % casein hydrolysate (casamino acid), 2 m L Vitamin Bi (thiamine) (2 m g / m L stock solution), 1 m L M g S O 4 (I m o l / L stock solution), 100 /JL Ca C l 2 (l m o l / L stock solution), I m L ferric ammonium citrate (10 m g / m L stock solution, filter sterilized), 250 uL A p (200 m g / m L stock solution, filter sterilized). (2) Each of the 4 shake flasks is inoculated with 10 m L of overnight cultures into I L M 9 complete medium. Shake at 37°C at approximately 200 r/m i n to an O D w m n value of 0-3. Add I m L 0-4 m o l / L I P T G to each shaker (final concentration). (3) Add the bacterium into a 9 L bottle and pass it through argon gas at 4°C for 14~16 h. The bacterium was then incubated in a 9 L bottle for 14~16 h. (4) Prepare the solution and glassware for the next day's use in an anaerobic chamber (usually the anaerobic chamber is equipped with standard pipetting equipment and tip heads, a centrifuge, vortexer, and centrifuge tubes). ① Solution. Buffer A: Prepare 600 mL in 500 mL and 100 mL glass bottles with screw caps (see Table 42.1). Buffer B: Prepare 600 mL in 500 mL and 100 mL bottles with screw cap glass (see Table 42.1 for formulation). Buffer C: 100 mL into screw cap glass bottles (see Table 42.1 for formulation). Buffer E: 100 m L in screw cap glass vial (see Table 42.1 for formulation). Water :100 m L bottle . Adjust the argon gas flow to produce a steady stream, and pass 20 mIn of argon gas into the solution through the customized venting system described in Section 2.1 of this chapter (Figure 42.1), using a manifold (Upcycled Scientific) placed at the bottom of the buffer solution. Glass tubes and plastic items placed in the anaerobic chamber include: two ultrahigh-speed centrifuge tubes and caps, component collection tubes, beakers for waste liquids, plungers and caps with sealing rings, glass vials (5 or 6 small, 2 medium) with butyl rubber caps, glass Pasteur pipettes, centrifuge vials with 4-6 caps with ◦-rings, 500 mL graduated measuring cylinders, 2 L and 4 L plastic beakers, 25 mL glass pipettes, and a Pasteur pipette. pipettes, Pasteur pipette ear washers, French press needles and test tubes. These items were placed into the anaerobic chamber through an air valve, uncapped and equilibrated for 12 hours. Day 3 (1) Pour aerated organisms from a 9 L shaker into a 4 L plastic beaker and immediately seal with plastic wrap and tape. Place the beaker in an anaerobic chamber and dispense 450 m L per portion into 500 m L centrifuge flasks and cover. (2) Remove the centrifuge bottles from the anaerobic chamber and weigh and balance them. Unbalanced centrifuge bottles are taken back to the anaerobic chamber to adjust the mass of the contents. Centrifuge at 8000 rpm at 4°C for 15 millimeters. (3) After centrifugation, return the bottle to the anaerobic chamber and remove the supernatant. Repeat the centrifugation steps until all organisms have been centrifuged. The bacteriophage precipitate should be an army green color. (4) During centrifugation, weigh 154 mg of DTT and 150 mL of DTH into separate I.5 mL EP tubes and cap them immediately. Place them in the anaerobic chamber with the centrifuged bottle. (5) In the anaerobic chamber, add I.5 mL of Buffer E to D T H and I.0 mL of water to D T T . Add 300 ul of DTH (final concentration 1.7 mmol/L). Add 300ul of DTH (final concentration 1.7 mmol/L) and 100ul of DTT (final concentration 1.1.0 mmol/L) to 100 ml of Buffer A. Add 1.0 ml of water to DTT. Resuspend the organisms in 20 mL of Buffer A containing I.7 mmol/L DTH and I.0 m m o l /L of DTT. The suspension can be used immediately for lysis of the organisms or can be transferred to a medium-sized glass vial, sealed with a butyl rubber stopper, frozen on dry ice, and then removed from the anaerobic chamber and stored in a freezer at 80 °C until ready for purification. Day 4 (1) To 500 mL of Buffer A and Buffer B, add I.5 mL of DTH and 0.5 mL of DTT, freshly prepared as described above, respectively. (2) Place the pump lead (p u m p lead) into the buffer so that Buffer A is connected to Pump A, and Buffer B is connected to Pump B. (3) Connect the I3ioRex-70 column to the F P L C system, making sure