Horseradish peroxidase

from Wikipedia, the free encyclopedia
Horseradish Peroxidase CA1 ( Armoracia rusticana )
Horseradish Peroxidase CA1 (Armoracia rusticana)
according to PDB  1W4W

Existing structural data : 1YY1 , 1ATJ

Mass / length primary structure 308 amino acids, 44,174 daltons
Secondary to quaternary structure Monomer
Cofactor 2 calcium, heme B
Precursor (323 aas)
Identifier
External IDs
Enzyme classification
EC, category 1.11.1.7 ,  peroxidase
Substrate Donor + H 2 O 2
Products oxidized donor + H 2 O

The horseradish peroxidase - mostly as HRP, from English. horseradish , abbreviated - is a peroxidase from horseradish , which is considered one of the three most commonly used reporter enzymes in biochemistry is used.

properties

Horseradish peroxidase catalyzes the reduction of various peroxides , mostly hydrogen peroxide . It is a metalloenzyme from the ferroprotoporphyring group of peroxidases. It has a molar mass of around 44 kDa , which is made up of the protein component (33,890 Dalton), hemin and Ca 2+ ions (around 700 Dalton) and the glycosylation (9,400 Dalton). For the catalyzed redox reaction , heme is used as a cofactor in the active center .

The HRP is inactivated to a small extent by the dismutation of hydrogen peroxide, whereas nitroxides such as 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) or 4-OH-TPO are added. The stability of the HRP decreases in the presence of phosphates . The optimal pH range for maximum enzyme activity is between pH 6 and 6.5. At pH 7.5 the enzyme activity is 84% ​​of the maximum value. The protein folding and the enzyme activity is stable between pH 5 and. 9

Horseradish peroxidase exists in seven isoenzymes with isoelectric points between pH 3 and pH 9. The isoenzymes also differ in their glycosylation and the proportions of galactose , arabinose , xylose , fucose , mannose , mannosamine and galactosamine .

Inhibitors

HRP inhibitors are sodium azide , cyanide , L– cystine , dichromate , ethylene thiourea , hydroxylamine , sulfide , vanadate , p-aminobenzoic acid , Cd 2+ , Co 2+ , Cu 2+ , Fe 3+ , Mn 2+ , Ni 2+ and Pb 2+ .

Extraction

Horseradish peroxidase is industrially isolated from horseradish . In the protein purification of the HRP, the purity number (RZ) is determined as the ratio of the extinctions at the wavelengths 403 and 275 nm. This determines the heme content, which is not necessarily associated with high enzyme activity. The RZ should be above 3 for the HRP for coupling to antibodies. The coupling methods for horseradish peroxidase include oxidation with sodium periodate and reductive amination with sodium cyanoborohydride (on the glycosylations ), the reaction with sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) and 2-mercaptoethylamine or the reaction with SMCC, N-succinimidyl-S-acetylthioacetate (SATA) and succinimidyl 3- (2-pyridyldithio) propionate (SPDP) was used.

Production of the HRP as a recombinant protein in E. coli leads to yields that result in higher production costs.

use

HRP is used to produce optically active hydroperoxides . It is also used in immunostaining as part of an immunoconjugate that z. B. in immunohistochemical techniques in histology , in ELISA , in Western Blot and in ELISPOT . The HRP has six Lysine which the molecular marker can be used. In addition, a molecule can also be coupled to cysteines in four disulfide bridges of the HRP. Periodic acid and glutaraldehyde , among others, are used as crosslinkers between HRP and other proteins . The horseradish peroxidase is hydrogen peroxide offered as one of the two substrates. From the splitting of the hydrogen peroxide, the previously almost colorless chromogen is oxidized to its colored end product.

Alternatively, alkaline phosphatase (AP) and β-galactosidase (βGal) are used as reporter enzymes. Compared to the AP, the HRP is smaller, more stable and cheaper. The HRP also has a higher turnover rate than both alternatives, which results in higher product formation rates, which are often desired with chemiluminescence. Due to the glycosylation of the HRP, fewer unspecific protein-protein interactions arise due to hydrophobic effects .

Furthermore, the HRP for the oxidation of aromatic compounds with hydroxyl groups from industrial wastewater is investigated.

Substrates

different substrates of HRP

The chemiluminescence reaction of luminol or other dioxetanes can be catalyzed by this peroxidase. In addition, the HRP forms colored or fluorescent reaction products with various chromogens .

