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The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures
硫加氧还原酶活性测定: 高温条件下催化以元素硫为底物的反应   

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Journal of Bacteriology
Feb 2017

Abstract

The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S0), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not required. Usually, the SOR assay is performed in a milliliter scale in S0-containing Tris-buffer at high temperatures followed by colorimetric product quantification. In order to make the SOR assay more sensitive and better reproducible, several modifications were implemented compared to the original SOR assay (Kletzin, 1989). Here we present the modified SOR assay and the following quantification of the reaction products.

Keywords: Sulfur oxygenase reductase (硫加氧还原酶), Elemental sulfur (元素硫), Thermozyme (嗜热酶), Sulfur disproportionation (硫歧化), Thiosulfate (硫代硫酸盐), Sulfite (亚硫酸盐), Hydrogen sulfide (硫化氢)

Background

Sulfur oxygenase reductases (SOR) catalyze a dioxygen-dependent disproportionation of elemental sulfur with sulfite, thiosulfate and hydrogen sulfide as detectable products. The initially found SORs were derived from hyperthermophilic, sulfur-oxidizing Archaea and Bacteria. The reported temperature optima were between 65 °C and 85 °C and the pH optima between pH 5 and pH 7.4 (Emmel et al., 1986; Kletzin, 1989; Sun et al., 2003; Pelletier et al., 2008). Surprisingly, SORs derived from the mesophilic bacterium Halothiobacillus neapolitanus (Veith et al., 2012) and the alkalihalophilic Thioalkalivibrio paradoxus (Rühl et al., 2017) also had temperature optima of around 80 °C at pH 8.4 and pH 9.0, respectively. Most SORs can be produced in E. coli by heterologous gene expression and only the first descriptions of the enzyme were derived from proteins purified from their native sources (Emmel et al., 1986; Kletzin, 1989; Pelletier et al., 2008). Today, sor genes are found in approximately 35 different Bacteria and Archaea (Rühl et al., 2017).

Usually, SORs have a stoichiometry between 4:1 and 10:1 of oxidized (thiosulfate and sulfite) and reduced products (hydrogen sulfide). The oxidation and reduction reactions could not be separated by site-directed mutagenesis, although the product stoichiometries may vary (Veith et al., 2012). So far, the only exception is the Thioalkalivibrio paradoxus SOR with stoichiometries of 100-1,000:1, which makes the enzyme an oxygenase with almost no reductase activity (Rühl et al., 2017). The thiosulfate:sulfite ratio increases with temperature and pH (Kletzin, 1989; Veith et al., 2012; Rühl et al., 2017). Therefore, the bulk of the thiosulfate is most likely formed rapidly by a non-enzymatic reaction between sulfur and sulfite at pH values above 6 and temperatures exceeding 70 °C.

The original SOR activity assay (Kletzin, 1989; Urich et al., 2004) involves shaking of an aliquot of the enzyme in a buffer containing elemental sulfur at the given reaction temperature coupled to the colorimetric determination of the amount of products at different time points. Product determination is also possible using HPLC (e.g., Rethmeier et al., 1997). However the number of samples that can be processed per day is higher using the colorimetric assays because of the longer time required for each HPLC run. Specific SOR activities were in the range of 10 U/mg of protein for the Acidianus ambivalens SOR and 40 U/mg for the Halothiobacillus enzyme, both determined with the original enzyme assay.

After developing several modifications, the activity assay became more sensitive, resulting in higher product formation and lower amount of the required enzyme together with a reduced incubation time of the enzyme assay (Table 1). The original assay (Kletzin, 1989) was performed in stoppered glass vials with the enzyme being added at room temperature prior to incubation. All vials were transferred simultaneously to a shaking bath with preheated heating liquid. In order to stop the reaction, the vials were transferred sequentially into an ice bath. Urich et al. (2004) modified the procedure for the use of 1.5 ml plastic reaction vials and thermomixers but kept the order of the steps. In the modified procedure, the enzyme solution is added last to the 0 min time point immediately before transfer of the entire set of vials to an ice bath. Thus, all vials remain at the assay temperature for exactly the same time minimizing background effects. Here we describe the modified SOR activity assay and the quantification of its three reaction products.

