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Jan 2015

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Endpoint or Kinetic Measurement of Hydrogen Sulfide Production Capacity in Tissue Extracts
组织提取物中生成硫化氢的终点或动力学测定   

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Abstract

Hydrogen sulfide (H2S) gas is produced in cells and tissues via various enzymatic processes. H2S is an important signaling molecule in numerous biological processes, and deficiencies in endogenous H2S production are linked to cardiovascular and other health complications. Quantitation of steady-state H2S levels is challenging due to volatility of the gas and the need for specialized equipment. However, the capacity of an organ or tissue extract to produce H2S under optimized reaction conditions can be measured by a number of current assays that vary in sensitivity, specificity and throughput capacity. We developed a rapid, inexpensive, specific and relatively high-throughput method for quantitative detection of H2S production capacity from biological tissues. H2S released into the head space above a biological sample reacts with lead acetate to form lead sulfide, which is measured on a continuous basis using a plate reader or as an endpoint assay.

Keywords: Hydrogen sulfide production capacity (硫化氢生成能力), H2S (H2S), Endpoint assay (终点测定), Continuous assay (连续测定), Liver (肝脏), Lead acetate (醋酸铅), Lead sulfide (硫化铅)

Background

Hydrogen sulfide (H2S) gas is produced endogenously by at least three different enzymes in mammals (CGL, CBS, 3-MST) with a range of tissue and cell-type distributions. H2S functions as a gasotransmitter and effector molecule (Wang, 2012) in a wide range of biological functions related to metabolism (Módis et al., 2013), stress resistance (Hine et al., 2015), and redox biology (Dickhout et al., 2012). Reduced H2S is linked to cardiovascular problems including hypertension in rodents (Yang et al., 2008) and cardiac hypertrophy in man (Polhemus et al., 2014). Increased H2S can also cause pathology, for example in rodent pancreatitis (Bhatia et al., 2005). Thus, accurate and quantitative detection of H2S from biological sources could facilitate a better understanding of its biological effects as well as its potential use as a clinical biomarker.

Techniques to measure absolute concentrations of H2S present in biological samples, along with their pros and cons, have been reviewed extensively (Olson, 2012; Wang, 2012; Hartle and Pluth, 2016; Takano et al., 2016). For example, free and sulfane-bound H2S pools can be measured in biological samples including serum or tissue homogenates ex vivo using headspace GC-MS, which is highly sensitive and selective, but requires expensive equipment. Nonetheless, due to the volatility of H2S, its interaction with other biological macromolecules and its breakdown into different sulfur-containing compounds, quantitative detection of steady-state free H2S levels in vivo remains challenging (Olson, 2009).

An alternate approach is to measure the capacity of a tissue homogenate or extract to produce H2S in a reaction mixture containing optimized levels of substrate and cofactor, thus allowing for H2S detection methods that are specific but less sensitive. An example is the methylene blue method in which H2S in solution is trapped by lead acetate to form lead sulfide, which upon conversion to methylene blue can be easily read in a standard spectrophotometer (Stipanuk and Beck, 1982; Ikeda et al., 2017). The pros and cons that must be taken into account with each method are based on the question being asked, the biological system and tissue being studied, the relative need for sensitivity, selectivity, or speed, and the cost and resources of the investigator.

Here, we describe an inexpensive, rapid, and moderately high throughput methodology for measuring H2S production capacity in extracts of relatively small amounts of biological material. This method is based on the reaction of H2S present in the headspace above a biological sample with lead acetate to form the black precipitate lead sulfide, a technique used throughout the past 100 years to detect H2S and H2S-producing bacteria (McBride and Edwards, 1914; Kuester and Williams, 1964; Zhang and Weiner, 2014). Previously, we used this method to detect changes in H2S production capacity as a function of diet or genetic background in a variety of biological samples including yeast, worms, flies, and rodent tissues/organs including liver (Hine et al., 2015; Mitchell et al., 2016; Nikonorova et al., 2017). Here, we present an optimized procedure to measure H2S production capacity in mammalian liver via (B) an end-point assay using Whatman paper-embedded lead acetate, or (C) a kinetic assay using agar-embedded lead acetate. As the liver is a strong producer of H2S in mammalian systems via the enzyme cystathionine gamma lyase (CGL) (Kabil et al., 2011), we feel this is a good starting point for researchers to understand and confidently develop this protocol for their own research questions. Furthermore, this procedure can be easily adapted to other biological samples and organisms, although the procedure may need to be optimized by the investigator in order to obtain suitable results.

Materials and Reagents

  1. Petri dish
  2. 1.5 ml RNase-free disposable pellet pestles and 1.5 ml tubes (Fisher Scientific, catalog number: 12-141-368 )
  3. Disposable razor blades
  4. 8-strip well format tubes (Denville Scientific)
  5. Hard/rigid plastic dissecting platform/sheet
  6. Plastic wrap
  7. Filter paper (703 Style Whatman)
  8. 96-well plates with lid (Corning, catalog number: 3370 )
  9. Gloves and proper personal protective equipment
  10. 15 ml centrifuge tube
  11. Flash frozen mouse livers
  12. De-ionized water
  13. Phosphate buffered saline (PBS), pH 7.4 (Fisher Scientific, catalog number: BP24384 )
  14. Liquid nitrogen
  15. Ice
  16. Dry ice
  17. 5x passive lysis buffer (Promega, catalog number: E1941 )
  18. BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23227 )
  19. L-cysteine (Sigma-Aldrich, catalog number: C7352 )
  20. Pyridoxal 5’-phosphate hydrate (Sigma-Aldrich, catalog number: P9255 )
  21. Lead(II) acetate trihydrate (Sigma-Aldrich, catalog number: 316512 )
  22. Agarose (HS Molecular Biology Grade) (Denville Scientific, catalog number: CA3510-8 , or use similar)
    Note: This product has been discontinued.
  23. 1x passive lysis buffer (see Recipes)
  24. 20 mM lead(II) acetate trihydrate (see Recipes)
  25. H2S reaction mixture (see Recipes)
  26. 1% agarose gel with 100 mM lead(II) acetate trihydrate (see Recipes)

Equipment

  1. Liquid nitrogen flask (Thermo flask 2122) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2122 )
  2. Forceps
  3. Scale (OHAUS, catalog number: EP214C )
  4. Pipettes and tips (single use and multichannel for pipetting between 2 µl to 5 ml)
  5. -80 °C freezer
  6. Motorized tissue grinder (Fisher Scientific, catalog number: 12-1413-61 )
  7. 37 °C water bath
  8. Micro-centrifuge (VWR, model: Galaxy 16DH )
  9. UV-Vis plate reader (BioTek Instruments, model: Synergy 2 )
  10. Large glass Pyrex baking dish (> 100 ml)
  11. Glass flask (> 100 ml capacity)
  12. Vacuum oven (VWR, catalog number: 89508-424 )
  13. Incubator (VWR, model: 1500E )
  14. Digital camera (Kodak, model: KODAK EASYSHARE C182 )
  15. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
  16. Computers (HP Pavilion dv6 and Lenovo IdeaPad)
  17. Heat block cube (9.5 x 7.5 x 5 cm), or other heavy object with approximate dimensions

Software

  1. GraphPad Prism 7
  2. Microsoft Excel
  3. ImageJ
  4. Gen5

Procedure

Note: This protocol includes two related techniques to measure H2S production capacity via an endpoint assay (Procedure B) or a kinetic assay (Procedure C). Both techniques use the specific chemistry in which lead acetate (white in color) is converted to lead sulfide (brown to black in color) upon exposure the H2S. The techniques differ in their detection methods, with (Procedure B) utilizing endpoint densitometry to quantitate lead sulfide spots on the filter paper, while (Procedure C) utilizes the kinetic-capable absorbance of light in the 310 nm wavelength as well as endpoint densitometry of the lead sulfide spots on the agar gel. Shared common procedures and techniques are presented first (Procedure A) and then later split into the specifics that differentiate the two.

