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Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions

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Jul 2012


Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by heme, thus allowing correlation of heme content with activity. This requires the absolute heme concentration and an accurate protein concentration. Another use is to determine the extinction coefficients of a heme-bound protein. This assay is fast, easy, and reproducible if done correctly.

Keywords: heme (血红素), pyridine (吡啶), hemochromagen (血色素), hemoprotein (血红蛋白), porphyrin (卟啉)


G.G. Stokes was the first to prepare what we now refer to as hemochromagen. As early as 1863, he was monitoring changes in the hemoglobin absorbance spectrum upon reduction of the heme to the Fe(II) form Stokes (1863). Stokes had reduced blood in the presence of ammonia; what he was seeing were the intense α and β peaks of Fe(II) heme b from hemoglobin in complex with ammonia. Later authors (Anson and Mirsky, 1928) were able to show that the hemochromagen, as it had been called by Christian Bohr (Edsall, 1972) , was heme in complex with some nitrogenous ligand. It can be formed as well by simply reducing myoglobin under denaturing conditions, in which case histidines serve as the axial ligands. Hill demonstrated that the pyridine hemochromagen is formed from the nitrogen of two pyridine molecules binding to the axial position of the reduced heme (Hill, 1926; Hill, 1929); this was confirmed by Smith (1959) and Gallagher and Elliot (Gallagher et al., 1965; Gallagher et al., 1968).

With advances in spectroscopic techniques, later workers were able to use hemochromagen for an important analytical purpose (Hill, 1929). The regularity of the α peak of reduced pyridine hemochromagen, its high extinction coefficient, and the fact that it follows Beer's Law over a wide range allows its use for determining the total heme composition of a sample. De Duve (1948) published one of the first protocols for this, and determined the extinction coefficient (ε) at 557 nm to be 32 mM-1 cm-1. The method used relied on gravimetric determination of the standard heme samples, which could lead to an underestimate of the true heme concentration if the sample is impure. Paul et al. (1953) re-determined the extinction coefficient for pyridine hemochromogen from recrystallized heme b and myoglobin, using the iron content as an internal control. They found a value (34.7 mM-1 cm-1) significantly higher than the previous value of 32 mM-1 cm-1, representing a difference of roughly 9%. Both values have been in use comparatively recently: e.g. (Scott and Lecomte, 2000; Fushitani and Riggs, 1988; Miyoshi et al., 1997; Yachie et al., 1999; Lee et al., 2000; Huche et al., 2006), which use 32 mM-1 cm-1, and (Sono et al., 1984; Senturia et al., 2012; Sinclair et al., 2001; Berry and Trumpower, 1987), which use 34.7 mM-1 cm-1. In this assay we have taken the value given by Paul et al. (1953) to be most accurate.

[Principle of action] The heme-containing protein solution is first mixed with Solution I (see Recipes) containing pyridine, NaOH and potassium ferricyanide. Pyridine serves as a ligand for heme in the Fe(II) state. NaOH keeps sodium dithionite stable. Potassium ferricyanide ensures that all dissolved heme is in the Fe(III) state at first. Then, excess sodium dithionite in solution is added to reduce the heme from Fe(III) to Fe(II). Another option is to use a pipette tip to add a few grains of solid dithionite to the cuvette, and allow it to dissolve. Recipes similar to the current one have been recommended by Sinclair et al. (2001); Berry and Trumpower (1987); Antonini and Brunori (1971). The concentration of heme in the sample can be determined from the absorbance of the reduced sample; the extinction coefficient for reduced pyridine hemochromagen is 34.7 mM-1 cm-1 at 557 nm for heme b. It can also be done using the difference spectrum between the reduced and oxidized samples. Don't forget, in either case, to take your dilution factor into consideration. This protocol is written for heme b; however, other types of heme form hemochromagens as well and can be quantified using the same technique. See Berry and Trumpower (1987) or Table 1 for extinction coefficients for hemes a and c.