there are no air bubbles in the column. (4) Start the F P L C program and wash the column for I h with 100% Buffer B (in an anaerobic chamber) at a flow rate of 0.17 m L /m i n , then wash again for I h with 100 % Buffer A. The column should be washed with 100% Buffer B (in an anaerobic chamber) at a flow rate of 0.17 m L /m i n . (5) Place frozen organisms in an anaerobic chamber and thaw the suspension under anaerobic conditions (do not thaw the organisms outside the anaerobic chamber or oxygen will enter the glass vial). (6) Place the French press container into the anaerobic chamber. Rinse the sample container twice with Anaerobic Buffer A containing D TT and D TH. Fill the sample container with resuspended organisms, seal it, attach the sample outlet to a capped 1 8-gauge needle, and remove the sample container from the anaerobic chamber along with the glass vial with a butyl rubber cap used to collect the organism lysates. (7) Load the sample container into a French press, which is preferably placed in a cold room. To exclude oxygen from the lysate extract, the needle attached to the container is inserted into the collection vial and two additional syringes are lodged in the vial, one to gently add argon gas and the other to vent the gas. The organisms were lysed at 4°C and 20,000 psi. (8) Remove the syringes and place the bacterial lysate in the anaerobic chamber, dispensing the lysate into two ultracentrifuge tubes. Take them out of the anaerobic chamber and weigh and balance them before centrifugation. The unbalanced tubes are placed in the anaerobic chamber for mass adjustment. (9) Centrifuge for 60 m i n with a 70.1 Ti rotor at 4°C, 45,000 r/min . (10) After centrifugation, place the tube in an anaerobic chamber and pour the supernatant into a superloop and seal. The other end of the superloop is filled with Buffer A and sealed. Remove the superloop from the anaerobic chamber and connect it to the FPC system. (11) Start the FPLC sampling program and add the appropriate buffers in the proper sequence to elute the FNr from the BioRex-70 column. (12) Set the component collector in the anaerobic chamber to collect the column eluate. Day 5 Concentration and Storage. (1) The 4Fe-FNR-containing components are easily identified by their color. These components are poured into medium-sized flasks and diluted with Buffer C at a ratio of 1:4. (2) Construct an I mL gravity column of BioRex-70 and wash with 2x column volume of Buffer B, followed by 6x column volume of Buffer C. The column should be washed with Buffer B and Buffer C. (3) Protein solution is loaded onto the column, washed with 2x column volume of Buffer A, followed by elution with ∼0.8 mol/L KCl buffer (made up of Buffer B and Buffer C). Only colored droplets were collected. (4) Dispense the concentrated 4Fe-FNR into 0.5 mL EP tubes with the caps cut off and place the EP tubes into small glass vials. The glass vial is capped with a butyl rubber stopper and sealed with an aluminum cap (Bellco). The protein samples stored in the glass vials are immediately frozen on dry ice-ethanol and placed in an anaerobic chamber through an air lock. In this way, the samples can be stored safely in an 80°C refrigerator for several months. Before thawing, it is crucial to place the sealed glass bottles on dry ice-ethanol during transfer to the anaerobic chamber. This step prevents the introduction of O2 into the glass bottles during the thawing process. 2.3 Isolation of proteins for special purposes 2.3.1 Removal of Trace R N A Enzymes from Purified 4F e-F N R for In Vitro Transcription Analysis After passing through two B i oRex-70 cation exchange columns, the purified F N R protein was estimated to be more than 95 % pure by S D S--P A G E . However, this preparation contained a large amount of RNAase, which was sufficient to degrade the RNA product during in vitro transcription analysis. The RNAase activity was subsequently analyzed by the RNAase enzyme, which is an essential component of the RNA product. The RNAase activity is subsequently removed by separation on a size exclusion column (HR-12 Superose, GE Healthcare) connected to the Beckman HPC system (Mettert and Kiley, 2007). The entire H P L C system was placed in an anaerobic chamber and configured with P E E K tubing ( U p c h u r c h Scientific). Since the F N R isolated in this way was to be used for in vitro transcriptional analysis, all buffer solutions and component collection tubes were subjected to diethylpyrocarbonate (D E P C ) and/or high pressure treatment. Purification by volumetric exclusion chromatography also yields close to 1 0 0 % of dimerized 4Fe^F N R, as judged by sulfide analysis ( M o o r e and Kiley, 2001). 