  1. Shared procedures
    1. Tissue samples
      1. Excise mouse liver tissue following euthanasia according to the procedures detailed in the animal use protocol that has been approved by your institutional animal welfare committee.
        1. Before euthanasia and harvesting, prepare 10 ml PBS in a Petri dish on ice to use as a wash for removing superficial blood/debris from the excised liver tissues. Also, fill the liquid nitrogen flask with liquid nitrogen.
        2. After euthanasia, quickly remove the large left lobe of the liver, rinse in PBS, place into a 1.5 ml centrifuge tube and flash freeze in liquid nitrogen.
        3. Leave samples in liquid nitrogen until all livers have been harvested.
      2. For previously harvested liver samples, store at -80 °C until ready for homogenization.
    2. Homogenize mouse livers
      1. With liver samples in 1.5 ml centrifuge tubes, remove from liquid nitrogen or -80 °C freezer and place on dry ice.
      2. Using a sterile or clean disposable razor and forceps, quickly remove the frozen liver from the tube, cut off segments on a hard plastic or other dissecting platform/sheet that is placed on dry ice, and weigh on a scale to approximately 100 mg.
        Note: This weight does not need to be accurate, just close enough so that the weight of liver sample to volume of lysis buffer is relatively constant between samples.
      3. While still frozen, place the ~100 mg of liver tissue into a new 1.5 ml centrifuge tube precooled on dry ice.
      4. Make up a fresh 1x solution of passive lysis buffer (see Recipes) from the 5x passive lysis buffer stock (Promega) with deionized H2O.
        Note: 1x passive lysis buffer should be kept on regular ice and made fresh with each assay. The 5x passive lysis buffer should always be stored at -20 °C when not in use. Unfortunately the recipe is proprietary.
      5. One 1.5 ml tube at a time, add 250 µl of 1x passive lysis buffer to each approximate 100 mg sample.
      6. Immediately, start homogenizing with the Motorized tissue grinder (Fisherbrand) with Pellet Pestle (Fisherbrand) until the sample has been thoroughly homogenized. This usually takes 30 sec to 1 min to perform.
      7. Remove the grinder and pestle from the tube, secure the cap, and place the homogenized sample and 1.5 ml tube into liquid nitrogen to flash freeze.
      8. Continue to homogenize all samples, keeping those completed in the liquid nitrogen.
      9. Once all samples have been homogenized, remove all samples at the same time from liquid nitrogen and thaw at room temperature for approximately 5-10 min or in a 37 °C water bath for approximately 1-5 min.
        Caution: Take care to use tubes suitable to freeze/thaw with liquid N2 to avoid loss of samples and potential injury from exploding tubes.
      10. Repeat freeze-thaw an additional two times, ensuring at least 1 min of freezing in the liquid N2 and keeping the same times for thawing as described above.
      11. Spin down the tubes in a microcentrifuge for 5 min at 4 °C at 5,000 x g.
      12. Remove and save the supernatants (~200 µl) in 8-strip well format tubes and place on ice if immediately going onto the next step (protein concentration determination). If not, flash freeze the tubes and place into a -80 °C freezer until ready to do protein concentration determination.
    3. Protein concentration determination and normalization of protein concentrations
      1. In fresh 8-trip well tubes, place 198 µl of deionized water and 2 µl of the liver homogenate supernatant to make a 1:100 dilution.
      2. Follow the directions of the Pierce BCA Protein Assay Kit for preparing the working solutions and standard BSA solutions (between 0 µg/µl to 2 µg/µl) that are contained in the kit for the protein concentration assay.
      3. Pipette 200 µl of the assay solution in each needed well, and 5 µl of the standard or 1:100 diluted sample.
      4. Cover with plastic wrap and incubate at room temperature in the dark for 30 min.
      5. Unwrap the plate and place it onto a UV-Vis spectrophotometer.
        Note: We use a Synergy 2 multimode plate reader running the Gen5 software, but other plate readers can be used.
      6. Measure the absorbance of light at 562 nm.
      7. Determine the protein concentration of each sample based on the standard curve equation obtained from the absorbance at 562 nm from each of the standards.
      8. In new 8 strip well tubes, normalize the protein concentration of each sample to 20 µg/µl using 1x passive lysis buffer. Bring up the final volume to 100 µl.
        Note: If 20 µg/µl is not possible, then dilute each sample to 10 µg/µl or other suitable concentration.

  2. Filter paper-embedded lead acetate endpoint assay
    1. Prepare lead acetate filter papers
      1. In a large glass Pyrex dish, pour 100 ml of 20 mM lead(II) acetate trihydrate (see Recipes) in deionized H2O.
      2. Cut the 703 style Whatman filter papers into approximate size dimensions of a 96-well plate (8 x 12.5 cm).
      3. Place the cut filter papers into the lead(II) acetate trihydrate solution and let soak for at least 20 min.
      4. Embed and dry the lead(II) acetate trihydrate into the filter papers by placing the wet papers into the vacuum oven set at 110 °C and the vacuum turned on. Let the papers sit in the vacuum oven at these settings for 20-30 min.
      5. Store the finished papers at room temperature in a dark and dry area until ready for use. Although we have not empirically tested the shelf-life and stability of these papers, they are good for at least one month after their production.
    2. Prepare H2S production capacity reaction solution.
      Note: This must be done fresh for each assay.
      1. Calculate the volume needed to run all of the samples. Each sample requires 150 µl of reaction solution (see Recipes).
      2. Prepare working stock of L-cysteine (100 mM in PBS) and pyridoxal 5’-phosphate (PLP) (10 mM in PBS). The cysteine must be made fresh each time. For PLP, warming to 37 °C and vortexing may be necessary to completely solubilize the powder. The PLP stock solution can be stored in aliquots at -20 °C for several months.
      3. In an adequate amount of PBS to cover the needed volume of reaction mixture, add L-cysteine to a final concentration of 10 mM and pyridoxal 5’-phosphate hydrate to a final approximate concentration of 1 mM.
      4. Keep at room temperature and use immediately.
    3. Setting up the assay
      1. In each well of a 96-well plate, pipette 150 µl of the reaction mixture.
      2. In each well, pipette 0-500 µg of sample protein. We have found 100 µg (or 5 µl when samples are diluted to 20 µg/µl) produces a detectable and non-saturating signal within 1 h with liver lysates. Other tissues may require more protein.
      3. Once all samples have been added, ensure there is no residual or spilled liquid on the surface of the 96-well plate by gently wiping with tissue paper.
      4. Place the dry lead acetate embedded filter paper directly over the 96-well plate.
      5. Place a similar sized piece of hard/rigid plastic or other non-flexible cover over the paper to keep it in place and ensure direct contact of the lead acetate paper with the lips of the wells on the plate.
      6. Place the heavy heating block cube or other heavy object with similar dimensions (9.5 x 7.5 x 5 cm) over the piece of hard plastic.
      7. Making sure not to shake or spill any of the liquid, place the ‘sandwich’ into a non-shaking/rocking 37 °C incubator.
      8. Over time, brown to black circles will form on the filter paper over the wells (see Figure 1 for visual example). This is the formation of lead sulfide due to the interaction of lead acetate with hydrogen sulfide.


        Figure 1. Filter paper-embedded lead acetate endpoint assay for H2S production capacity determination from liver extracts. A. Schematic overview of the assay. Liver is excised from the mouse, homogenized and lysed in passive lysis buffer followed by protein determination and normalization. In a 96-well plate, the reaction mixture is added first, followed by the liver lysate samples. The lead acetate paper, rigid plastic, and weight are then placed on top of the 96-well plate. Incubation occurs at 37 °C for 1 to 2 h followed by imaging and analysis of the lead sulfide dots on the paper. B. Photographic image of the lead acetate embedded filter paper after a 2-h incubation at 37 °C over a 96-well plate containing increasing amounts (between 0 to 500 µg) of mouse liver protein in each in a 150 µl reaction in PBS containing 10 mM L-cysteine and 1 mM pyridoxal 5’-phosphate hydrate. The dark circles are due to the formation of lead sulfide on the filter paper. C. Quantification of the image in (B) using the Integrated Density (IntDen) function of the software program ImageJ and subtracting out the background. Notice that this two hour exposure leads to a saturation plateau between the 300 to 400 µg reactions.

      9. After 45 min, take a look at the paper by looking from the underside of the plate. Do not take apart the ‘sandwich’.
      10. Once strong circles have formed, which usually takes 1 h to 2 h, remove the filter paper and place off to the side. A new filter paper can be placed onto the same plate and the ‘sandwich’ reassembled and placed back into the 37 °C incubator for a second or even third exposure. These secondary exposures will take less time to reach a saturation point, so care must be used to not incubate too long. Approximately 15 min to 30 min is all that is needed to obtain a signal for the secondary/tertiary incubations/exposures when using strong H2S producing tissues like the liver.
      11. To each finished paper, image with a digital camera or scanner.
        Note: Over time, the brown to black circles can become photobleached if exposed to light. Thus, if some samples may have hit a saturation point on the filter paper, the paper can be left out in ambient light for several hours to decrease the intensity of all the bands and bring the circles to a non-saturating point in order to more accurately analyze differences between strong and weak producing samples. However, it is recommended that a new, shorter incubation/exposure be performed (such as the 15-30 min secondary/tertiary exposure) to obtain an image with signals in the linear range.
      12. The image file can then be analyzed by densitometry of each circle via ImageJ using the IntDen (Integrated Density) as the readout. Make sure to also analyze the same size area of a portion of the filter paper where there was no sample to be used for background subtraction.
      13. The results will give a relative H2S production capacity when compared to a standard, control, or known amount of protein added. It is recommended to run all samples in duplicate or triplicate. Results can be presented as average H2S production capacity relative to a control group or standard, or can be plotted as the raw arbitrary IntDen units given from the ImageJ analysis subtracting the background reading.
      14. An example of the experimental setup, data output and analysis of the lead acetate/lead sulfide filter paper method and data analysis using a single liver homogenate but with various amounts of protein input (from 0 to 500 µg) is given in Figure 1.