Materials and Reagents

  1. Heme-containing sample
    Note: The concentration of heme in your stock solution should be at least 10 μM and not much greater than 80 μM. Dithiothreitol (DTT) and other reducing agents can reduce potassium ferricyanide and may interfere with the oxidized sample, but not with the reduced sample. Any common biological buffer should be compatible with this procedure so long as it has no significant absorbance in the 500 nm to 600 nm range and does not form a complex with heme, as should be the case with all Good's buffers. We have personally used phosphate, tris, HEPES, EPPS, MES, and CHES without issue.
  2. 0.5 M NaOH
  3. Pyridine (Sigma-Aldrich, catalog number: 360570 )
  4. Potassium ferricyanide(III) (Sigma-Aldrich, catalog number: 702587 )
  5. Sodium dithionite (Sigma-Aldrich, catalog number: 157953-5 G )
  6. Deionized water
  7. 0.2 M NaOH, 40% (v/v) pyridine, 500 μM potassium ferricyanide (see Recipes)
  8. 0.1 M potassium ferricyanide (K3[Fe(CN)6]) (see Recipes)
  9. 0.5 M sodium dithionite in 0.5 M NaOH (see Recipes)


  1. Spectrophotometer with a bandwidth ≤ 2 nm
  2. Fume hood
  3. Quartz or glass cuvette , 1 cm length
  4. Pipettes
    Note: It is generally good to have a spectrophotometer capable of relatively low spectral bandwidth (SBW) in order to get highly accurate measurements. It is suggested in the literature that the ratio of SBW to the natural bandwidth (NBW) should be 0.1 or lower in order to get an error of less than 0.5% (Surles and Erickson, 1974; Brodersen, 1954). The α band of reduced pyridine hemochromagen has a NBW of around 20 nm, hence the recommendation of 2 nm or less for SBW. Check with your manufacturer if you are unsure of your SBW. Most modern spectrophotometers have SBW less than 4 nm, corresponding to an error of less than 2%. It is also important to note that this error is not random, but results in an underestimate of the true absorbance; having a higher SBW/NBW ratio leads to a 'flattening' of absorbance peaks. If possible, use a scanning spectrophotometer. This allows you to verify that your spectrum looks similar to the spectrum shown in Figure 1.

    Figure 1. Example spectrum of reduced and oxidized pyridine hemochromagen (heme b). The bandwidth is set to 1 nm, with data interval 1 nm.


The volumes shown below can be scaled up or down as desired, as long as the concentrations stay the same.

  1. Set the spectrophotometer to ≤ 2 nm SBW (if adjustable) and data interval at 1 nm between 500 and 600 nm (at least). The assay is not strongly temperature sensitive; room temperature works well.
  2. Blank the spectrophotometer with Solution I mixed 1:1 with whichever buffer your protein sample is in.
  3. Empty cuvette. Add 0.5 ml Solution I to 0.5 ml of your heme-containing sample and mix well.
  4. Scan this mixture; this is your oxidized sample.
  5. Add 10 μl Solution III to your oxidized sample and mix well. The sample should turn a reddish color.
  6. Scan immediately and again every minute until the absorbance peak no longer increases; this should take no more than 5 min. The scan with the highest peak is the reduced spectrum.
  7. Using the extinction coefficients in Table 1, and taking into account your dilution factors, calculate the concentration of heme in your original sample using Beer's law, A = ε c l (Absorbance = extinction coefficient x concentration x pathlength). You can use the absolute absorbance of the reduced sample, or the difference spectrum.

    Table 1. Extinction coefficients for pyridine hemochromogens
    ε (mM-1 cm-1)
    Pyr2-heme b Reduced
    557 nm
    NaOH, 10-40% pyr.
    Paul et al., 1953
    Pyr2-heme b, Red. - Oxid.
    557 nm min 540 nm
    NaOH, 10-40% pyr.
    Berry and Trumpower, 1987
    Pyr2-heme a, Red. - Oxid.
    587 nm min 620 nm
    NaOH, 10-40% pyr.
    Berry and Trumpower, 1987
    Pyr2-heme c Reduced
    550 nm
    NaOH, 10-40% pyr.
    Berry and Trumpower, 1987
    Pyr2-heme c, Red. - Oxid.
    550 nm min 535 nm
    NaOH, 10-40% pyr.
    Berry and Trumpower, 1987

  8. To determine the extinction coefficient of the heme when it is bound to your native protein, you will also need a high quality scan of the protein sample in a suitable buffer. Using Beer's law and the sample heme concentration, calculate ε for the heme absorbance peaks of your native protein.