2.3.2 Purification of 57Fe-labeled 4Fe-FNR for Ntossbauer spectroscopy M s s s b a u e r spectroscopy is a powerful method for qualitative and quantitative analysis of iron-sulfur clusters (Beinert etal., 1997). It distinguishes iron-sulfur cluster types on the basis of the energy level of the hyperfine splitting mode of the iron nucleus and the energy required for the iron-nucleus to jump from the ground state to the excited state in the γ-ray excitation or absorption spectra. 57Fe is by far the most common element studied by MSSBUR spectroscopy, and because iron-sulfur-containing proteins play an important role in biological systems, MSSBUR has been widely used for the analysis of iron-sulfur clusters. u e r spectroscopy has been widely used to characterize such proteins. To prepare 4Fe-F N R enriched in 57F e , we flowed 1X M 9 medium through a C h e k x 100 (200 to 4,0 0 0 mesh, B i o R a d ) column (40 g of resin per 1 L of medium) to remove the naturally enriched 56F e in the growth medium, and treated the growth medium with ferrous ethylenediammonium sulfate (ferric). ethylenediammonium sulfate) in the growth medium to remove the naturally enriched 56F e , and to supplement 57F e in the form of ferrous ethylenediammonium sulfate. Preparation of iron-free growth cultures (1) Prepare the Chelex 100 column by adding 200 g of C helex resin and approximately 200 m L of water to a beaker. After 30 m in of incubation at room temperature, pour the resin into a 250 m L gravity column. Rinse with 5 times the column volume of water before use. Regeneration of the column requires a rinse with 2x column volume of Imol/L HCl, followed by a rinse with 5x column volume of water; this is followed by a rinse with 2x column volume of Imol/L NaOH and another 5x column volume of water until the p H of the column effluent is approximately 7.0. (2) Removal of Iron from Growth Medium: Pass 5 L of 1 X M 9 medium through a new or remanufactured Chelex100 column. Collect the effluent and autoclave. After use, store the columns in 0.5 m o l /L ammonium sulfate. (3) Bacteriophage growth and isolation of F N R containing 57F e-labeled [ 4 F H S ] 24 clusters. (1) Inoculate strain P K 8 72 in 100 mL of L B medium. Add I mL of 20% dextrose and 50 uL of 200 m g/m L ampicillin to the medium, and incubate at 37°C overnight. ② Centrifuge overnight cultures to remove L B medium. Resuspend the bacterial precipitate with 100 mL of iron removal IX M 9 medium, centrifuge at 6000 rpm at 4°C, and pour off the supernatant. ③ Resuspend the precipitate in 100 m L of I X M 9 medium, inoculate 10 m L of bacterial suspension into I L of iron-excluding M 9 basal medium with the added components as described above except that the iron is replaced with 15 uL of vinyl diammonium ferrous bisulfate containing 57F e (I mol/L stock solution). Cultures were incubated under conditions similar to those described above for purification of non57F e-labeled F N R -, including passage of argon gas to allow assembly of 57Fe-labeled [4Fe-4S ] 2+ clusters into F N R . ④ Because we observed that once 57Fe was incorporated into 4Fe -FNR, exchange of 57Fe in the [4Fe--4S ] clusters with 57F e in solution was difficult to occur, the buffers (A, B, and C) used for subsequent purification were not iron-removing and the purification steps were similar to those of the 4F & amp; F N R without 57Fe labeling (Section 2.2 of this chapter). A number of analytical experiments can be used to characterize purified 4F e-F N R , including protein concentration measurements, iron and sulfur content measurements, cluster retention and type, and cluster oxidation status. The purity