Table 1. Incubation conditions for the original and modified SOR enzyme assays

Materials and Reagents

  1. Safe-lock reaction vials, 1.5 ml (SARSTEDT, catalog number: 72.690.001 )
  2. Micro cuvettes, polystyrene (SARSTEDT, catalog number: 67.742 )
  3. 37% [wt/vol] formaldehyde (Merck, catalog number: 104003 )
  4. Double deionized water (ddH2O; 18.2 MΩ at 25 °C)
  5. Tris(hydroxymethyl)aminomethane (Tris base) (Carl Roth, catalog number: 5429.2 )
  6. 32% [wt/vol] hydrochloric acid (HCl) (Carl Roth, catalog number: P074.4 )
  7. Tween 20 (Carl Roth, catalog number: 9127.2 )
  8. Sulfur flower (AppliChem, catalog number: A1687 )
    Note: This product has been discontinued; alternative product: Merck, catalog number: 107983 .
  9. Methylene blue (Merck, catalog number: 115943 )
  10. Sodium thiosulfate pentahydrate (Merck, catalog number: 106513 )
  11. Fuchsine (Merck, catalog number: 105226 )
  12. Sulfuric acid (Carl Roth, catalog number: 9316.2 )
  13. Sodium sulfite (Merck, catalog number: 106657 )
  14. Zinc acetate dihydrate (Merck, catalog number: 108802 )
  15. Acetic acid (Carl Roth, catalog number: 3738.5 )
  16. Dimethyl-4-phenylenediamine dihydrochloride (Merck, catalog number: 103067 )
  17. Iron-(III)-chloride hexahydrate (Carl Roth, catalog number: 7119.1 )
  18. 20% [wt/vol] ammonium sulfide solution (Sigma-Aldrich, catalog number: A1925 )
    Note: This product has been discontinued; alternative product: Merck, catalog number: 105442 .
  19. SOR assay buffer (see Recipes)
  20. Methylene blue solution (see Recipes)
  21. Sodium thiosulfate solution (1 mM) (see Recipes)
  22. Fuchsine solution (see Recipes)
  23. Sodium sulfite solution (1 mM) (see Recipes)
  24. Zinc acetate solution (see Recipes)
  25. Dimethyl-4-phenylenediamine dihydrochloride solution (see Recipes)
  26. Iron-(III)-chloride solution (see Recipes)
  27. Ammonium sulfide solution (1 mM) (see Recipes)

Equipment

  1. Polypropylene Griffin beaker 250 ml (Carl Roth, catalog number: 2875.1 )
  2. Pipettes L20, L200, L1000 (Abimed LABMATE Optima)
    Note: This product has been discontinued; alternative product: Gilson, catalog number: F167350 .
  3. Magnetic stirrer (e.g., IKAMAG RET; IKA)
  4. Thermomixer basic (shaking heat block; CellMedia, Elsteraue, Germany)
  5. pH meter (Xylem, WTW, model: inoLab pH 720 ) with SenTix 41 pH electrode (Xylem, WTW, catalog number: 103635 )
    Note: The product “Xylem, WTW, model: inoLab pH 720 ” has been discontinued.
  6. Centrifuge Heraeus Pico 17 Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 , catalog number: 75002410)
  7. Spectrophotometer (e.g., Beckmann Coulter, model: DU-640 )
  8. Ultrasound device (Emerson Electric, Branson, model: Sonifier 250 , catalog number: 100-132-868) equipped with a macrotip

Software

  1. Microsoft Excel 365 (Microsoft)

Procedure

  1. Preparation of the SOR assay buffer
    1. Prepare a 70 mM Tris solution in ddH2O in a plastic beaker and adjust the pH value with 32% [wt/vol] hydrochloric acid, usually to 7.2.
      Note: The actual pH depends on the pH optimum of the respective SOR.
    2. Add 2% [wt/vol] elemental sulfur (sulfur flower) and 0.1% [vol/vol] Tween 20 for a better dispersal of the sulfur. Stir mixture on a magnetic stirrer for approximately 1 min.
    3. Transfer the beaker to an ice bath and disperse the elemental sulfur by sonication for 5 min with a macrotip at level 10 and 100% duty cycle.
      Note: The ultrasonic treatment of the almost insoluble sulfur flower in the presence of detergent will result in a finely dispersed yellow sulfur suspension.

  2. SOR activity assay
    1. Stir the sonicated enzyme buffer on a magnetic stirrer and transfer 6 x 1 ml to 1.5 ml reaction tubes while continuing to stir.
    2. Preheat the reaction tubes with the enzyme buffer to the appropriate temperature for 5 min in a thermomixer with continuous shaking at 800 rpm.
      Note: The incubation temperature usually depends on the optimum of the respective SOR and is mostly in the range between 65 °C and 85 °C.
    3. Add 2 µg of enzyme to the first reaction tube and repeat this step every 30 sec for each consecutive tube (Figure 1).
      Notes:
      1. The first addition of your enzyme to the reaction buffer corresponds to the 150 sec time point, the last addition corresponds to the 0 sec value of the enzyme assay.
      2. The amount of enzyme can be raised up to 100 µg/ml or lowered to 0.5 µg/ml depending on the specific activity of the individual SOR preparation, the assay temperature or pH in order to provide a suitable amount of product for the colorimetric assays (see below).
    4. Immediately after the enzyme addition to the last reaction tube, transfer all reaction tubes to an ice/water bath to quench the reaction. Keep the tubes for approximately 2-3 min on ice.
    5. Transfer the reaction tubes to a microcentrifuge and sediment the elemental sulfur (13,000 x g, 1 min).
    6. Use the supernatant for the three colorimetric assays (Figures 1 and 2) to quantify the reaction products.
    7. As a negative control, perform the same incubation with enzyme reaction buffer by adding water instead of enzyme solution to the reaction vials and perform the colorimetric assays accordingly.