  1. Solution I [0.2 M NaOH, 40% (v/v) pyridine, 500 μM potassium ferricyanide]
    Add 2/5 volume 0.5 M NaOH to 2/5 volume pyridine in fume hood or well-ventilated space and dilute to final volume with water
    Add 1/200 volume 0.1 M potassium ferricyanide (Solution II)
    Paul et al. (1953) reported that NaOH concentrations between 0.02 and 0.5 M and pyridine concentrations between 10% and 50% make no difference to the molar absorptivities of the hemochromagen.
    Hazards: Pyridine is toxic, flammable, and has what the Merck Index calls a "characteristic disagreeable odor." Mix solutions in a fume hood or well-ventilated area.
    Warnings: Pyridine eventually oxidizes; when this happens it turns yellow. Do not use if yellow, and seal the pyridine bottle after use.
  2. Solution II {0.1 M potassium ferricyanide (K3[Fe(CN)6])}
    Hazards: Releases cyanide if mixed with strong acids.
    Warnings: Potassium ferricyanide must be made fresh and used within 24 h. Dispose of in a separate container.
  3. Solution III (0.5 M sodium dithionite in 0.5 M NaOH)
    Sodium dithionite, off the shelf, is usually no more than 85% pure, no matter the supplier (McKenna et al., 1991).
    Note: It should always be dissolved and diluted in neutral or basic solution; acidic conditions cause it to release hydrogen sulfide, a poisonous gas. It must be made fresh every time.
    Storage: Sodium dithionite should be kept away from water. Fresh sodium dithionite is powdery and free-flowing. It is best to purchase it in small amounts and open a fresh bottle periodically, due to degradation upon exposure to air (McKenna et al., 1991). Sodium dithionite has a redox potential of -0.66 V at pH 7.0, 25 °C (Mayhew, 1978), and doesn’t absorb strongly in the > 400 nm region.


This work was funded in part by a grant from the NIH (GM080563) to F. G. and a UCLA Dissertation Year Fellowship to I. B. We would also like to thank Aaron T. Smith and Judith N. Burstyn for advice.


  1. Anson, M. L. and Mirsky, A. E. (1928). On Hemochromogen. J Gen Physiol 12(2): 273-288.
  2. Antonini, E. and Brunori, M. (1971). Hemoglobin and myoglobin in their reactions with ligands. North-Holland Pub. Co.
  3. Berry, E. A. and Trumpower, B. L. (1987). Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal Biochem 161(1): 1-15.
  4. Brodersen, S. (1954). Slit-width effects. J Opt Soc Am 44:22.
  5. De Duve, C. (1948). A spectrophotometric method for the simultaneous determination of myoglobin and hemoglobin in extracts of human muscle. Acta Chem Scand 2(3): 264-289.
  6. Edsall, J. T. (1972). Blood and hemoglobin: the evolution of knowledge of functional adaptation in a biochemical system, part I: The adaptation of chemical structure to function in hemoglobin. J Hist Biol 5(2): 205-257.
  7. Fushitani, K. and Riggs, A. F. (1988). Non-heme protein in the giant extracellular hemoglobin of the earthworm Lumbricus terrestris. Proc Natl Acad Sci U S A 85(24): 9461-9463.
  8. Gallagher, W. A. and Elliott, W. B. (1965). The formation of pyridine haemochromogen. Biochem J 97(1): 187-193.
  9. Gallagher, W. A. and Elliott, W. B. (1968). Alkaline haematin and nitrogenous ligands. Biochem J 108(1): 131-136.
  10. Hill, R. (1926). The chemical nature of haemochromogen and its carbon monoxide compound. Proc R Soc London B Biol Sci 419-430.
  11. Hill, R. (1929). Reduced haematin and haemochromogen. Proc R Soc London Ser B Contain Pap a Biol 112-130.
  12. Huche, F., Delepelaire, P., Wandersman, C. and Welte, W. (2006). Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA-HasR from Serratia marcescens. Acta Crystallogr Sect F Struct Biol Cryst Commun 62(Pt 1): 56-60.
  13. Lee, Y. C., Martin, E. and Murad, F. (2000). Human recombinant soluble guanylyl cyclase: expression, purification, and regulation. Proc Natl Acad Sci U S A 97(20): 10763-10768.
  14. Mayhew, S. G. (1978). The redox potential of dithionite and SO-2 from equilibrium reactions with flavodoxins, methyl viologen and hydrogen plus hydrogenase. Eur J Biochem 85(2): 535-547.
  15. McKenna, C. E., Gutheil, W. G. and Song, W. (1991). A method for preparing analytically pure sodium dithionite. Dithionite quality and observed nitrogenase-specific activities. Biochim Biophys Acta 1075(1): 109-117.
  16. Miyoshi, S., Inami, Y., Moriya, Y., Kamei, T., Rahman, M. M., Yamamoto, S., Tomochika, K. and Shinoda, S. (1997). Characterization of a mutant of Vibrio vulnificus for heme utilization. FEMS Microbiol Lett 148(1): 101-106.
  17. Paul, K. G., Theorell, H. and Åkeson, A. (1953). The molar light absorption of pyridine ferroprotoporphyrin (pyridine haemochromogen). Acta Chem Scand 7 1284-1287.
  18. Scott, N. L. and Lecomte, J. T. (2000). Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803. Protein Sci 9(3): 587-597.
  19. Senturia, R., Laganowsky, A., Barr, I., Scheidemantle, B. D. and Guo, F. (2012). Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8. PLoS One 7(7): e39688.
  20. Sinclair, P. R., Gorman, N. and Jacobs, J. M. (2001). Measurement of heme concentration. Curr Protoc Toxicol Chapter 8: Unit 8 3.
  21. Smith, M. H. (1959). Kinetics and equilibria in systems containing haem, carbon monoxide and pyridine. Biochem J 73: 90-101.
  22. Sono, M., Dawson, J. H. and Hager, L. P. (1984). The generation of a hyperporphyrin spectrum upon thiol binding to ferric chloroperoxidase. Further evidence of endogenous thiolate ligation to the ferric enzyme. J Biol Chem 259(21): 13209-13216.
  23. Stokes, G. G. (1863). On the reduction and oxidation of the colouring matter of the blood. Proc R Soc 13: 355-364.
  24. Surles, T. and Erickson, J. O. (1974). Absorbance measurements at various spectral bandwidths. Clin Chem 1243-1244.
  25. Yachie, A., Niida, Y., Wada, T., Igarashi, N., Kaneda, H., Toma, T., Ohta, K., Kasahara, Y. and Koizumi, S. (1999). Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest 103(1): 129-135.