of each protein preparation was estimated by S D ^ P A G E , usually greater than 95%. Protein concentration was determined by Coomassie Pluss Protein Analytical Reagent (Pierce Company) and corrected by dividing by the factor 1.33 to standardize the protein concentration to the results of the amino acid analytical assay (Protein Center, Iowa State University). Iron and sulphur content of protein were analyzed by the method developed by Beinert and colleagues (Beinert, 1983; Kennedy U 1984). The content of [4Fe-4S] 2+ clusters in purified protein is calculated from the number of moles of sulfur per mole of FNR protein. The [4Fe-4S] 2+ clusters can also be determined from the photoabsorption value of [4Fe-4S] 2- clusters at 405 mn, which has been found to have an extinction coefficient of 16,125 mOl/(L. Cm) (Suttlet ). Cm ) (Sutton et al., 2004). The retention rate of [4Fe-4S] 2+ clusters was determined by the percentage of F N R subunits containing a [4Fe-4S] 2+ cluster. Of the more than 100 FNR preparations we measured, we specifically observed a slightly higher concentration of iron than sulfur, probably due to a small amount of iron contamination in the buffer solution, but this iron was not bound to the clusters. Therefore, we used the sulfur concentration to determine the concentration of [4Fe--4S] 2- clustered proteins in the methionine preparations, which allowed for less introduction of human factors. Although the [4Fe--4S] 2+ clusters exhibit characteristics of visible absorption spectra (Khoroshilova et al., 1995), the type and oxidation state of the Fe-S clusters can only be determined by using the M s s s b a u e r spectra of 57Fe-labeled F N R proteins (Khoroshilovaetal., 1997). Recently, Fourier transform ion cyclotron resonance (F T -I C R ) mass spectrometry has been used to characterize adenylyl sulfate reductase in the form of the auxin-free (apo-)-, [2Fe-2S ]-, and [4F e 4S ]- proteins isolated from Mycobacterium tuberculosis (Carroll et al., 2005), although it is not yet clear how versatile this approach is. 3.1 Analysis of Iron and Sulfur1 Iron content was determined by a rapid process without ashing described by K ennedy et al. (I 9S4 ). The principle of this method is to reduce all the iron in the sample proteins to Fe2+, and then add the Fe2- specific chelator ferene{5,5'-[3-(2-pyridyl)-l ,2,4-triazine--5,6-diyl] bis-2-furansulfonic acid, Sigma} to form the Fe2- ferene complex, which has a high extinction coefficient at 593 nm. Solutions a, b, and c (Table 42.2) were prepared prior to the measurements (K ennedyetal., 1984). Protein dilution buffer (buffer D, Table 42.1) and a semimicro colorimetric cup (path length I cm ) were placed in an anaerobic chamber at least one day before the measurement. Normally, 5 protein samples and 95 Buffer D are added to the cuvette in the anaerobic chamber; the cuvette is covered with parafilm and removed from the anaerobic chamber. Then 100 pL of reagent a was added to the cuvette and mixed well; 100 pL of reagent b was added and mixed well; the cuvette was covered with parafilm and incubated at 30°C for 15 m in. Finally, 5 buckets of reagent c were added and mixed well, and the absorbance value was measured at 593 nm. In this assay, the molecular absorbance value of 1 ng of iron atoms was 0.119. Sulfur was determined using the reagent JV,IV-dimethyl-p-phenylenediamine (N ,N-dimethyl-p-phenylenediamine, DMPD) with sulfur forming methylene blue in a semi-micro-analytical method invented by Beinert in 1983. In this method, precautions are taken to avoid the loss of S2- in the form of hydrogen sulfide gas, a side reaction that usually occurs in the presence of strong acids and/or oxidizing agents (e.g., ferric chloride). Preventive measures include: (i) the entire process is carried out in a small glass tube of 10 mm diameter and L 7 m L total volume, with a conical bottom and fitted with a stirring bar (10 mmX 3 mmX 3 mm) and a glass stopper; (ii) gentle mixing when the solution is acidic or oxidizing agents are added, which reduces agitation of the liquid in the tube and the formation of new air-water interfaces, and thus the escape of the hydrogen sulfide gas