Amino Acids

, Volume 49, Issue 4, pp 715–723 | Cite as

Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions

Original Article


We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.


Urea Guanidine hydrochloride Conformational stability Guinier analysis Porod analysis 


  1. Akita M, Tsutsumi D, Kobayashi M, Kise H (2001) Structural change and catalytic activity of horseradish peroxidase in oxidative polymerization of phenol. Biosci Biotechnol Biochem 65(7):1581–1588. doi:10.1271/bbb.65.1581 CrossRefPubMedGoogle Scholar
  2. Azevedo AM, Martins VC, Prazeres DM, Vojinovic V, Cabral JM, Fonseca LP (2003) Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol Annu Rev 9:199–247CrossRefPubMedGoogle Scholar
  3. Carvalho AS, Melo EP, Ferreira BS, Neves-Petersen MT, Petersen SB, Aires-Barros MR (2003) Heme and pH-dependent stability of an anionic horseradish peroxidase. Arch Biochem Biophys 415(2):257–267CrossRefPubMedGoogle Scholar
  4. Cha HJ, Jang DS, Kim YG, Hong BH, Woo JS, Kim KT, Choi KY (2013) Rescue of deleterious mutations by the compensatory Y30F mutation in ketosteroid isomerase. Mol Cells 36(1):39–46. doi:10.1007/s10059-013-0013-1 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cha HJ, Jang DS, Jin KS, Lee HJ, Hong BH, Kim ES, Kim J, Lee HC, Choi KY, Lee M (2014) Three-dimensional structures of a wild-type ketosteroid isomerase and its mutant in solution. Sci Adv Mater 6(11):2325–2333CrossRefGoogle Scholar
  6. Chattopadhyay K, Mazumdar S (2000) Structural and conformational stability of horseradish peroxidase: effect of temperature and pH. Biochemistry 39(1):263–270CrossRefPubMedGoogle Scholar
  7. Cockle SA, Epand RM, Moscarello MA (1978) Resistance of lipophilin, a hydrophobic myelin protein, to denaturation by urea and guanidinium salts. J Biol Chem 253(22):8019–8026PubMedGoogle Scholar
  8. Feng JY, Liu JZ, Ji LN (2008) Thermostability, solvent tolerance, catalytic activity and conformation of cofactor modified horseradish peroxidase. Biochimie 90(9):1337–1346. doi:10.1016/j.biochi.2008.03.010 CrossRefPubMedGoogle Scholar
  9. Franke D, Svergun DI (2009) DAMMIF, a program for rapid ab initio shape determination in small-angle scattering. J Appl Crystallogr 42:342–346CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gajhede M, Schuller DJ, Henriksen A, Smith AT, Poulos TL (1997) Crystal structure of horseradish peroxidase C at 2.15 A resolution. Nat Struct Biol 4(12):1032–1038CrossRefPubMedGoogle Scholar
  11. Glatter O, Kratky O (1982) Small angle X-ray scattering. Academic Press, LondonGoogle Scholar
  12. Greenfield NJ (2006) Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 1(6):2527–2535. doi:10.1038/nprot.2006.204 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Haque E, Debnath D, Basak S, Chakrabarti A (1999) Structural changes of horseradish peroxidase in presence of low concentrations of urea. Eur J Biochem 259(1–2):269–274CrossRefPubMedGoogle Scholar
  14. Hargrove MS, Olson JS (1996) The stability of holomyoglobin is determined by heme affinity. Biochemistry 35(35):11310–11318. doi:10.1021/bi9603736 CrossRefPubMedGoogle Scholar
  15. Howes BD, Feis A, Raimondi L, Indiani C, Smulevich G (2001) The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase. J Biol Chem 276(44):40704–40711. doi:10.1074/jbc.M107489200 CrossRefPubMedGoogle Scholar
  16. Hsu MC, Woody RW (1971) The origin of the heme Cotton effects in myoglobin and hemoglobin. J Am Chem Soc 93(14):3515–3525CrossRefPubMedGoogle Scholar