      Figure 1. Schematic overview for the SOR activity assay and the colorimetric reactions


      Figure 2. Representative calibrations curves for colorimetric assays. Polynomial (A) and linear (B) fits of thiosulfate calibration , polynomial (C) and linear (D) fits of sulfite calibration and linear fit of sulfide calibration (E); error bars from 3 replicates.

  3. Thiosulfate determination
    The quantification of thiosulfate is based on the discoloration of methylene blue (Pachmayr, 1960).
    1. Label one micro cuvette for each of the time points to be analyzed.
    2. Transfer 250 µl of the enzyme reaction mixture into an empty cuvette.
    3. Add 750 µl methylene blue solution (see Recipes).
    4. Incubate at room temperature for 30 min.
    5. Determine the absorption spectrophotometrically at 670 nm against ddH2O as reference.
    6. Prepare a standard curve based on a 1 mM freshly prepared sodium thiosulfate solution (see Recipes, see Figures 2A and 2B). Perform the colorimetric assay as described above and replace the reaction mixture with different volumes of the sodium thiosulfate solution (0, 1, 5, 10, 20, 50, 75 and 100 µl; these values correspond to the same amount of thiosulfate in nmol). Make up the volume to 250 µl with ddH2O.
      Note: Note that there is a decrease in absorbance with an increase of the amount of thiosulfate (or concentration).

  4. Sulfite determination
    The quantification of sulfite is based on the reaction of fuchsine with sulfite. The addition of formaldehyde leads to the formation of a stable purple compound (Pachmayr, 1960).
    1. Label one 1.5 ml reaction tube for each of the time points.
    2. Transfer 50 µl fuchsine solution (see Recipes) into each reaction tube and add 200 µl ddH2O.
    3. Add 250 µl of the enzyme reaction mixture and incubate for 5 min at room temperature.
    4. Pipet exactly 5 µl of 37% [wt/vol] formaldehyde into the inner lid of the reaction tube.
    5. Carefully close the reaction tube and briefly spin down the formaldehyde.
    6. Transfer the whole mixture to a cuvette using an L1000 pipette and incubate for 60 min at room temperature.
    7. Determine the absorption spectrophotometrically at 570 nm against ddH2O as reference.
    8. Prepare a standard curve based on a freshly prepared 1 mM sodium sulfite solution (see Recipes, see Figures 2C and 2D). Perform the colorimetric assay as described above and replace the reaction mixture with different volumes of the sodium sulfite solution as described above for the thiosulfate calibration curve (0, 1, 5, 10, 20, 50, 75 and 100 µl).

  5. Hydrogen sulfide determination
    The quantification of sulfide is based on the formation of methylene blue (King and Morris, 1967).
    1. Label one micro cuvette for each of the time points.
    2. Transfer 250 µl zinc acetate solution (see Recipes) into each of the cuvettes for fixation of the volatile hydrogen sulfide.
    3. Add 350 µl reaction mixture, 125 µl dimethyl-4-phenylenediamine dihydrochloride solution (see Recipes) and 50 µl iron-(III)-chloride solution (see Recipes) in the order provided.
    4. Incubate at room temperature for at least 12 h.
      Note: The incubation time can be extended up to 72 h if desired, e.g., over the weekend.
    5. Determine the absorption spectrophotometrically at 670 nm against ddH2O as reference.
    6. Prepare a standard curve based on a freshly prepared 1 mM ammonium sulfide solution (see Recipes, see Figure 2E). Perform the colorimetric assay as described above and replace the reaction mixture with different volumes of the ammonium sulfide solution as described above for the thiosulfate calibration curve (0, 1, 2.5, 5, 7.5, 10, 15 and 20 µl).

Data analysis

The data analysis was performed using Microsoft Excel. Specific enzymatic activities are determined from the range of the linear increase/decrease of the optical densities of the colorimetric assays (Figure 3). The corresponding amounts of the respective products are calculated and plotted against the time. The gradient corresponds to the product formation per minute and the amount of enzyme added to each reaction mixture (= enzyme activity). The specific SOR activities are defined as 1 µmol of sulfite plus thiosulfate (oxygenase activity) or 1 µmol of hydrogen sulfide (reductase activity) formed per minute and mg of protein (= 16.7 µkat/g).


Figure 3. Representative data of Acidianus ambivalens SOR enzyme reaction. The formation of thiosulfate (A), sulfite (B) and sulfide (C) at different time points were followed at 85 °C and pH 7.2 and with 2 µg of purified enzyme in 1 ml SOR assay buffer; the error bars are from triplicate measurements. Note the differences in the scaling of the y-axes.

Notes

  1. The volume of reaction mixture during colorimetric assays can be scaled down to 50 µl, if the coloration or discoloration is too strong for the linear range of the calibration curve.
  2. The amount of enzyme can be increased up to 100 µg/ml during the enzymatic assay to enhance product formation and sensitivity of the colorimetric assays, e.g., with low temperature measurements and/or high/low pH values.
  3. Sulfur flower can contain minor amounts of thiosulfate and/or form thiosulfate non-enzymatically during the pre-heating step of the assay at alkaline pH. The background levels in the colorimetric assay were 8-16 nmol/ml at pH 7.2 and 28-40 nmol/ml at pH 9.
  4. Ammonium sulfide and sodium sulfite solutions should be always prepared with freshly boiled water (microwave) in order to remove dissolved oxygen, which chemically oxidizes the sulfur compounds.