formed; (iii) the formation of a new air-water interface, and the formation of the new air-water interface; and (iv) the formation of the new air-water interface. escape of the hydrogen sulfide gas formed; (iii) add a few drops of phenolphthalein to the solution to monitor the mixing efficiency of the gentle mixing method; and (iv) add the strongly acidic DMFD solution to the zinc hydroxide suspension using a micropipette so that the heavier DMPD solution is in the lower layer and the lighter solution is in the upper layer. The usual method is as follows. The glass tubes and water were equilibrated in an anaerobic chamber for 24 h prior to measurement. 10ul of protein sample and 90ul of water were then added to each glass tube , and the samples were removed from the anaerobic chamber with the lid on. The tubes were placed on a stir plate and 300ul of freshly prepared 1 % acetic acid cast [Zn( C2H3O2 ) 2+ was added and stirred gently. Then, quickly add 15ul of 12% Na0H and stir vigorously until the schlieren disappears or the phenolphthalein color is uniform. After 5 min of incubation at room temperature, 75 uL1 % DMPD solution (in 5 mol/L H Cl) was placed in the layer below the protein sample layer. The solution was gently stirred until only the top 2 mm layer contained insoluble zinc hydroxide or was pink in color. Then add 10 ul 23 mmol/L FeCl3 (in 1.2 mol/L H Cl) directly to the solution and stir rapidly until the solution is homogeneous (colorless). After incubation at 20~25°C for 30 min, centrifuge for 15~20 min until the protein clumps. The supernatant was transferred to a cuvette and the absorption values were measured at 670 nm, 710 nm and 750 nm. The ratio of absorption values should be approximately A670nm: A750nm: A710nm = 3 : 2 : I . The sulfur content was measured by an extinction coefficient of 34,500 m ol/(L . cm) at 670 nm. The sulfur content is measured by an extinction coefficient of 34 500 m ol/(L . cm) at 670 nm (Beinert, 1983). 3.2 ICP-MS analysis: 57Fe in 7Fe-labeled 4Fe - FNR Inductively coupled plasma mass spectrometry (ICP-MS) is a class of mass spectrometry methods that is highly sensitive for analyzing a range of metals and can resolve isotopic ionic forms. It is an ideal method for analyzing57F e in a large number of natural and experimental samples. Our protein preparations were recently analyzed using the Wisconsin State Health Laboratory's -'Thermoscientific E L E M E N T 2 high-resolution I C P-M S . Specifically, 50 u L I M m ol/L protein samples were transferred into ironless test tubes, sealed and removed from the anaerobic chamber, and submitted for analysis. Proteins were isolated according to the steps described above, with yields typically ranging from 8 0 % to 9 0 % and about 8 0 % of the protein clusters containing 57Fe. 3.3 UV-visible spectrophotometric analysis of [4Fe-4S] 2+ Xiang UV-visible spectra were typically obtained using a L a m b d a 2 UV-visible spectrophotometer (Perkin-Elmer Corporation) (Sutton etal., 2004). Quartz cuvettes with screw caps (I c m path length, Star n a Inc.) were placed in the anaerobic chamber, uncovered, and equilibrated for 24 h. The cuvettes were then incubated in the anaerobic chamber with the lid open. These cuvettes are fitted with screw caps with 〇 rings to prevent oxygen ingress and can therefore be used in standard spectrophotometers. In the anaerobic chamber, appropriately diluted protein samples are added to the cuvette, sealed with the screw cap and removed from the anaerobic chamber. The samples were scanned at wavelengths from 250 to 700 nm to obtain a complete absorbance spectrum of the proteins, and the absorbance value at 405 nm was used to calculate the amount of protein in the sample using an extinction coefficient of 16,125 m ol/(L . c m ). The absorbance value at 405 nm was used to calculate the concentration of [4F e-4S] For more product details, please visit Aladdin Scientific website
Induce F N R synthesis by adding I m L of 0-4 m m o l / L I P T G to each shaker (final concentration to 0.4 m m o l /L ) and shake for another hour.
The steps for preparing iron-free growth medium and supplementing ^F e are described below.