  17. Jang DS, Lee HJ, Lee B, Hong BH, Cha HJ, Yoon J, Lim K, Yoon YJ, Kim J, Ree M, Lee HC, Choi KY (2006) Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase. FEBS Lett 580(17):4166–4171. doi:10.1016/j.febslet.2006.06.069 CrossRefPubMedGoogle Scholar
  18. Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751(2):119–139. doi:10.1016/j.bbapap.2005.06.005 CrossRefPubMedGoogle Scholar
  19. Kim ES, Jang DS, Yang SY, Lee MN, Jin KS, Cha HJ, Kim JK, Sung YC, Choi KY (2013) Controlled release of human growth hormone fused with a human hybrid Fc fragment through a nanoporous polymer membrane. Nanoscale 5(10):4262–4269. doi:10.1039/c3nr00474k CrossRefPubMedGoogle Scholar
  20. Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr 36:1277–1282CrossRefGoogle Scholar
  21. Koua D, Cerutti L, Falquet L, Sigrist CJ, Theiler G, Hulo N, Dunand C (2009) PeroxiBase: a database with new tools for peroxidase family classification. Nucleic Acids Res 37(Database issue):D261–D266. doi:10.1093/nar/gkn680 CrossRefPubMedGoogle Scholar
  22. Kozin MB, Svergun DI (2001) Automated matching of high- and low-resolution structural models. J Appl Crystallogr 34:33–41CrossRefGoogle Scholar
  23. Krainer FW, Glieder A (2015) An updated view on horseradish peroxidases: recombinant production and biotechnological applications. Appl Microbiol Biotechnol 99(4):1611–1625. doi:10.1007/s00253-014-6346-7 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kundu J, Kar U, Gautam S, Karmakar S, Chowdhury PK (2015) Unusual effects of crowders on heme retention in myoglobin. FEBS Lett 589(24 Pt B):3807–3815. doi:10.1016/j.febslet.2015.11.015 CrossRefPubMedGoogle Scholar
  25. Mao L, Luo S, Huang Q, Lu J (2013) Horseradish peroxidase inactivation: heme destruction and influence of polyethylene glycol. Sci Rep 3:3126. doi:10.1038/srep03126 PubMedPubMedCentralGoogle Scholar
  26. Mogharrab N, Ghourchian H, Amininasab M (2007) Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation. Biophys J 92(4):1192–1203. doi:10.1529/biophysj.106.092858 CrossRefPubMedGoogle Scholar
  27. Monhemi H, Housaindokht MR, Moosavi-Movahedi AA, Bozorgmehr MR (2014) How a protein can remain stable in a solvent with high content of urea: insights from molecular dynamics simulation of Candida antarctica lipase B in urea: choline chloride deep eutectic solvent. Phys Chem Chem Phys 16(28):14882–14893. doi:10.1039/c4cp00503a CrossRefPubMedGoogle Scholar
  28. Myer YP (1968) Conformation of cytochromes. III. Effect of urea, temperature, extrinsic ligands, and pH variation on the conformation of horse heart ferricytochrome c. Biochemistry 7(2):765–776CrossRefPubMedGoogle Scholar
  29. Pappa HS, Cass AE (1993) A step towards understanding the folding mechanism of horseradish peroxidase. Tryptophan fluorescence and circular dichroism equilibrium studies. Eur J Biochem 212(1):227–235CrossRefPubMedGoogle Scholar
  30. Poulos TL (1993) Peroxidases. Curr Opin Biotechnol 4(4):484–489CrossRefPubMedGoogle Scholar
  31. Rambo RP, Tainer JA (2011) Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95(8):559–571. doi:10.1002/bip.21638 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Ryan BJ, O’Fagain C (2007) Arginine-to-lysine substitutions influence recombinant horseradish peroxidase stability and immobilisation effectiveness. BMC Biotechnol 7:86. doi:10.1186/1472-6750-7-86 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Semenyuk AV, Svergun DI (1991) GNOM—a program package for small-angle scattering data processing. J Appl Crystallogr 24:537–540CrossRefGoogle Scholar