  3. Agar-embedded lead acetate kinetic assay for H2S production capacity determination from liver extracts and data analysis
    This adaptation of the filter-paper embedded lead acetate endpoint assay described above was inspired by the use of a Nafion polymer embedded silver ion microplate for H2S production (Jarosz et al., 2013). This assay offers the potential for kinetic analysis, making it much easier to confirm that the data obtained are in the linear range and below the saturation point. Drawbacks compared to the filter paper method include increased set up time and decreased number of samples that can be run at once. The method is described below:
    1. Prepare the lead acetate agar plate
      1. Make 50 ml of 1% agarose gel (Agarose HS Molecular Grade from Denville) (see Recipes) in double deionized H2O in a glass Pyrex flask by microwaving until all of the agarose has dissolved.
      2. Let it cool slightly, but do not let it start solidifying.
      3. Add lead(II) acetate trihydrate to the agarose to a final concentration of 100 mM. Hand mix the flask until all of the lead(II) acetate trihydrate is dissolved.
        Note: Under normal conditions, lead acetate is not volatile under room temperature or in room temperature solutions with water. However, care should be taken, such as wearing a face mask, when putting it into the hot/warm agar. Additionally, proper disposal of lead containing compounds into appropriate chemical waste containers should be completed as per the regulations of each respective research institution.
      4. Pour the 1% agarose/100 mM lead acetate solution onto the top cover of a 96-well culture plate.
      5. Allow the solution to solidify and cool for 30 min in the dark on a flat surface.
        Note: The volume of the agarose/lead acetate solution added to the top cover is approximately 50 ml. However, due to differences in the shapes and styles of 96-well format plates and tops, this volume may need to be adjusted in order to cover the entire surface of the inside of the lid and have enough volume so that the well edges touch the gel in a way that they leave a slight impression into the gel without breaking the gel.
    2. After the gel has solidified, place it in the dark uncovered while the H2S production capacity reaction solutions and plate are prepared. It should be used immediately and not stored long term in order to prevent drying/cracking of the gel.
    3. Set up the H2S production assay reactions
      1. Prepare H2S production capacity reaction solution. This must be freshly done with each assay.
      2. Calculate the volume needed to run all of the samples. Each sample requires 150 µl of reaction solution.
      3. In an adequate amount of PBS to cover the needed volume of reaction mixture, add L-cysteine to a final concentration of 10 mM and pyridoxal 5’-phosphate hydrate to a final concentration of 1 mM from stocks as described previously.
      4. Keep at room temperature and use immediately.
      5. In each well of a 96-well plate, pipette 150 µl of the reaction mixture.
        Note: To prevent cross contamination of signal between wells, it is recommended to skip a space in all directions from each well used.
      6. In each well, pipette 0-300 µg of sample protein.
      7. Once all samples have been added, ensure there is no residual or spilled liquid on the surface of the 96-well plate by gently wiping with tissue paper.
      8. Directly place the solidified lead acetate agarose gel containing lid cover over the 96-well plate.
        Note: Make sure the lid is firmly in place to avoid cross contamination between wells, but do not press too hard so that the gel is broken. An illustrative description of the assay setup is given in Figure 2A.


        Figure 2. Agar-embedded lead acetate kinetic assay for H2S production capacity determination from liver extracts. A. Schematic overview of the assay. Liver is excised from the mouse, homogenized and lysed in passive lysis buffer followed by protein determination and normalization. In a 96-well plate, the reaction mixture is added first, followed by the liver lysate samples. The lead acetate gel-filled plate lid, which is prepared just prior to setting up the reaction, is placed on top of its compatible 96-well plate. Absorbance at 310 nm is at 0 min to establish a baseline prior to incubating the plate at 37 °C. Throughout the incubation, the absorbance at 310 nm is determined at various time points as lead sulfide gradually forms in the gel. At the completion of the assay, the lead sulfide dots in the gel-filled lid are imaged and analyzed. B. Photographic image of a 96-well plate with a lid cover containing a lead acetate embedded agarose gel after a 2-h incubation at 37 °C. Reaction wells contained various amounts of mouse liver protein (between 0 to 300 µg) as indicated in a 150 µl reaction in PBS containing 10 mM L-cysteine and 1 mM pyridoxal 5’-phosphate hydrate. The dark circles are due to the formation of lead sulfide in the agarose gel. The well highlighted by the red circle contained only the reaction mixture without any protein added and served for background subtraction in the Data analysis. C and D. Plots of the absorbance of light at 310 nm after subtracting background when the entire assembly (plate and lid) was analyzed in the plate reader as a function of time (C) or protein concentration (D) on the x axis. E. Photographic image of the lid cover containing a lead acetate embedded agarose gel after 5 h of incubation at 37 °C. The dark circles are due to the formation of lead sulfide in the agarose gel. The spot highlighted by the red circle corresponds to the well that did not have any protein added and served as background subtraction in the data analysis. F. Plot of the absorbance of light at 310 nm after subtracting background when using just the lid, after a 5 h incubation at 37 °C, was analyzed in the plate reader as a function of protein concentration on the x axis. G. Quantification of the image in (D) using the Integrated Density (IntDen) function of the software program ImageJ and subtracting out the background and plotting the average IntDen for each sample set as a function of protein concentration on the x axis.

      9. Take baseline absorbance at 310 nm reading on the UV-Vis spectrophotometer.
      10. Incubate the plate at 37 °C in the dark for 0-120 min. At various timepoints between 0-120 min, take the plate out and analyze the absorbance at 310 nm on the plate reader.
        1. Visual data obtained during the kinetic/timecourse portion of this assay that can be used for endpoint analysis if so desired is done by taking an image of the plate with the lid to visualize the formation of lead sulfide (Figure 2B). Quantitative and numerical data obtained during the kinetic/timecourse portion of this assay is accomplished by subtracting the background absorbance at 310 nm from the 0 µg protein reaction and plotting the absorbance data at 310 nm for the experimental samples. This can be done as a function of time on the x axis (Figure 2C) or as a function of protein concentration on the x axis (Figure 2D).
        2. Visual data obtained during the endpoint analysis of this assay is done by removing the lid and taking a digital image of the underside of the lid to capture the dark lead sulfide circles formed from H2S exposure to the lead acetate in the gel (Figure 2E). Quantitative and numerical data obtained during the endpoint analysis of this assay is accomplished by placing just the lid upside down into the plate reader and reading the absorbance at 310 nm, subtracting the background, and plotting the values on a graph as a function of protein concentration on the x axis (Figure 2F). Additionally, the digital image of the gel can be analyzed for densitometry of each circle via the software program ImageJ (similar to the procedure described for the lead acetate/lead sulfide filter paper method) using the IntDen (Integrated Density) as the readout. Make sure to also analyze the same size area of a portion of the gel where there was no protein sample added to be used for background subtraction. The values are plotted on a graph as a function of protein concentration on the x axis (Figure 2G).

Data analysis

  1. Analysis and interpretation of data obtained in the endpoint assays are as follows:
    1. Incubate the reaction with the overlaying lead acetate embedded filter paper or agar gel to a point in which detectable but non-saturating lead sulfide circles form.
    2. Remove the filter paper or agar gel and image with the digital camera or other imaging device, such as a digital image scanner or gel/blot imager.
    3. The image file can then be analyzed by densitometry of each circle via ImageJ using the IntDen (Integrated Density) as the readout. Make sure to also analyze the same size area of a portion of the filter paper or agar gel where there was no sample to be used for background subtraction.
    4. The results will give a relative H2S production capacity when compared to a standard, control, or known amount of protein added. It is recommended to run all individual samples in technical duplicates or triplicates and have at least three biological replicates for each condition.
    5. Results can be presented as average H2S production capacity relative to a control group or standard, or can be plotted as the average raw arbitrary IntDen units given from the ImageJ analysis subtracting the background reading.
  2. Analysis and interpretation of data obtained in the kinetic assays are as follows:
    1. Take a baseline absorbance at 310 nm reading on the UV-Vis spectrophotometer.
    2. Incubate the plate at 37 °C for 0-120 min. At various timepoints between 0-120 min, take the plate out and analyze the absorbance at 310 nm on the plate reader.
    3. Quantitative and numerical data obtained during the kinetic/timecourse portion of this assay is accomplished by subtracting the background absorbance at 310 nm from the 0 µg protein reaction and plotting the absorbance data at 310 nm for the experimental samples. This can be done as a function of time on the x axis or as a function of protein concentration on the x axis.
    4. The results will give a relative H2S production capacity when compared to a standard, control, or known amount of protein added at various timepoints. It is recommended to run all individual samples in technical duplicates or triplicates and have at least three biological replicates for each condition.
    5. Results can be presented as average absorbance at 310 nm for each experimental condition as a function of a specific time or calculated using the area under the curve for all time points.
  3. With all three of these tests, standard statistical analysis using Student’s t-test can be used when comparing between two groups or an ANOVA with an appropriate multiple comparisons test can be used when comparing values from multiple experimental groups.