Recipes

Notes:

  1.  Use ddH2O for all solutions unless stated otherwise.
  2.  Sodium thiosulfate solution and especially the sodium sulfite and ammonium sulfide solutions are unsuitable for long-term storage and should be prepared freshly.
  3. All other solutions could be stored at room temperature for up to one month and even longer, if the calibration curves are done. Although the solutions are not especially light-sensitive, direct sunlight should be avoided.
  1. SOR assay buffer (250 ml)
    70 mM Tris (2.1 g) adjusting the pH value with 32% HCl
    0.1% [vol/vol] Tween 20 (250 µl)
    2% [wt/vol] elemental sulfur (sulfur flower; 5 g)
  2. Methylene blue solution (1 L)
    12 mg methylene blue dissolved in 5 N HCl
  3. Sodium thiosulfate solution (1 mM)
    Dissolve 0.248 g sodium thiosulfate pentahydrate in 10 ml ddH2O for a 100 mM solution
    Prepare a 1:100 dilution to give the final 1 mM thiosulfate solution (0.1 ml 100 mM sodium thiosulfate solution with 9.9 ml ddH2O)
  4. Fuchsine solution (100 ml)
    40 mg fuchsine dissolved in 87.5 ml ddH2O and 12.5 ml concentrated sulfuric acid
  5. Sodium sulfite solution (1 mM)
    Dissolve 0.126 g sodium sulfite in 10 ml freshly boiled (microwave) ddH2O for a 100 mM solution
    Prepare a 1:100 dilution with freshly boiled ddH2O to give the final 1 mM sulfite solution (0.1 ml 100 mM sodium sulfite solution with 9.9 ml ddH2O)
  6. Zinc acetate solution (250 ml)
    6.5 g zinc acetate dissolved in 249.75 ml ddH2O and 250 µl 100% [wt/vol] acetic acid
  7. Dimethyl-4-phenylenediamine dihydrochloride solution (100 ml)
    0.1 g dimethyl-4-phenylenediamine dihydrochloride dissolved in 5 M HCl
  8. Iron-(III)-chloride solution (50 ml)
    0.16 g iron-(III)-chloride dissolved in 0.6 M HCl
  9. Ammonium sulfide solution (1 mM)
    Add 3.4 µl 20% [wt/vol] ammonium sulfide solution to 10 ml freshly boiled ddH2O

Acknowledgments

Patrick Rühl was supported by a fellowship of the Carlo und Karin Giersch-Stiftung an der TU Darmstadt, Darmstadt, Germany and by a grant of the Deutsche Forschungsgemeinschaft to Arnulf Kletzin (Az Kl885-7/1). This protocol was adapted from Kletzin (1989). The original enzyme assay for sulfite formation from sulfur was developed by Emmel et al. (1986).

References

  1. Emmel, T., Sand, W., König, W. A. and Bock, E. (1986). Evidence for the existence of a sulfur oxygenase in Sulfolobus brierleyi. J Gen Microbiol 132: 3415-3420.
  2. King, T. E. and Morris, R. (1967). Determination of acid-labile sulfide and sulfhydryl groups. Meth Enzymol 10: 634-641.
  3. Kletzin, A. (1989). Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purification and properties of a sulfur oxygenase reductase from the facultatively anaerobic archaebacterium Desulfurolobus ambivalens. J Bacteriol 171(3): 1638-1643.
  4. Pachmayr, F. (1960). Vorkommen und Bestimmung von Schwefelverbindungen in Mineralwasser. Dissertation, Justus-Maximilian Universität, München.
  5. Pelletier, N., Leroy, G., Guiral, M., Giudici-Orticoni, M. T. and Aubert, C. (2008). First characterisation of the active oligomer form of sulfur oxygenase reductase from the bacterium Aquifex aeolicus. Extremophiles 12(2): 205-215.
  6. Rethmeier, J., Rabenstein, A., Langer, M., and Fischer, U. (1997). Detection of traces of oxidized and reduced sulfur compounds in small samples by combination of different high-performance liquid chromatography methods. J Chromatogr A 760(2): 295-302.
  7. Rühl, P., Pöll, U., Braun, J., Klingl, A. and Kletzin, A. (2017). A sulfur oxygenase from the haloalkaliphilic bacterium Thioalkalivibrio paradoxus with atypically low reductase activity. J Bacteriol 199(4).
  8. Sun, C. W., Chen, Z. W., He, Z. G., Zhou, P. J. and Liu, S. J. (2003). Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, Acidianus strain S5. Extremophiles 7(2): 131-134.
  9. Urich, T., Bandeiras, T. M., Leal, S. S., Rachel, R., Albrecht, T., Zimmermann, P., Scholz, C., Teixeira, M., Gomes, C. M. and Kletzin, A. (2004). The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron centre. Biochem J 381(Pt 1): 137-146.
  10. Veith, A., Botelho, H. M., Kindinger, F., Gomes, C. M. and Kletzin, A. (2012). The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme. J Bacteriol 194(3): 677-685.