  34. Soh YM, Burmann F, Shin HC, Oda T, Jin KS, Toseland CP, Kim C, Lee H, Kim SJ, Kong MS, Durand-Diebold ML, Kim YG, Kim HM, Lee NK, Sato M, Oh BH, Gruber S (2015) Molecular basis for SMC rod formation and its dissolution upon DNA binding. Mol Cell 57(2):290–303. doi:10.1016/j.molcel.2014.11.023 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Strickland EH (1968) Circular dichroism of horseradish peroxidase and its enzyme-substrate compounds. Biochim Biophys Acta 151(1):70–75CrossRefPubMedGoogle Scholar
  36. Strickland EH, Kay E, Shannon LM, Horwitz J (1968) Peroxidase isoenzymes from horseradish roots. 3. Circular dichroism of isoenzymes and apoisoenzymes. J Biol Chem 243(13):3560–3565PubMedGoogle Scholar
  37. Svergun D, Barberato C, Koch MHJ (1995) CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773CrossRefGoogle Scholar
  38. Tamura M, Asakura T, Yonetani T (1972) Heme-modification studies on horseradish peroxidase. Biochim Biophys Acta 268(2):292–304CrossRefPubMedGoogle Scholar
  39. Tanford C (1968) Protein denaturation. Adv Protein Chem 23:121–282CrossRefPubMedGoogle Scholar
  40. Uppal S, Salhotra S, Mukhi N, Zaidi FK, Seal M, Dey SG, Bhat R, Kundu S (2015) Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin. J Biol Chem 290(4):1979–1993. doi:10.1074/jbc.M114.603225 CrossRefPubMedGoogle Scholar
  41. Veitch NC (2004) Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry 65(3):249–259CrossRefPubMedGoogle Scholar
  42. Volkov VV, Svergun DI (2003) Uniqueness of ab initio shape determination in small-angle scattering. J Appl Crystallogr 36:860–864CrossRefGoogle Scholar
  43. Wagner M, Nicell JA (2002) Detoxification of phenolic solutions with horseradish peroxidase and hydrogen peroxide. Water Res 36(16):4041–4052CrossRefPubMedGoogle Scholar
  44. Wang C, Chen Z, Hong X, Ning F, Liu H, Zang J, Yan X, Kemp J, Musselman CA, Kutateladze TG, Zhao R, Jiang C, Zhang G (2014) The structural basis of urea-induced protein unfolding in beta-catenin. Acta Crystallogr D Biol Crystallogr 70(Pt 11):2840–2847. doi:10.1107/S1399004714018094 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Won K, Kim YH, An ES, Lee YS, Song BK (2004) Horseradish peroxidase-catalyzed polymerization of cardanol in the presence of redox mediators. Biomacromolecules 5(1):1–4. doi:10.1021/bm034325u CrossRefPubMedGoogle Scholar
  46. Yonetani T (1967) Studies on cytochrome c peroxidase. X. Crystalline apo-and reconstituted holoenzymes. J Biol Chem 242(21):5008–5013PubMedGoogle Scholar
  47. Yu H, Huang H (2014) Engineering proteins for thermostability through rigidifying flexible sites. Biotechnol Adv 32(2):308–315. doi:10.1016/j.biotechadv.2013.10.012 CrossRefPubMedGoogle Scholar
  48. Zakharova GS, Uporov IV, Tishkov VI (2011) Horseradish peroxidase: modulation of properties by chemical modification of protein and heme. Biochemistry (Mosc) 76(13):1391–1401. doi:10.1134/S0006297911130037 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2017

Authors and Affiliations

  1. 1.Pohang Accelerator LaboratoryPohang University of Science and Technology (POSTECH)PohangKorea
  2. 2.Department of Life SciencesPOSTECHPohangKorea
  3. 3.Huons Co., Ltd.SeongnamKorea

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