Notes

  1. Although we focus on mammalian liver in this protocol, most of the materials, reagents, equipment and procedures are applicable to other tissue types and organisms. Concentrations of protein, sample volume, L-cysteine concentration, pyridoxal 5’-phosphate hydrate concentration, and incubation times may need to be adjusted dependent on what tissue types and/or organisms are used. We have observed that weaker H2S producing tissues and organisms need longer incubation times (up to 8 h or even overnight), increased protein amounts loaded (up to 1 mg), increased L-cysteine concentrations (up to 100 mM), and/or increased pyridoxal 5’-phosphate hydrate concentrations (up to 10 mM) in order to achieve suitable and quantifiable lead sulfide signals. Additionally, muscle and connective tissue require stronger forms of homogenization than liver, such as the use of bead/bullet homogenization or mechanical knife homogenization. Otherwise, the majority of steps taken in this protocol are the same for both strong and weak H2S producing mammalian tissues. However, tissues/samples from non-mammals, invertebrates, and yeast may need additional and/or alternative steps to obtain optimum results. For example, homogenization of worms (C. elegans) in passive lysis buffer requires more mechanical force via a sonicator or bead/bullet homogenization compared to what is required for liver homogenization and the use of the insoluble/cuticle portion of the worm homogenate can be used in the H2S assay. A recent publication showing a successful C. elegans H2S production capacity assay using the lead acetate to lead sulfide filter paper method can be found here: (Wei and Kenyon, 2016). For fruit flies, whole fly homogenates (including the soluble and insoluble portions) can be used for the H2S assay and normalization is possible by adding the same number of flies (20 to 25 is a suitable number) into the reaction. For yeast, the lead acetate paper can simply be placed on the underside of the plastic/cork/rubber stopper at the top of the culture flask to capture the H2S produced from live yeast cultures.
  2. Most of the materials, reagents and equipment listed are given with a specific brand and catalog number. This is not meant to be seen as a specific endorsement of that brand and/or product. Many of these can be substituted with other brands and/or catalog numbers that the investigator sees fit to use or with materials that are currently present in the investigator’s lab. Additionally, as time progresses, some of these catalog numbers for certain vendors may change or products may not become available from that vendor, so substitution may be necessary. While we have had success with the proprietary passive lysis buffer (Promega), other non-ionic lysis buffers of known composition could also be used. The lead acetate should be kept away from light and liquid solutions should be made fresh with each use. Gloves and proper personal protective equipment such as safety glasses and lab coat should be worn to protect against contact with the lead and exposure to potential biohazards when dealing with cells and tissues. Proper disposal of lead and cellular and tissue biohazards should be followed as determined by the respective intuition.
  3. The method of tissue homogenization and lysis was chosen due to its ease of use and ability to maintain proteins in the natural state and preserve enzymatic activity of H2S producing enzymes. However, other methods, such as the use of bead beaters or rotor-stators, could also potentially be used and may be more appropriate for hard-to-homogenize tissues such as muscle and connective tissue.
  4. To prevent protein degradation and denaturation and to maintain proper enzymatic function, harvest of the liver from mice should be done as soon as possibly post-euthanasia, and all homogenization steps and steps leading up to the enzymatic assay should be done with the protein homogenates/lysates on ice or kept cold (~4 °C to 10 °C).
  5. Ideal incubation times are always approximate and depend on the amount of protein added as well as the source of the protein, and should stop prior to the saturation point of the lead sulfide spot for the strongest producing sample in that group.
  6. The amount, in µg, of protein added to each of the reactions is up to the investigator to decide as long as the protein is the limiting factor and not the substrate or co-factor. The more protein added, the quicker the assay will run. However, if too much protein is added, it may cause for the substrate (L-cysteine) or the co-factor (pyridoxal 5’-phosphate hydrate) to become limiting factors, thus compromising the assay and giving false results. In our hands, 100 µg of protein lysate added to the reaction tends to give reliable results.
  7. L-cysteine is the preferred substrate or ‘fuel’ (Singh et al., 2009) and pyridoxal 5’-phosphate hydrate (PLP) the co-factor (Zhu et al., 2008) for CGL-dependent generation of H2S. L-cysteine working stocks in PBS should be made fresh with each assay. Working stocks of 10 mM PLP in PBS can be prepared ahead of time, aliquoted, and saved at -20 °C for at least several months.
  8. There are multiple potential substrates for H2S generation by CGL and CBS that could in principle be added used to monitor H2S production capacity. While L-Cys is the preferred substrate for CGL-dependent H2S production, CBS-dependent H2S production condensation of L-Cys together with L-homocysteine is 50x more efficient than L-Cys alone (Chen et al., 2004). Enzyme inhibitors can be further used to distinguish the enzymatic source of H2S. For example, 3 mM propargylglycine, a suicide inhibitor specifically of CGL, can be added prior to the addition of substrate in order to determine the specific contribution of CGL to H2S generation (Kabil et al., 2011).
  9. Even though the generic cofactor term for what cystathionine beta synthase and cystathionine gamma lyase utilize for their enzymatic activities and production of H2S is called Vitamin B6, it should be noted that for these ex vivo/in vitro H2S assays that only the bioactive form of Vitamin B6 (pyridoxal 5’-phosphate, aka PLP) should be used and not the more common form of Vitamin B6 (pyridoxine).
  10. A comparison between the two quantification methods (filter paper-based endpoint assay with image-based density quantification vs. agar-based lead acetate method with kinetic absorbance) revealed saturation with high protein concentrations in the endpoint assay but not with the kinetic assay in the given time frame. This could be due to the increased surface for interaction between H2S and lead acetate in the 3-dimensional agar overlay vs. the 2-dimensional filter paper. Additionally, the optical tools for quantification via the plate reader/absorbance method (spectrophotometer and Gen5 analysis software) are more sophisticated than simply analyzing a flat digital image for density. Thus, more information can be extracted from the plate reader/absorbance method than the image/density method at higher protein input levels. However, it seems useful to at least document and analyze the image of the lead sulfide produced on the gel for archival purposes.
  11. The sensitivity/ limit of detection of the filter paper method (Figure 1) is a function of both protein concentration and incubation/exposure time. Theoretically, if the incubation time is extended (on the order of 4 to 24 h), even a small amount of protein from the liver or other strong H2S producing tissue should give a detectable signal. However, using short incubation/exposure times as described here, we found that even 50 µg of protein gives an adequate signal in the filter paper method. The kinetic agar-based method should theoretically have improved sensitivity over the filter paper method, and here we were able to detect H2S production from 37.5 µg of protein at the 60, 90, and 120 min time points.

Recipes

  1. 1x passive lysis buffer
    8 ml deionized H2O
    2 ml 5x passive lysis buffer (Promega) (store at -20 °C)
    Make up fresh for each assay and keep on ice during its use
    Discard unused 1x passive lysis buffer
  2. 20 mM lead(II) acetate trihydrate
    100 ml deionized H2O
    759 mg lead(II) acetate trihydrate (Sigma-Aldrich)
    Make up fresh at room temperature for each batch of lead acetate filter papers produced
  3. H2S reaction mixture (10 mM L-cysteine, 1 mM pyridoxal 5’-phosphate hydrate, in PBS)
    10 ml phosphate buffered saline (aka PBS), without calcium and without magnesium, pH 7.4 (Fisher Scientific)
    11.9 mM phosphates
    137 mM sodium chloride
    2.7 mM potassium chloride
    12.1 mg L-cysteine (Sigma-Aldrich)
    2.5 mg pyridoxal 5’-phosphate hydrate (Sigma-Aldrich)
    Mix all reagents in a 15 ml centrifuge tube using a vortex mixer
    Notes:
    1. Not all of the pyridoxal 5’-phosphate hydrate (PLP) will go into solution. After you have mixed for 1-2 min, briefly spin down in a centrifuge to pellet the undissolved PLP and save the supernatant for use.
    2. This must be made fresh for each assay. Once made, use immediately and do not put on ice
  4. 1% agarose gel with 100 mM lead(II) acetate trihydrate
    1. 50 ml double deionized H2O
      500 mg HS molecular biology grade agarose (Denville)
      Mix in a large flask and microwave until all of the agar is dissolved
      Let it cool slightly, but do not let it start solidifying
    2. Add 1.9 g lead(II) acetate trihydrate (Sigma-Aldrich) to the agarose
    3. Hand mix the flask until all of the lead(II) acetate trihydrate is dissolved. Make sure to do this quickly before the agarose solidifies
    4. Pour the 1% agarose/100 mM lead acetate solution onto the top cover of a 96-well culture plate and let it cool and solidify in the dark on a flat surface
      Note: This must be done fresh just prior to running the assay.

Acknowledgments

The authors thank Marissa Granitto for testing of the protocols and providing critical feedback on the manuscript. This work was supported by grants and funds provided by DK090629 and AG036712 (JRM), and AG050777 (CH). The authors declare that there are no conflicts of interests or competing interests related to the design and implementation of this protocol.