[历史背景] G.G.斯托克斯是第一个准备我们现在称为血色素的。早在1863年,他正在监测血红蛋白吸收光谱的变化,血红素还原为Fe(II)形式斯托克斯(1863)。斯托克斯在氨存在下减少了血液;他所看到的是来自与氨络合的血红蛋白的Fe(II)血红素的强烈α和β峰。后来的作者(Anson和Mirsky,1928)能够证明血红素原是由Christian Bohr(Edsall,1972)所调用的,是一种含有一些含氮配体的血红素。它也可以通过在变性条件下简单地还原肌红蛋白而形成,在这种情况下组氨酸用作轴向配体。 Hill证明吡啶血色素由结合还原血红素的轴向位置的两个吡啶分子的氮形成(Hill,1926; Hill,1929);这由Smith(1959)和Gallagher和Elliot(Gallagher等人,1965; Gallagher等人,1968)证实。
随着光谱学的发展技术,后来的工人能够使用血色素作为重要的分析目的(Hill,1929)。还原的吡啶血色素的α峰的规律性,其高消光系数,以及其在宽范围内遵循比尔定律的事实允许其用于确定样品的总血红素组成。 De Duve(1948)公布了其中的第一个方案之一,并确定在557nm处的消光系数(ε)为32mM -1 cm -1 。所使用的方法依赖于重量法测定标准血红素样品,如果样品不纯,这可能导致真实血红素浓度的低估。 Paul等人(1953)使用铁含量作为内部对照,重新确定了来自重结晶血红素和肌红蛋白的吡啶血色素的消光系数。他们发现了显着高于以前的32mM血浆浓度的值(34.7mM < - > cm <-1> -1) 1 ,代表大约9%的差异。这两个值最近都在使用中:例如(Scott和Lecomte,2000; Fushitani和Riggs,1988; Miyoshi等人,1997; Yachie等人。,1999; Lee等人,2000; Huche等人,2006),其使用32mM的sup- cm -1 ,和(Sono等人,1984; Senturia等人,2012; Sinclair等人, 2001; Berry和Trumpower,1987),其使用34.7mM -1 cm -1 。在该测定中,我们采用由Paul等人(1953)给出的值最准确。