简介

硫加氧酶还原酶(SOR)反应是元素硫的二氧依赖性歧化(S 0 ),在65℃至85℃之间的最佳温度下催化。 硫代硫酸盐和亚硫酸盐作为还原产物形成氧化产物以及硫化氢。 不需要外部辅助因素。 通常,SOR测定在高温下以含有S 0含有Tris缓冲液的毫升标度进行,随后进行比色产物定量。 为了使SOR测定更灵敏和更好的再现性,与原始SOR测定相比,实施了几个修改(Kletzin,1989)。 在这里,我们提供修饰的SOR测定和以下反应产物的定量。
【背景】硫氧化酶还原酶(SOR)催化与亚硫酸盐,硫代硫酸盐和硫化氢作为可检测产物的元素硫的二氧依赖性歧化。最初发现的SOR来自超嗜热,硫氧化古菌和细菌。报道的温度最佳值在65℃和85℃之间,pH值在pH5和pH7.4之间是最佳的(Emmel等人,1986; Kletzin,1989; Sun等人, ,2003; Pelletier等人,2008)。令人惊讶的是,源自嗜温细菌的新生儿烟草属(SOUTH)等(2012)的嗜碱性细菌和碱性嗜碱性硫代弧菌悖论的SORs(Rühlet al。还有在pH8.4和pH9.0下分别具有约80℃的温度最佳值。大多数SOR可以在E中生产。大肠杆菌通过异源基因表达,并且只有该酶的第一个描述源自从其天然来源纯化的蛋白质(Emmel等人,1986; Kletzin,1989; Pelletier et al。 et al。,2008)。今天,在大约35种不同的细菌和古细菌(Rühlet al。,2017)中发现了基因。
 通常,SOR的氧化(硫代硫酸盐和亚硫酸盐)和还原产物(硫化氢)的化学计量比为4:1和10:1。尽管产物化学计量可能不同,但是通过定点诱变不能分离氧化和还原反应(Veith等人,2012)。到目前为止,唯一的例外是化学计量为100-1,000:1的硫代阿维菌素悖论,这使酶几乎没有还原酶活性的加氧酶(Rühlet al。 ,2017)。硫代硫酸盐:亚硫酸盐比率随着温度和pH的增加而增加(Kletzin,1989; Veith等人,2012;Rühl等人,2017)。因此,大部分硫代硫酸盐很可能通过硫和亚硫酸盐之间的非酶反应快速形成,pH值高于6,温度超过70℃。
 原始的SOR活性测定(Kletzin,1989; Urich等人,2004)涉及在与比色测定相结合的给定反应温度下将酶的等分试样振荡在含有元素硫的缓冲液的产品数量在不同的时间点。使用HPLC(例如,Rethmeier等人,1997)也可以进行产物测定。然而,由于每个HPLC运行所需的时间较长,使用比色测定法,每天可处理的样品数量较高。特异性SOR活性在酸性酸对照组SOR中为10U / mg蛋白质的量级,对于硫杆菌属酶为40U / mg,均用原始酶测定法测定。
 在进行了几项修改后,活性测定变得更加敏感,导致更高的产物形成和更少量的所需酶以及酶测定的孵育时间减少(表1)。在封闭的玻璃小瓶中进行原始测定(Kletzin,1989),在温育之前,在室温下加入酶。将所有小瓶同时转移到具有预热的加热液体的摇动浴中。为了停止反应,将小瓶依次转移到冰浴中。 Urich等人(2004)修改了使用1.5ml塑料反应瓶和温热混合器的程序,但保持了步骤的顺序。在修改的程序中,将酶溶液在将整组小瓶转移到冰浴之前立即加入0分钟的时间点。因此,所有小瓶保持在测定温度下完全相同的时间使背景效应最小化。在这里我们描述修饰的SOR活性测定和其三个反应产物的定量。