References

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  2. Chen, X., Jhee, K. H., and Kruger, W. D. (2004). Production of the neuromodulator H2S by cystathionine β-synthase via the condensation of cysteine and homocysteine. J Biol Chem 279: 52082-52086.
  3. Dickhout, J. G., Carlisle, R. E., Jerome, D. E., Mohammed-Ali, Z., Jiang, H., Yang, G., Mani, S., Garg, S. K., Banerjee, R., Kaufman, R. J., Maclean, K. N., Wang, R. and Austin, R. C. (2012). Integrated stress response modulates cellular redox state via induction of cystathionine γ-lyase: cross-talk between integrated stress response and thiol metabolism. J Biol Chem 287(10): 7603-7614.
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  5. Hine, C., Harputlugil, E., Zhang, Y., Ruckenstuhl, C., Lee, B. C., Brace, L., Longchamp, A., Trevino-Villarreal, J. H., Mejia, P., Ozaki, C. K., Wang, R., Gladyshev, V. N., Madeo, F., Mair, W. B. and Mitchell, J. R. (2015). Endogenous hydrogen sulfide production is essential for dietary restriction benefits. Cell 160(1-2): 132-144.
  6. Ikeda, M., Ishima, Y., Shibata, A., Chuang, V. T. G., Sawa, T., Ihara, H., Watanabe, H., Xian, M., Ouchi, Y., Shimizu, T., Ando, H., Ukawa, M., Ishida, T., Akaike, T., Otagiri, M. and Maruyama, T. (2017). Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach. Anal Chim Acta 969: 18-25.
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  8. Kabil, O., Vitvitsky, V., Xie, P. and Banerjee, R. (2011). The quantitative significance of the transsulfuration enzymes for H2S production in murine tissues. Antioxid Redox Signal 15(2): 363-372.
  9. Kuester, E. and Williams, S. T. (1964). Production of hydrogen sulfide by Streptomycetes and methods for its detection. Appl Microbiol 12, 46-52.
  10. McBride, R. S. and Edwards, J. D. (1914). Lead acetate test for hydrogen sulphide in gas. United States Department of Commerce 41: 4-46.
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简介

通过各种酶法在细胞和组织中产生硫化氢(H 2 S)。 H 2 S是许多生物过程中重要的信号分子,内源性H 2 S生产的缺陷与心血管和其他健康并发症有关。稳态H 2 S水平的定量是由于气体的挥发性和专门设备的需要而具有挑战性的。然而,器官或组织提取物在优化的反应条件下产生H 2 S的能力可以通过许多灵敏度,特异性和通过能力变化的现有测定来测量。我们开发了用于从生物组织定量检测H 2 S生产能力的快速,廉价,特异性和相对高通量的方法。释放到生物样品上方的头部空间中的H 2 S 2与乙酸铅反应形成硫化铅,其使用平板阅读器或作为终点测定法连续测量。
【背景】通过哺乳动物(CGL,CBS,3-MST)中的至少三种不同的酶以一定范围的组织和细胞类型分布内源性地产生硫化氢(H 2 S)。 H 2 S作为与代谢相关的广泛生物学功能的气体发射器和效应分子(Wang,2012)起作用(Módiset al。,2013),应力抵抗(Hine等人,2015)和氧化还原生物学(Dickhout et al。,2012)。减少的H 2 S与心血管问题有关,包括啮齿动物中的高血压(Yang等人,2008)和人心脏肥大(Polhemus等人, ,2014)。增加的H 2 S也可引起病理学,例如啮齿动物胰腺炎(Bhatia et al。,2005)。因此,从生物来源准确和定量地检测H 2 S可以有助于更好地了解其生物效应以及其作为临床生物标志物的潜在用途。
已经广泛地回顾了测量生物样品中存在的H 2 O 2绝对浓度的技术及其优缺点(Olson,2012; Wang,2012; Hartle and Pluth,2016; Takano 等,,2016)。例如,可以使用高度敏感的顶空GC-MS在生物样品(包括离体血清或组织匀浆)中测量游离和硫化结合的H 2 S池,选择性,但需要昂贵的设备。然而,由于H 2 S的挥发性,其与其他生物大分子的相互作用及其分解成不同的含硫化合物,定量检测稳态游离H 2体内的S水平仍然具有挑战性(Olson,2009)。
另一种方法是测量组织匀浆或提取物在含有最佳水平的底物和辅因子的反应混合物中产生H 2 S的能力,从而允许H 2 sub> S检测方法具体但不太敏感。一个例子是其中溶液中的H 2 S被乙酸铅捕获以形成硫化铅的亚甲基蓝法,其可以在标准分光光度计(Stipanuk和Beck, 1982; Ikeda等人,2017)。每种方法必须考虑的利弊是基于所要求的问题,正在研究的生物系统和组织,敏感性,选择性或速度的相对需求以及调查员的成本和资源。
这里,我们描述了一种便宜,快速和适中的高通量方法,用于测量相对少量生物材料的提取物中的H 2 S生产能力。该方法基于存在于生物样品上方的顶部空间中的H 2 O 3与乙酸铅形成黑色沉淀硫化铅的反应,这是在过去100年中用于检测H 2 S和H 2 S产生细菌(McBride和Edwards,1914; Kuester和Williams,1964; Zhang和Weiner,2014)。以前,我们使用这种方法来检测各种生物样品(包括酵母,蠕虫,苍蝇和包括肝脏的啮齿动物组织/器官)在内的H 2 O 3 S生产能力的变化,作为饮食或遗传背景的函数(Hine等人,2015; Mitchell等人,2016; Nikonorova等人,2017)。在这里,我们提出了一种通过(B)使用Whatman纸包埋的醋酸铅的终点测定来测量哺乳动物肝脏中2 H 2 S生产能力的优化方法,或(C)使用琼脂的动力学测定内置醋酸铅。由于肝脏是哺乳动物系统中通过酶胱硫醚γ裂解酶(CGL)(Kabil等人,2011))的强力生产者H 2 N,所以我们认为这是是研究人员理解并自信地开发本协议的自身研究问题的良好起点。此外,该过程可以容易地适应于其他生物样品和生物体,尽管该方法可能需要由研究者优化以获得合适的结果。

关键字:硫化氢生成能力, H2S, 终点测定, 连续测定, 肝脏, 醋酸铅, 硫化铅

材料和试剂

  1. 培养皿
  2. 1.5毫升无RNase的一次性颗粒杵和1.5毫升管(Fisher Scientific,目录号:12-141-368)
  3. 一次性剃刀刀片
  4. 8条井格式管(Denville Scientific)
  5. 硬/硬塑料解剖平台/板
  6. 塑料包装
  7. 滤纸(703 Style Whatman)
  8. 带盖的96孔板(Corning,目录号:3370)
  9. 手套和适当的个人防护装备
  10. 15 ml离心管
  11. 快速冷冻的老鼠肝脏
  12. 去离子水
  13. 磷酸盐缓冲盐水(PBS),pH 7.4(Fisher Scientific,目录号:BP24384)
  14. 液氮

  15. 干冰
  16. 5x被动裂解缓冲液(Promega,目录号:E1941)
  17. BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23227)
  18. L-半胱氨酸(Sigma-Aldrich,目录号:C7352)
  19. 吡哆醛5'-磷酸盐水合物(Sigma-Aldrich,目录号:P9255)
  20. 乙酸铅(II)三水合物(Sigma-Aldrich,目录号:316512)
  21. 琼脂糖(HS分子生物学级)(Denville Scientific,目录号:CA3510-8,或使用相似的)
    注意:本产品已停产。
  22. 1x被动裂解缓冲液(见配方)
  23. 20mM醋酸铅(II)三水合物(参见食谱)
  24. H 2 S反应混合物(参见食谱)
  25. 1%琼脂糖凝胶,100mM醋酸铅(II)三水合物(参见食谱)

设备

  1. 将液氮烧瓶(Thermo flask 2122)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2122)
  2. 镊子
  3. 比例(OHAUS,目录号:EP214C)
  4. 移液器和提示(单次使用和多通道移液2μl至5 ml)
  5. -80°C冰箱
  6. 电动磨碎机(Fisher Scientific,目录号:12-1413-61)
  7. 37°C水浴
  8. 微型离心机(VWR,型号:Galaxy 16DH)
  9. UV-Vis读卡器(BioTek Instruments,型号:Synergy 2)
  10. 大玻璃Pyrex烤盘(> 100毫升)
  11. 玻璃烧瓶(> 100 ml容量)
  12. 真空烘箱(VWR,目录号:89508-424)
  13. 孵化器(VWR,型号:1500E)
  14. 数码相机(柯达,型号:柯达EASYSHARE C182)
  15. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2,目录号:SI-0236)
  16. 电脑(惠普Pavilion dv6和联想IdeaPad)
  17. 热块立方体(9.5 x 7.5 x 5厘米)或其他重物,大小尺寸为

软件

  1. GraphPad Prism 7
  2. Microsoft Excel
  3. ImageJ
  4. Gen5

程序

注意:该协议包括通过终点测定(方法B)或动力学测定(方法C)测量H 2 S生产能力的两种相关技术。两种技术都使用特定的化学方法,其中乙酸铅(白色)在暴露于H 2 S时被转化成硫化铅(棕色至黑色)。这些技术的检测方法不同,(方法B)利用端点密度测定来定量滤纸上的硫化铅点,而(程序C)利用310nm波长的光的动能吸收以及终点光密度测定琼脂凝胶上的硫化铅斑点。首先介绍共享的常见过程和技术(过程A),然后再分为两个区别的细节。