[原理] 首先将含有血红素的蛋白质溶液与含有吡啶,NaOH和铁氰化钾的溶液I(参见Recipes)混合。吡啶在Fe(II)态中用作血红素的配体。 NaOH使连二亚硫酸钠稳定。铁氰化钾确保所有溶解的血红素首先处于Fe(III)态。然后,加入过量的连二亚硫酸钠溶液以将血红素从Fe(III)还原成Fe(II)。另一种选择是使用移液管尖端向试管中加入几个固体连二亚硫酸盐,并允许其溶解。与目前的食谱相似的食谱已由Sinclair等人(2001)推荐; Berry和Trumpower(1987); Antonini和Brunori(1971)。样品中血红素的浓度可以从还原样品的吸光度确定;对于血红素b,在557nm,还原的吡啶血色素的消光系数为34.7mM cm -1 -1 。它也可以使用还原和氧化样品之间的差谱进行。不要忘记,无论如何,考虑你的稀释倍数。此协议是为heme b 编写的;然而,其他类型的血红素也形成血色素,并且可以使用相同的技术定量。参见Berry和Trumpower(1987)或表1中hemes的消光系数 a 和 c 。

关键字:血红素, 吡啶, 血色素, 血红蛋白, 卟啉


  1. 含血红素的样品
    注意:您的储备溶液中血红素的浓度应至少为10μM,而不要大于80μM。 二硫苏糖醇(DTT)和其他还原剂可以还原铁氰化钾并且可以干扰氧化的样品,但不干扰还原的样品。 任何生物缓冲液应当与该程序相容,只要其在500nm至600nm范围内没有显着的吸光度并且不与血红素形成复合物,如所有的 好的缓冲区。 我们亲自使用磷酸盐,tris,HEPES,EPPS,MES和CHES没有问题。
  2. 0.5 M NaOH
  3. 吡啶(Sigma-Aldrich,目录号:360570)
  4. 铁氰化钾(III)(Sigma-Aldrich,目录号:702587)
  5. 连二亚硫酸钠(Sigma-Aldrich,目录号:157953-5G)
  6. 去离子水
  7. 0.2M NaOH,40%(v/v)吡啶,500μM铁氰化钾(参见配方)
  8. 0.1M铁氰化钾(K 3 [Fe(CN)6]])(参见配方)
  9. 在0.5M NaOH中的0.5M连二亚硫酸钠(参见配方)


  1. 分光光度计带宽≤2 nm
  2. 通风橱
  3. 石英或玻璃比色皿,1厘米长
  4. 移液器
    ote:通常,具有能够具有相对低的光谱带宽(SBW)的分光光度计以获得高精度的测量是好的。在文献中建议SBW与自然带宽(NBW)的比率应为0.1或更低,以便得到小于0.5%的误差(Surles和Erickson,1974; Brodersen,1954)。还原的吡啶血色素的α带具有约20nm的NBW,因此推荐SBW为2nm或更小。如果您不确定SBW,请与制造商联系。大多数现代分光光度计具有小于4nm的SBW,对应于小于2%的误差。同样重要的是注意这个误差不是随机的,而是导致真实吸光度的低估;具有较高的SBW/NBW比率导致吸收峰的"平坦化"。如果可能,使用扫描分光光度计。这允许您验证您的光谱看起来类似于图1所示的光谱。

    图1.还原和氧化的吡啶血色素(血红素b)的示例光谱。 带宽设置为1 nm,数据间隔为1 nm。



  1. 将分光光度计设置为≤2 nm SBW(如果可调节)和500 nm至600 nm之间的1 nm数据间隔(至少)。 该测定不是强烈温度敏感; 室温工作良好。
  2. 将溶液I与1:1混合的分光光度计与您的蛋白质样品所在的缓冲液混合。
  3. 空的比色杯。 向0.5ml含血红素的样品中加入0.5ml溶液I,混匀。
  4. 扫描此混合物;这是您的氧化样本。
  5. 加入10μl溶液III到您的氧化样品,并混匀。样品应变为微红色。
  6. 每分钟立即再次扫描,直到吸光度峰不再增加;这应该不超过5分钟。具有最高峰的扫描是降低的光谱。
  7. 使用表1中的消光系数,并考虑到您的稀释因子,使用比尔定律计算原始样品中血红素的浓度,其中吸光度=消光系数 x 浓度 x 路径长度)。您可以使用缩减样品的绝对吸光度或差谱。