表1.原始和修改的SOR酶测定的温育条件

关键字:硫加氧还原酶, 元素硫, 嗜热酶, 硫歧化, 硫代硫酸盐, 亚硫酸盐, 硫化氢

材料和试剂

  1. 安全锁反应瓶,1.5毫升(SARSTEDT,目录号:72.690.001)
  2. 微量比色皿,聚苯乙烯(SARSTEDT,目录号:67.742)
  3. 37%[wt / vol]甲醛(Merck,目录号:104003)
  4. 双去离子水(ddH 2 O; 25℃18.2MΩ)
  5. 三羟甲基氨基甲烷(Tris碱)(Carl Roth,目录号:5429.2)
  6. 32%[wt / vol]盐酸(HCl)(Carl Roth,目录号:P074.4)
  7. 吐温20(Carl Roth,目录号:9127.2)
  8. 硫磺花(AppliChem,目录号:A1687)
    注意:本产品已停产;替代产品:默克,目录号:107983。
  9. 亚甲基蓝(Merck,目录号:115943)
  10. 硫代硫酸钠五水合物(Merck,目录号:106513)
  11. Fuchsine(默克,目录号:105226)
  12. 硫酸(Carl Roth,目录号:9316.2)
  13. 亚硫酸钠(Merck,目录号:106657)
  14. 醋酸锌二水合物(Merck,目录号:108802)
  15. 乙酸(Carl Roth,目录号:3738.5)
  16. 二甲基-4-苯二胺二盐酸盐(Merck,目录号:103067)
  17. 氯化铁(III)六水合物(Carl Roth,目录号:7119.1)
  18. 20%[wt / vol]硫化铵溶液(Sigma-Aldrich,目录号:A1925)
    注意:本产品已停产;替代产品:默克,目录号:105442。
  19. SOR测定缓冲液(参见食谱)
  20. 亚甲基蓝溶液(见食谱)
  21. 硫代硫酸钠溶液(1 mM)(见食谱)
  22. 富士山解决方案(见食谱)
  23. 亚硫酸钠溶液(1 mM)(见食谱)
  24. 醋酸锌溶液(见配方)
  25. 二甲基-4-苯二胺二盐酸盐溶液(见配方)
  26. 氯化铁(III)溶液(参见食谱)
  27. 硫化铵溶液(1 mM)(见配方)

设备

  1. 聚丙烯格里芬烧杯250毫升(Carl Roth,目录号:2875.1)
  2. 移液器L20,L200,L1000(Abimed LABMATE Optima)
    注意:本产品已停产;替代产品:Gilson,目录号:F167350。
  3. 磁力搅拌器(例如,,IKAMAG RET; IKA)
  4. Thermomixer基础(震动热块; CellMedia,Elsteraue,德国)
  5. 使用SenTix 41 pH电极(Xylem,WTW,目录号:103635)的pH计(Xylem,WTW,型号:inoLab pH720)
    注意:产品“Xylem,WTW,型号:inoLab pH值为720”已停产。
  6. 离心机Heraeus Pico 17微量离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Pico TM,目录号:75002410) />
  7. 分光光度计(例如,Beckmann Coulter,型号:DU-640)
  8. 超声波装置(Emerson Electric,Branson,型号:Sonifier 250,目录号:100-132-868)配备了一个宏尖端

软件

  1. Microsoft Excel 365(Microsoft)

程序

  1. SOR测定缓冲液的制备
    1. 在塑料烧杯中制备ddH 2 O中的70mM Tris溶液,并用32%[wt / vol]盐酸调节pH值至7.2。
      注意:实际的pH值取决于相应SOR的pH最佳值。
    2. 添加2%[wt / vol]元素硫(硫花)和0.1%[vol / vol]吐温20以更好地分散硫。在磁力搅拌器上搅拌约1分钟
    3. 将烧杯转移到冰浴中,通过超声处理5分钟,将元素硫分散在10级和100%占空比的宏观提示。
      注意:在洗涤剂存在下对几乎不溶的硫磺花的超声处理将导致细分散的黄色硫悬浮液。

  2. SOR活性测定
    1. 将超声处理酶缓冲液搅拌在磁力搅拌器上,并继续搅拌6×1ml至1.5ml反应管。
    2. 将反应管用酶缓冲液预热至温热分离器中适当温度5分钟,连续以800rpm振荡。
      注意:培养温度通常取决于相应SOR的最佳值,并且大部分在65℃和85℃之间的范围内。
    3. 向第一个反应管中加入2μg酶,每隔30秒对每个连续管重复此步骤(图1)。
      注意:
      1. 首先将酶加入到反应缓冲液中对应于150秒的时间点,最后一次加入对应于酶测定的0秒值。
      2. 根据各个SOR制剂的比活性,测定温度或pH值,酶的量可以提高至100μg/ ml或降低至0.5μg/ ml,以提供合适量的产品用于比色测定(见下文)。
    4. 将酶加到最后一个反应管后立即将所有的反应管转移到冰/水浴中以淬灭反应。在冰上保持管约2-3分钟。
    5. 将反应管转移到微量离心机并沉淀元素硫(13,000 x g,1分钟)。
    6. 使用上清液进行三种比色测定(图1和图2)来量化反应产物。
    7. 作为阴性对照,通过向反应小瓶中加入水代替酶溶液,与酶反应缓冲液进行相同的温育,并相应地进行比色测定。



      图1. SOR活性测定和比色反应的示意图


      图2.比色测定的代表性校准曲线。 硫酸铵校准,硫酸盐校准和硫化物校准线性拟合(E)的硫酸铵校准,多项式(C)和线性(D)拟合的多项式(A)和线性(B)拟合。来自3个重复的错误栏。