  1. 共享程序
    1. 组织样品
      1. 根据您的机构动物福利委员会批准的动物使用方案详述的程序,在安乐死后消除小鼠肝脏组织。
        1. 在安乐死和收获之前,在冰上的陪替氏培养皿中制备10ml PBS,以用作清除从切除的肝组织中的表面血液/碎片的洗涤液。另外,用液氮填充液氮烧瓶
        2. 安乐死后,迅速取出肝左大叶,在PBS中冲洗,放入1.5ml离心管中,并在液氮中快速冷冻。
        3. 将样品放在液氮中,直到所有肝脏都被收获
      2. 对于以前收获的肝脏样品,储存在-80°C直到准备好均匀化
    2. 使老鼠肝脏匀浆
      1. 用1.5ml离心管中的肝脏样品,从液氮或-80°C冰箱中取出,放在干冰上。
      2. 使用无菌或干净的一次性剃刀和镊子,快速从管中取出冷冻的肝脏,切下放在干冰上的硬塑料或其他解剖平台/片材上的片段,并将其称重至约100mg。
        注意:这种重量不需要准确,只需足够接近,以便样本之间的肝脏样本与体积的裂解缓冲液的重量相对恒定。
      3. 在仍然冷冻的同时,将约100毫克的肝组织置于新的1.5毫升离心管中,预冷在干冰上。
      4. 从5x被动裂解缓冲液(Promega)与去离子H 2 O组成一个新鲜的1x溶液的被动溶解缓冲液(参见食谱)。
        注意:1x被动裂解缓冲液应保存在常规冰上,并通过每次测定进行新鲜处理。 5x被动裂解缓冲液在不使用时应始终保存在-20°C。不幸的是,食谱是专有的。
      5. 一次一个1.5 ml管,每个近似100 mg样品中加入250μl1x被动裂解缓冲液。
      6. 立即用带有Pellet Pestle(Fisherbrand)的电动组织研磨机(Fisherbrand)开始均质,直到样品完全均匀。这通常需要30秒至1分钟才能执行。
      7. 从管中取出研磨机和研杵,固定盖子,并将均匀的样品和1.5 ml试管放入液氮中闪光冷冻。
      8. 继续均匀化所有样品,将其保存在液氮中
      9. 一旦所有样品均匀化,同时从液氮中除去所有样品,并在室温下解冻约5-10分钟,或在37℃水浴中约1-5分钟。
        注意:注意使用适合冷冻/解冻的液体N 2的管子,以避免样品损失和爆炸管的潜在伤害。
      10. 重复冻融另外两次,确保在液体N 2中至少1分钟的冷冻,并保持与上述相同的解冻时间。
      11. 将微量离心机的管子在4℃下以5,000×g的速度旋转5分钟。
      12. 取出并将上清液(〜200μl)保存在8条样品管形式中,如果立即进入下一步骤(蛋白质浓度测定),置于冰上。如果没有,则将管冷冻并置于-80°C冷冻箱中,直到准备进行蛋白质浓度测定。
    3. 蛋白质浓度测定和蛋白质浓度正常化
      1. 在新鲜的8管井管中,放置198μl去离子水和2μl肝匀浆上清液,进行1:100稀释。
      2. 按照Pierce BCA蛋白测定试剂盒的指示,准备用于蛋白质浓度测定的试剂盒中所含的工作溶液和标准BSA溶液(0μg/μl至2μg/μl)。
      3. 在每个需要的孔中移取200μl测定溶液,5μl标准品或1:100稀释样品。
      4. 盖上塑料包装,并在室温下在黑暗中孵育30分钟。
      5. 打开平板并将其放在UV-Vis分光光度计上。
        注意:我们使用运行Gen5软件的Synergy 2多模板阅读器,但可以使用其他读卡器。
      6. 测量562 nm光线的吸光度
      7. 根据从每个标准562 nm处的吸光度获得的标准曲线方程,确定每个样品的蛋白质浓度。
      8. 在新的8条试管中,使用1x被动裂解缓冲液将每个样品的蛋白质浓度归一化至20μg/μl。将最终体积提高至100μl。
        注意:如果不能使用20μg/μl,则将每个样品稀释至10μg/μl或其他合适浓度。

  2. 过滤纸 - 醋酸铅终点测定
    1. 准备醋酸铅滤纸
      1. 在大型玻璃Pyrex盘中,将100毫升20毫克醋酸铅(II)三水合物(见食谱)倒入去离子H 2 O中。
      2. 将703样式Whatman滤纸切成96孔板(8 x 12.5厘米)的大小尺寸尺寸。
      3. 将切割的滤纸放入乙酸铅(II)三水合物溶液中,并浸泡至少20分钟
      4. 通过将湿纸放入设置在110℃的真空炉中并将真空开启,将乙酸铅(II)三水合物嵌入并干燥到滤纸中。让这些纸放在真空烘箱中20-30分钟。
      5. 将成品纸在室温下储存在黑暗和干燥的区域,直至准备使用。虽然我们没有经验性地测试这些文件的保质期和稳定性,但它们在生产至少一个月后是有利的。
    2. 制备H 2 S生产能力反应溶液。
      注意:对于每个测定,这必须是新鲜的。
      1. 计算运行所有样品所需的体积。每个样品需要150μl反应溶液(参见食谱)
      2. 制备L-半胱氨酸(PBS中100mM)和吡哆醛5'-磷酸(PLP)(10mM在PBS中)的工作原料。半胱氨酸每次必须新鲜。对于PLP,升温至37℃,涡旋可能是完全溶解粉末所必需的。 PLP储存液可以在-20°C等分几个月储存。
      3. 在足量的PBS中以覆盖所需体积的反应混合物,加入L-半胱氨酸至终浓度为10mM,并且将吡哆醛5'-磷酸水合物加至最终约1mM浓度。
      4. 保持室温并立即使用。
    3. 设置测定
      1. 在96孔板的每个孔中,移液150μl反应混合物
      2. 在每个孔中,移液0-500μg样品蛋白。我们已经发现100μg(当样品稀释到20μg/μl时为5μl),在肝裂解物1小时内产生可检测和非饱和信号。其他组织可能需要更多的蛋白质
      3. 一旦添加了所有样品,请确保在96孔板的表面上没有残留或溢出的液体,用薄纸轻轻擦拭。
      4. 将干醋酸铅包埋滤纸直接放在96孔板上。
      5. 将类似尺寸的硬/硬塑料或其他非柔性盖板放在纸上,以保持其位置,并确保醋酸铅纸与板上孔的嘴唇直接接触。
      6. 将重型加热块立方体或其他类似尺寸(9.5 x 7.5 x 5厘米)的重物放置在硬质塑料上。
      7. 确保不要晃动或溅出任何液体,将“三明治”放入不振动/摇摆的37℃培养箱中。
      8. 随着时间的推移,在孔上的滤纸上将形成棕色至黑色的圆圈(参见图1的视觉示例)。这是由于乙酸铅与硫化氢的相互作用而形成的硫化铅

        图1.从肝脏提取物测定H 2 S生产能力的滤纸 - 醋酸铅终点测定。A.测定的示意图。将肝脏从小鼠切除,匀浆并在被动裂解缓冲液中裂解,随后蛋白质测定和归一化。在96孔板中,首先加入反应混合物,然后加入肝裂解液样品。然后将醋酸铅纸,刚性塑料和重量放在96孔板的顶部。孵育在37℃下进行1至2小时,然后对纸上的硫化铅点进行成像和分析。 B.在37℃下在含有增加量(0-500μg)的小鼠肝脏蛋白质的PBS中的150μl反应物中的PBS中孵育2小时后的乙酸铅包埋滤纸的照相图像含有10mM L-半胱氨酸和1mM吡哆醛5'-磷酸盐水合物。黑眼圈是由于在滤纸上形成硫化铅。 C.使用软件程序ImageJ的集成密度(IntDen)函数对(B)中的图像进行定量,并减去背景。请注意,这两小时的暴露导致300至400μg反应之间的饱和平衡。