    表1.吡啶血色素的消光系数 化合物
    ε(mM -1 cm -1 )
    Pyr 2 -heme b 减少
    557 nm
    Paul 等人,1953年
    Pyr <2> ,红色。 - 氧化。
    557nm min 540nm
    Pyr <2> ,红色。 - 氧化。
    587nm min 620nm
    Pyr 2 -heme c 减少
    550 nm
    Pyr <2> ,红色。 - 氧化。
    550nm min 535nm

  8. 要确定血红素与天然蛋白结合时的消光系数,您还需要在合适的缓冲液中对蛋白样品进行高质量扫描。 使用Beer定律和样品血红素浓度,计算您的天然蛋白的血红素吸收峰的ε


  1. 溶液I [0.2M NaOH,40%(v/v)吡啶,500μM铁氰化钾] 在通风橱或通风良好的空间中加入2/5体积的0.5M NaOH至2/5体积吡啶,用水稀释至最终体积
  2. 溶液II {0.1M铁氰化钾(K 3 [Fe(CN)6]])} / 危害:如果与强酸混合,释放氰化物。
  3. 溶液III(0.5M NaOH中的0.5M连二亚硫酸钠) 无论供应商如何,连续测定的连二亚硫酸钠通常不超过85%纯度。(
    储存:连二亚硫酸钠应远离水。新鲜的连二亚硫酸钠是粉状和自由流动的。最好是少量购买,并定期打开新鲜的瓶子,因为暴露于空气下降解(McKenna等人,1991)。连二亚硫酸钠在pH 7.0,25℃下具有-0.66V的氧化还原电位(Mayhew,1978),并且在> 400 nm区域。


这项工作的部分资金来自NIH(GM080563)授予F. G.和UCLA论文年奖学金给I. B.我们也要感谢亚伦·T·史密斯和朱迪思N.布斯特廷的建议。


  1. Anson,M.L。和Mirsky,A.E。(1928)。 On Hemochromogen。 J Gen Physiol 12(2) :273-288。
  2. Antonini,E。和Brunori,M.(1971)。 血红蛋白和肌红蛋白与配体的反应。<北>荷兰 Pub。 Co.
  3. Berry,E.A。和Trumpower,B.L。(1987)。 从吡啶血红素光谱中同时测定血红素a,b和c。 Anal Biochem 161(1):1-15
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引用:Barr, I. and Guo, F. (2015). Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions. Bio-protocol 5(18): e1594. DOI: 10.21769/BioProtoc.1594.



Bezalel Bacon
Stony Brook University
I have been trying to use this assay and have had a few questions about unusual peaks and peak shifts.
1.Both in myoglobin control and in my protein of interest, I see an additional peak at 578nm. What accounts for this? This peak is not seen when I dissolve myoglobin in water, only when I use my protein buffer.
2. The peak for my protein of interest appears at 550nm, which is strange because my protein doesn't purify bound strongly to heme, so I supplement with large excess of hemin (Heme B), which itself should have a peak at 557nm. Is there a reason this shift would happen? Regardless, would you suggest I use extinction coefficients for heme B or heme C?
Please see attached photos.
6/16/2017 11:18:32 AM Reply
Ian Barr
Department of Biological Chemistry, David Geffen School of Medicine, UCLA, USA

1. So , what's in your protein buffer? Imidazole, DTT, TCEP, anything that could be binding to the heme? That's the best guess I have. Something might be out-competing the pyridine for binding to the heme. And have you scanned it before you add the dithionite to reduce the heme? Is the band there originally?
2. From the spectrum, it looks like you have heme c in your protein. Mass spectrometry might be one way to show this. There is also an old paper I found to separate heme c from your protein. KG Paul (Acta Chemica Scandinavica (1950) 4, 239-240 ) for how to do this; essentially, 0.2 volumes of glacial acetic acid and 1 volume 25 mM siver sulfate, and they keep it at 60 C for 4 hours. You could then use HPLC or something similar to identify it. I've never used it though. If you have any cytochrome c lying around, give that a try; the spectra should be the same.

6/16/2017 3:43:35 PM

bezalel bacon
Stony Brook University
I've been trying to work with this assay for the last few weeks and I've found that my reduced pyridine absorbance peak isn't cleanly falling at 557nm or 550nm. Its usually right around 552-553nm (variation depending on some buffer conditions). Is this expected? What would be the reason for this variability?
6/9/2017 1:27:43 PM Reply