  3. 硫代硫酸盐测定
    硫代硫酸盐的定量是基于亚甲蓝的变色(Pachmayr,1960)。
    1. 为每个要分析的时间点标记一个微量比色皿。
    2. 将250μl酶反应混合物转移到空的比色皿中
    3. 加入750μl亚甲基蓝溶液(参见食谱)。
    4. 在室温下孵育30分钟
    5. 通过分光光度法测定在670nm处的ddH 2 O作为参考。
    6. 制备基于1mM新鲜制备的硫代硫酸钠溶液的标准曲线(参见食谱,参见图2A和2B)。进行如上所述的比色测定,并用不同体积的硫代硫酸钠溶液(0,1,5,10,20,50,75和100μl)代替反应混合物;这些值对应于相同量的硫代硫酸盐(nmol)), 。用ddH 2 O补足体积至250μl。
      注意:注意,随着硫代硫酸盐(或浓度)的增加,吸光度降低。

  4. 亚硫酸盐测定
    亚硫酸盐的定量是基于紫苏碱与亚硫酸盐的反应。加入甲醛导致形成稳定的紫色化合物(Pachmayr,1960)。
    1. 为每个时间点标记一个1.5 ml反应管。
    2. 将50μl紫红色溶液(参见食谱)转移到每个反应管中并加入200μlddH 2 O。
    3. 加入250μl酶反应混合物,室温孵育5 min
    4. 将5μl37%[wt / vol]甲醛吸入反应管的内盖。
    5. 仔细关闭反应管,并短暂地旋下甲醛。
    6. 使用L1000移液管将整个混合物转移到比色皿,并在室温下孵育60分钟。
    7. 通过分光光度法测定在570nm处的ddH 2 O作为参考。
    8. 根据新制备的1mM亚硫酸钠溶液制备标准曲线(参见食谱,参见图2C和2D)。进行如上所述的比色测定,并用不同体积的亚硫酸钠溶液替代反应混合物,如上所述的硫代硫酸盐校准曲线(0,1,5,10,20,50,75和100μl)。 >
  5. 硫化氢测定
    硫化物的定量是基于亚甲蓝的形成(King and Morris,1967)。
    1. 为每个时间点标记一个微量比色杯。
    2. 将250μl醋酸锌溶液(参见食谱)转移到每个比色杯中,以固定挥发性硫化氢。
    3. 按照提供的顺序加入350μl反应混合物,125μl二甲基-4-苯二胺二盐酸盐溶液(参见食谱)和50μl氯化铁(III)盐酸盐溶液(参见食谱)。
    4. 在室温下孵育至少12小时。
      注意:如果需要,孵育时间可以延长至72小时,例如在周末。
    5. 通过分光光度法测定在670nm处的ddH 2 O作为参考。
    6. 根据新制备的1mM硫化铵溶液制备标准曲线(参见食谱,参见图2E)。如上所述进行比色测定,并用不同体积的硫化铵溶液替代反应混合物,如上述硫代硫酸盐校准曲线(0,1,2.5,5,7.5,10,15和20μl)。 >

数据分析

数据分析使用Microsoft Excel执行。比色测定的光密度的线性增加/减少的范围确定特异性酶活性(图3)。计算各个产品的相应数量并绘制时间。梯度对应于每分钟的产物形成和每个反应混合物中加入的酶的量(=酶活性)。具体的SOR活性定义为每分钟形成1μmol亚硫酸盐加硫代硫酸盐(加氧酶活性)或1μmol硫化氢(还原酶活性),mg蛋白质(= 16.7μkat/ g)。


图3.酸性双歧杆菌的代表性数据SOR酶反应在不同时间点形成硫代硫酸盐(A),亚硫酸盐(B)和硫化物(C) 85℃和pH 7.2,并用1μgSOR测定缓冲液中的2μg纯化酶;误差棒来自一式三份的测量。请注意y轴缩放比例的差异。

笔记

  1. 如果在校准曲线的线性范围内着色或变色太强,则比色测定中反应混合物的体积可以缩小到50μl。
  2. 在酶测定期间,酶的量可以增加至100μg/ ml,以增强低温测量和/或高/低pH值的比色测定法(例如)的产物形成和敏感性。
  3. 在碱性pH下在测定的预热步骤期间,硫花可以含有少量硫代硫酸盐和/或非酶促形成硫代硫酸盐。比色测定中的背景浓度在pH7.2时为8-16nmol / ml,pH9为28-40nmol / ml。
  4. 硫化铵和亚硫酸钠溶液应始终用新鲜开水(微波)制备,以除去化学氧化硫化合物的溶解氧。

食谱

注意:

  1. 除非另有说明,否则对所有解决方案使用ddH 2 O。
  2. 硫代硫酸钠溶液,特别是亚硫酸钠和硫化铵溶液不适合长期储存,应当新鲜制备。
  3. 如果校准曲线完成,所有其他解决方案可以在室温下储存长达一个月甚至更长时间。虽然解决方案不是特别光敏,但应避免阳光直射。
  1. SOR测定缓冲液(250ml)
    70mM Tris(2.1g)用32%HCl调节pH值
    0.1%[vol / vol]吐温20(250μl)
    2%[wt / vol]元素硫(硫花; 5g)
  2. 亚甲蓝溶液(1升)
    12mg亚甲蓝溶于5N HCl中
  3. 硫代硫酸钠溶液(1 mM)
    将0.248g硫代硫酸钠五水合物溶解在10ml ddH 2 O中,用于100mM溶液
    制备1:100稀释液,得到最终的1mM硫代硫酸盐溶液(0.1ml 100mM含9.9ml ddH 2 O的硫代硫酸钠溶液)
  4. 紫红色溶液(100毫升)
    溶于87.5毫升ddH 2 O和12.5毫升浓硫酸的40毫克fuchsine
  5. 亚硫酸钠溶液(1 mM)
    将0.126g亚硫酸钠溶解在10ml新鲜沸腾(微波)ddH 2 O中,用于100mM溶液
    用新鲜沸腾的ddH 2 O制备1:100稀释液,得到最终的1mM亚硫酸盐溶液(0.1ml 100mM亚硫酸钠溶液,9.9ml ddH 2 O) br />
  6. 醋酸锌溶液(250毫升)
    将6.5g溶于249.75ml ddH 2 O的乙酸锌和250μl100%[wt / vol]乙酸的乙酸溶液
  7. 二甲基-4-苯二胺二盐酸盐溶液(100ml) 溶解在5M HCl中的0.1g二甲基-4-苯二胺二盐酸盐
  8. 氯化铁(III)溶液(50ml) 溶解在0.6M HCl中的0.16g铁 - (III)盐酸盐
  9. 硫化铵溶液(1 mM)
    加入3.4μl20%[wt / vol]硫化铵溶液至10ml新鲜沸腾的ddH 2 O -

致谢

帕特里克·鲁尔得到了德国达姆施塔特的卡洛和卡林·吉尔施特施塔特(Tlo Darmstadt)的奖学金和德意志民主共和国(Arnulf Kletzin)的赞助(Az Kl885-7 / 1)的支持。这个协议是从Kletzin(1989)改编的。 Emmel等人开发了用于硫形成亚硫酸盐的原始酶测定法。 (1986)。

参考

  1. Emmel,T.,Sand,W.,König,WA和Bock,E。(1986)。在Sulfolobus brierleyi中存在硫氧化酶的证据。 J Gen Microbiol 132:3415-3420。
  2. King,TE and Morris,R。(1967)。  酸不稳定硫化物和巯基的测定。 Meth Enzymol 10:634-641。
  3. Kletzin,A。(1989)。  将酶促生产亚硫酸盐,硫代硫酸盐和来自硫的硫化氢:纯化和来自兼性厌氧古细菌的硫磺氧化还原酶的性质。脱氧沙门菌二恶英。 :1638-1643。
  4. Pachmayr,F.(1960)。  Mineralwasser的Vorkommen und Bestimmung von Schwefelverbindungen 慕尼黑的Justus-Maximilian大学的论文。
  5. Pelletier,N.,Leroy,G.,Guiral,M.,Giudici-Orticoni,MT和Aubert,C。(2008)。&lt; a class =“ke-insertfile”href =“http://www.ncbi .nlm.nih.gov / pubmed / 18060346“target =”_ blank“>来自细菌Aquifex aeolicus的硫加氧酶还原酶的活性寡聚体形式的首次表征极端亲本< / em> 12(2):205-215。
  6. Rethmeier,J.,Rabenstein,A.,Langer,M.,and Fischer,U.(1997)。&nbsp; 通过不同的高效液相色谱方法的组合检测小样品中氧化和还原的硫化合物的痕量。 Chromatogr A 760 (2):295-302。
  7. Rühl,P.,Pöll,U.,Braun,J.,Klingl,A.and Kletzin,A.(2017)。&lt; a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/27920296“target =”_ blank“>来自具有非常低的还原酶活性的卤代碱性细菌硫代胆碱菌悖论的硫氧化酶。细菌> 199(4)。
  8. Sun,CW,Chen,ZW,He,ZG,Zhou,PJ and Liu,SJ(2003)。&nbsp; 来自酸性嗜热古细菌,Acidianus菌株S5的硫加氧酶/还原酶的纯化和性质极端亲本 7 2):131-134。
  9. Urich,T.,Bandeiras,TM,Leal,SS,Rachel,R.,Albrecht,T.,Zimmermann,P.,Scholz,C.,Teixeira,M.,Gomes,CM和Kletzin,A。(2004)。 &nbsp; Acidiaus ambivalens中的硫加氧酶还原酶em>是含有低电位单核非血红蛋白中心的多聚体蛋白质。生物化学J 381(Pt 1):137-146。
  10. Veith,A.,Botelho,HM,Kindinger,F.,Gomes,CM和Kletzin,A.(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm。 nih.gov/pubmed/22139503“target =”_ blank“>来自嗜温细菌的硫氧还原酶是一种高活性的热酶。 194(3):677-685。
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引用:Rühl, P. and Kletzin, A. (2017). The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures. Bio-protocol 7(14): e2403. DOI: 10.21769/BioProtoc.2403.
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