      9. 45分钟后,从纸板的下面看,看纸。不要分开'三明治'。
      10. 一旦形成了强烈的圆圈,通常需要1小时至2小时,则取出滤纸并将其放在一边。新的滤纸可以放在同一块板上,“三明治”重新组装并放回37℃的孵化器中进行第二次或第三次曝光。这些二次曝光将花费更少的时间来达到饱和点,因此必须注意不要孵育太久。大约15分钟到30分钟是所有需要的,以获得二级/三级孵化/暴露的信号,当使用强烈的H 2/2 S生产组织像肝脏。
      11. 对于每张成品纸,使用数码相机或扫描仪的图像。
        注意:随着时间的推移,棕色至黑色的圆圈如果暴露在光线下,可以变得漂白。因此,如果一些样品可能已经达到滤纸上的饱和点,则纸张可以在环境光下放置数小时,以降低所有带的强度,并将圆圈带到非饱和点,以便更多准确分析强弱生产样本之间的差异。然而,建议进行新的较短的孵化/暴露(例如15-30分钟的二次/三次曝光)以获得具有线性范围信号的图像。
      12. 然后可以通过使用IntDen(集成密度)作为读数的ImageJ,通过每个圆的光密度分析图像文件。确保还要分析滤纸的一部分相同尺寸的区域,在那里没有样本用于背景扣除。
      13. 当与添加的蛋白质的标准,对照或已知量相比时,结果将产生相对的H 2 S 2 S生产能力。建议一式两份运行所有样品。结果可以相对于对照组或标准表示为平均H 2 N 2 S生产能力,或者可以绘制为从ImageJ分析给出的原始任意IntDen单位减去背景读数。
      14. 乙酸铅/硫化铅滤纸方法的实验设置,数据输出和分析以及使用单一肝匀浆,但具有不同数量的蛋白质输入(0至500μg)的数据分析示例如图1所示。 br />
  3. 琼脂包埋乙酸铅动力学测定从肝脏提取物和数据分析确定H 2 S生产能力
    通过使用Nafion聚合物嵌入的银离子微孔板用于H 2 S生产(Jarosz等人, >,2013)。该测定提供动力学分析的潜力,使得更容易确认获得的数据处于线性范围并低于饱和点。与过滤纸方法相比的缺点包括增加设置时间和减少可以一次运行的样品数量。该方法如下所述:
    1. 准备醋酸铅琼脂板
      1. 通过微波将玻璃Pyrex烧瓶中的250毫升1%琼脂糖凝胶(来自Denville的琼脂糖HS分子级)(参见食谱)在双去离子H 2 O 2中,直到所有琼脂糖溶解。 />
      2. 让它稍微凉一些,但不要让它开始凝固。
      3. 将乙酸铅(II)三水合物加入到琼脂糖中至终浓度为100mM。将手混合,直到所有的乙酸铅(II)三水合物溶解。
        注意:在正常条件下,醋酸铅在室温下或室温下不溶于水。然而,当把它放入热/温热的琼脂时,应该小心佩戴面罩。此外,应根据各研究机构的规定,妥善处理含铅化合物进入适当的化学废物容器。
      4. 将1%琼脂糖/ 100mM醋酸铅溶液倒入96孔培养板的顶盖上
      5. 让溶液在平坦的表面上在黑暗中固化和冷却30分钟。
        注意:加入顶盖的琼脂糖/乙酸铅溶液的体积约为50ml。然而,由于96孔格式板和顶部的形状和样式的差异,可能需要调节该体积以覆盖盖的内部的整个表面并具有足够的体积,使得边缘接触凝胶,使得它们在凝胶中留下轻微的印象,而不会破坏凝胶。
    2. 凝胶凝固后,将其置于黑色未被覆盖的同时制备H 2 S 2 S生产能力的反应溶液和板。应立即使用并长期不保存,以防止凝胶的干燥/破裂。
    3. 建立H 2 N 2 S生产测定反应
      1. 准备H 2 S生产能力反应解决方案。每次测定必须新鲜完成。
      2. 计算运行所有样品所需的体积。每个样品需要150μl的反应溶液
      3. 在足够量的PBS中以覆盖所需体积的反应混合物,加入L-半胱氨酸至最终浓度为10mM,并将吡哆醛5'-磷酸氢盐水合物从原液中加入到最终浓度为1mM。
      4. 保持室温并立即使用。
      5. 在96孔板的每个孔中,移液150μl反应混合物。
        注意:为了防止井之间信号的交叉污染,建议您从每个井中的所有方向跳过一个空格。
      6. 在每个孔中,移液0-300μg样品蛋白。
      7. 一旦添加了所有样品,请确保在96孔板的表面上没有残留或溢出的液体,用薄纸轻轻擦拭。
      8. 将含有盖子的凝固醋酸铅琼脂糖凝胶直接放在96孔板上。
        注意:确保盖子牢固就位,以避免井间交叉污染,但不要太紧,以致凝胶破裂。测定设置的说明性描述在图2A中给出。


        图2.琼脂包埋乙酸铅动力学测定从肝提取物测定H 2 S生产能力。 A.测定的示意图。将肝脏从小鼠切除,匀浆并在被动裂解缓冲液中裂解,随后蛋白质测定和归一化。在96孔板中,首先加入反应混合物,然后加入肝裂解液样品。在安装反应之前准备的醋酸铅凝胶板盖放置在其兼容的96孔板的顶部。在310nm处的吸光度为0分钟,以在37℃下孵育该板之前建立基线。在整个孵化过程中,在各种时间点测定310nm处的吸光度,因为硫化氢在凝胶中逐渐形成。在测定完成后,将凝胶填充的盖中的硫化铅点成像并分析。 B.在37℃下孵育2小时后,具有含有醋酸铅包埋琼脂糖凝胶的盖子的96孔板的照相图像。如在含有10mM L-半胱氨酸和1mM吡哆醛5'-磷酸盐水合物的PBS中的150μl反应中所示,反应孔含有各种量的小鼠肝脏蛋白(0至300μg)。黑眼圈是由于在琼脂糖凝胶中形成硫化铅。由红色圆圈突出的孔仅包含没有添加任何蛋白质的反应混合物,并在数据分析中用于背景减除。 C和D.当在板读数器中整个组装(板和盖)在x轴上作为时间(C)或蛋白质浓度(D)的函数分析后,在减去背景之后减去背景的光的吸光度的曲线图。 E.在37℃孵育5小时后,盖子盖的照相图像含有醋酸铅包埋的琼脂糖凝胶。黑眼圈是由于在琼脂糖凝胶中形成硫化铅。由红色圆圈突出的斑点对应于没有添加任何蛋白质的孔,并在数据分析中用作背景减除。 F.在平板阅读器中,在37℃下孵育5小时后,仅使用盖子减去背景之后减去背景的光的吸光度的图谱作为x轴上的蛋白质浓度的函数。 G.使用软件程序ImageJ的集成密度(IntDen)函数对(D)中的图像进行定量,并减去背景,并将每个样本集合的平均IntDen绘制为x轴上蛋白质浓度的函数。 />
      9. 在紫外 - 可见分光光度计上,以310 nm的基线吸光度读取。
      10. 将培养板在37℃下在黑暗中孵育0-120分钟。在0-120分钟之间的各个时间点,取出板,并在平板阅读器上分析310nm处的吸光度。
        1. 如果需要,可以用于终点分析的该测定的动力学/时间段部分期间获得的视觉数据通过用盖子拍摄该板的图像来观察硫化铅的形成(图2B)。在该测定的动力学/时间段部分获得的定量和数值数据通过从0μg蛋白反应减去310nm处的背景吸光度并绘制实验样品的310nm处的吸光度数据来完成。这可以作为x轴上时间的函数(图2C)或作为x轴上蛋白质浓度的函数完成(图2D)。
        2. 在该测定的终点分析期间获得的视觉数据通过移除盖子并取盖盖的下侧的数字图像来捕获由暴露于铅的H 2 S形成的黑色硫化铅圆形凝胶中的乙酸盐(图2E)。在该测定的终点分析期间获得的定量和数值数据通过将盖子倒置放置在平板读数器中并读取310nm处的吸光度,减去背景并绘制作为蛋白质浓度的函数的图表上的值来实现在x轴上(图2F)。此外,可以使用IntDen(集成密度)作为读数,通过软件程序ImageJ(类似于用于醋酸铅/硫化铅滤纸方法描述的程序),分析凝胶的数字图像的每个圆的光密度测量。还要分析一部分凝胶中没有添加蛋白质样品用于背景减除的大小相同的区域。这些值作为x轴上蛋白质浓度的函数绘制在图上(图2G)

数据分析

  1. 在终点测定中获得的数据的分析和解释如下:
    1. 将反应与含醋酸铅的滤纸或琼脂凝胶反应到形成可检测但不饱和的硫化铅环的位置。
    2. 使用数码相机或其他成像设备(如数字图像扫描仪或凝胶/印迹成像仪)取出滤纸或琼脂凝胶和图像。
    3. 然后可以通过使用IntDen(集成密度)作为读数的ImageJ,通过每个圆的光密度分析图像文件。确保还要分析滤纸或琼脂凝胶的一部分相同尺寸的区域,在那里没有样品可用于背景扣除。
    4. 当与添加的蛋白质的标准,对照或已知量相比时,结果将产生相对的H 2 S 2 S生产能力。建议以技术重复或一式三份运行所有个别样品,并对每种条件至少进行三次生物复制。
    5. 结果可以相对于对照组或标准表示为平均H 2 N 2 S生产能力,或者可以绘制为从ImageJ分析减去背景读数给出的平均原始任意IntDen单位。 >
  2. 在动力学测定中获得的数据的分析和解释如下:
    1. 在UV-Vis分光光度计上取310 nm读数的基线吸光度。
    2. 将板在37℃下孵育0-120分钟。在0-120分钟之间的各个时间点,取盘,并在平板阅读器上分析310nm处的吸光度。
    3. 在该测定的动力学/时间段部分获得的定量和数值数据通过从0μg蛋白反应减去310nm处的背景吸光度并绘制实验样品的310nm处的吸光度数据来完成。这可以作为x轴上时间的函数或作为x轴上蛋白质浓度的函数完成。
    4. 当与在各种时间点加入的标准,对照或已知量的蛋白质相比时,结果将产生相对的H 2 S 2 S生产能力。建议以技术重复或一式三份运行所有个别样品,并对每种条件至少进行三次生物复制。
    5. 结果可以作为每个实验条件的310nm平均吸光度,作为特定时间的函数或使用曲线下所有时间点的面积计算。
  3. 通过所有这三个测试,当比较两组之间时,可以使用Student's -test的标准统计分析,或者当比较来自多个实验组的值时,可以使用具有适当多重比较检验的方差分析。

笔记

  1. 尽管我们在这个方案中专注于哺乳动物肝脏,但大多数材料,试剂,设备和程序都适用于其他组织类型和生物体。蛋白质浓度,样品体积,L-半胱氨酸浓度,吡哆醛5'-磷酸盐水合物浓度和孵育时间可能需要根据使用的组织类型和/或生物体进行调整。我们已经观察到较弱的H 2 S生产组织和生物体需要更长的孵育时间(高达8小时甚至过夜),加载的蛋白质量(高达1mg),L-半胱氨酸浓度增加高达100mM),和/或增加的吡哆醛5'-磷酸盐水合物浓度(高达10mM),以便实现合适和可量化的硫化铅信号。此外,肌肉和结缔组织需要比肝脏更强的均匀形式,例如使用珠子/子弹匀浆或机械刀匀浆。否则,在本方案中采取的大多数步骤对于产生高和弱H 2 N 2 S的哺乳动物组织是相同的。然而,来自非哺乳动物,无脊椎动物和酵母的组织/样品可能需要额外的和/或替代的步骤来获得最佳结果。例如,被动裂解缓冲液中的蠕虫(电解质)的均质化需要通过超声波器或珠子/子弹均质化所需的机械力,与肝匀浆所需要的和不溶性/角质层的使用相比较蠕虫匀浆物的一部分可用于H 2 S检测。最近的出版物显示了成功的C。使用醋酸铅对硫化铅滤纸方法的线虫生产能力测定可以在这里找到:(Wei和Kenyon,2016)。对于果蝇,可以将全匀浆(包括可溶和不溶部分)用于H 2 S测定,并且可以通过加入相同数量的苍蝇进行归一化(20至25是合适的数目)进入反应。对于酵母,醋酸铅纸可以简单地放置在培养瓶顶部的塑料/软木/橡胶塞的下侧,以捕获由活酵母培养物产生的H 2 S。 />
  2. 列出的大部分材料,试剂和设备均具有特定的品牌和目录号。这并不意味着被视为该品牌和/或产品的具体认可。其中许多可以替代研究者认为适合使用的其他品牌和/或目录号码,或与研究人员实验室中当前存在的材料。此外,随着时间的推移,某些供应商的某些目录号可能会更改,或者产品可能无法从该供应商处获得,因此可能需要进行替换。虽然我们已经使用专有的被动裂解缓冲液(Promega)取得了成功,但也可以使用其他已知组合物的非离子裂解缓冲液。每次使用时,应将醋酸铅放在远离光源和液体溶液的新鲜处。应佩戴手套和适当的个人防护装备,例如安全眼镜和实验室外套,以防止与铅接触并在处理细胞和组织时暴露于潜在的生物危害。铅和细胞和组织生物危害的适当处置应遵循相应的直觉方法。
  3. 选择组织均质和裂解的方法是由于其易于使用和保持蛋白质处于天然状态的能力,并保持产生H 2 S的酶的酶活性。然而,也可以使用其他方法,例如使用珠粒搅拌器或转子定子,并且可以更适合于硬组织如肌肉和结缔组织的均匀化。
  4. 为了防止蛋白质降解和变性并保持适当的酶功能,应尽快在安乐死后立即从小鼠收获肝脏,并且所有均匀化步骤和导致酶法测定的步骤均应进行蛋白质匀浆/裂解物在冰上或保持冷(约4℃至10℃)。
  5. 理想的孵育时间总是近似值,并且取决于蛋白质的添加量以及蛋白质的来源,并且应该在该组中最强生产样品的硫化铅点的饱和点之前停止。
  6. 每个反应中加入的蛋白质的量为μg,由研究者决定,只要蛋白质是限制因子,而不是底物或辅因子。添加的蛋白质越多,测定法将越快运行。然而,如果添加太多的蛋白质,则可能导致底物(L-半胱氨酸)或辅因子(吡哆醛5'-磷酸盐水合物)成为限制因素,从而损害测定并给出假结果。在我们手中,添加到反应中的100μg蛋白质裂解物往往给出可靠的结果
  7. L-半胱氨酸是优选的底物或“燃料”(Singh等人,2009)和吡哆醛5'-磷酸盐水合物(PLP)的辅因子(Zhu et al。 em>,2008)用于CGL依赖性产生H 2 S。每个测定应使PBS中的L-半胱氨酸工作原料新鲜。在PBS中的10mM PLP的工作储备可以提前准备,等分,并在-20℃下保存至少几个月。
  8. 通过CGL和CBS可以原理上添加用于监测H 2 S 2 S生产能力的多个用于H 2 S 2代的潜在底物。虽然L-Cys是用于CGL依赖性H 2 S生产的优选底物,但L-Cys与L-高半胱氨酸的CBS依赖性H 2 S 2 S生产缩合为50x比单独的L-Cys更有效(Chen et al。,2004)。酶抑制剂可以进一步用于区分H 2 S酶的酶源。例如,可以在加入底物之前加入3mM炔丙基甘氨酸,特别是CGL的自杀抑制剂,以确定CGL对H 2 S代的特异性贡献(Kabil等al。,2011)。
  9. 即使胱硫醚β合酶和胱硫醚γ裂解酶用于其酶活性和H 2 S生成的通用辅因子称为维生素B6,应当注意,对于这些离体只能使用生物活性形式的维生素B6(吡哆醛5'-磷酸酯,又称PLP),而不是维生素B6(吡哆醇)的更常见形式的体外 )
  10. 两种定量方法(基于图像的浓度定量法与基于琼脂的醋酸铅方法与动力学吸光度的基于滤纸的终点测定)之间的比较显示在终点测定中具有高蛋白浓度的饱和度,但不是在动力学测定中给定的时间框架。这可能是由于三维琼脂覆盖物中的H 2 S和乙酸铅与二维滤纸之间的相互作用增加的表面。另外,用于通过读板器/吸光度法(分光光度计和Gen5分析软件)定量的光学工具比简单地分析平面数字图像的密度更复杂。因此,在较高的蛋白质输入水平下,可以从平板阅读器/吸光度方法中提取比图像/密度法更多的信息。然而,至少记录和分析凝胶上产生的硫化铅的图像档案目的似乎是有用的。
  11. 滤纸方法(图1)的检测灵敏度/极限是蛋白质浓度和孵育/曝光时间的函数。理论上,如果培养时间延长(约4至24小时),即使少量来自肝脏或其他强H 2 S 2 S生成组织的蛋白质应当产生可检测的信号。然而,如本文所述使用短的孵育/暴露时间,我们发现甚至50微克的蛋白质在滤纸方法中给出足够的信号。理论上,基于动力学琼脂的方法理论上具有比滤纸方法更高的灵敏度,这里我们能够在60,90和120分钟的时间内从37.5微克的蛋白质中检测到H 2 S的产生分。

食谱

  1. 1x被动裂解缓冲液
    8ml去离子H 2 O
    2 ml 5x被动裂解缓冲液(Promega)(-20°C储存)
    在每次测定中补充新鲜物质,并在其使用期间保持冰上
    丢弃未使用的1x被动裂解缓冲液
  2. 20 mM醋酸铅(II)三水合物
    100ml去离子H 2 O
    759mg乙酸铅(II)三水合物(Sigma-Aldrich)
    每个批量生产的醋酸铅滤纸可以在室温下新鲜化妆
  3. H 2 S反应混合物(10mM L-半胱氨酸,1mM吡哆醛5'-磷酸盐水合物,在PBS中)
    10 ml磷酸盐缓冲盐水(又名PBS),不含钙,无镁,pH 7.4(Fisher Scientific)
    11.9mM磷酸盐
    137毫克氯化钠 2.7 mM氯化钾
    12.1mg L-半胱氨酸(Sigma-Aldrich)
    2.5mg吡哆醛5'-磷酸盐水合物(Sigma-Aldrich)
    使用涡旋混合器
    将所有试剂混合在15 ml离心管中 注意:
    1. 不是所有的吡哆醛5'-磷酸盐水合物(PLP)都将溶解。混合1-2分钟后,在离心机中短暂旋转以沉淀未溶解的PLP,并保存上清液使用。
    2. 对于每个测定,这必须是新鲜的。一旦制造,立即使用,不要放在冰上
  4. 1%琼脂糖凝胶,100mM醋酸铅(II)三水合物
    1. 50ml双去离子H 2 O
      500 mg HS分子生物学级琼脂糖(Denville)
      在大烧瓶和微波炉中混合,直至所有琼脂溶解 让它稍微凉一些,但不要让它开始固化
    2. 加入1.9g醋酸铅(II)三水合物(Sigma-Aldrich)至琼脂糖
    3. 将手混合,直到所有的乙酸铅(II)三水合物溶解。确保在琼脂糖固化之前快速进行
    4. 将1%琼脂糖/ 100mM乙酸铅溶液倒入96孔培养板的顶盖上,使其在黑色中冷却并固化在平坦的表面上
      注意:在运行化验之前,必须做好新鲜事。

致谢

作者感谢Marissa Granitto测试了协议,并对稿件提供了重要的反馈意见。这项工作由DK090629和AG036712(JRM)和AG050777(CH)提供的赠款和资金支持。作者声明,与本议定书的设计和实施没有任何利益冲突或相互竞争的利益。

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引用:Hine, C. and Mitchell, J. R. (2017). Endpoint or Kinetic Measurement of Hydrogen Sulfide Production Capacity in Tissue Extracts. Bio-protocol 7(13): e2382. DOI: 10.21769/BioProtoc.2382.
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