Substrates for chemiluminescence

Substrates for fluorescence

Substrates for color reactions

literature

  • NC Veitch: Horseradish peroxidase: a modern view of a classic enzyme. In: Phytochemistry . 65 (3), February 2004, pp. 249-259. doi: 10.1016 / j.phytochem.2003.10.022 . PMID 14751298 .
  • AM Azevedo, VC Martins, DM Prazeres, V. Vojinović, JM Cabral, LP Fonseca: Horseradish peroxidase: a valuable tool in biotechnology. In: Biotechnology annual review. Volume 9, 2003, pp. 199-247, PMID 14650928
  • JA Akkara, KJ Senecal, DL Kaplan: Synthesis and characterization of polymers produced by horseradish peroxidase in dioxane. In: Journal of Polymer Science. 29 (11), October 1991, pp. 1561-1574. doi: 10.1002 / pola.1991.080291105 .
  • YP Chau, KS Lu: Investigation of the blood-ganglion barrier properties in rat sympathetic ganglia by using lanthanum ion and horseradish peroxidase as tracers. In: Acta Anatomica . 153 (2), 1995, pp. 135-144. doi: 10.1159 / 000313647 . PMID 8560966 .
  • S. Avrameas, T. Ternynck: Peroxidase labeled antibody and Fab conjugates with enhanced intracellular penetration. In: Immunochemistry. Volume 8, Number 12, December 1971, pp. 1175-1179, PMID 5150003 .
  • PK Nakane, A. Kawaoi: Peroxidase-labeled antibody. A new method of conjugation. In: The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society. Volume 22, Number 12, December 1974, pp. 1084-1091, doi : 10.1177 / 22.12.1084 , PMID 4443551 .
  • JW Lichtman, D. Purves: Cell marking with horseradish peroxidase . Principles of Neural Development. Sinauer Associates, Sunderland, Mass 1985, ISBN 0-87893-744-7 , p. 114.

Individual evidence

  1. ↑ Carlsson GH, Nicholls P, Svistunenko D, Berglund GI, Hajdu J: Complexes of horseradish peroxidase with formats, acetate, and carbon monoxide . In: Biochemistry . 44, No. 2, Jan 2005, pp. 635-42. doi : 10.1021 / bi0483211 . PMID 15641789 .
  2. ↑ Gajhede M, Schuller DJ, Henriksen A, Smith AT, Poulos TL: Crystal structure of horseradish peroxidase C at 2.15 A resolution . In: Nature Structural Biology . 4, No. 12, Dec 1997, pp. 1032-8. doi : 10.1038 / nsb1297-1032 . PMID 9406554 .
  3. ↑ Henry Delincee, Bertold J. Radola: Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion exchange chromatography. In: European Journal of Biochemistry. 52, 1975, p. 321, doi : 10.1111 / j.1432-1033.1975.tb04000.x .
  4. ↑ A. Samuni, E. Maimon, S. Goldstein: Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity. In: Biochimica et Biophysica Acta . [electronic publication before printing] March 2017, doi : 10.1016 / j.bbagen.2017.03.021 , PMID 28365302 .
  5. ^ L. Haifeng, L. Yuwen, C. Xiaomin, W. Zhiyong, W. Cunxin: Effects of sodium phosphate buffer on horseradish peroxidase thermal stability. In: Journal of Thermal Analysis and Calorimetry. 93, 2008, p. 569, doi : 10.1007 / s10973-007-8407-y .
  6. ↑ Sedigheh Asad, Seyed-Fakhreddin Torabi, Mehrnoosh Fathi-Roudsari, Nasser Ghaemi, Khosro Khajeh: Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase. In: International Journal of Biological Macromolecules. 48, 2011, p. 566, doi : 10.1016 / j.ijbiomac.2011.01.021 .
  7. ↑ D. Schomburg, M. Salzmann, D. Stephan (editors): Enzyme Handbook 7 , (1993). Chapter EC 1.11.1.7:1–6. ISBN 978-3-642-48964-8 .
  8. ↑ a b L. M. Shannon, E. Kay, JY Lew: Peroxidase isozymes from horseradish roots. I. Isolation and physical properties. In: The Journal of biological chemistry. Volume 241, Number 9, May 1966, pp. 2166-2172, PMID 5946638 .
  9. ↑ Helmward Zollner: Handbook of Enzyme Inhibitors , 2nd Edition, Part A, pp. 367-368 (1993).
  10. ↑ T. Gundinger, O. Spadiut: A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli. In: Journal of biotechnology. [electronic publication before printing] March 2017, doi : 10.1016 / j.jbiotec.2017.03.003 , PMID 28288816 .
  11. ^ VG Grigorenko, IP Andreeva, MY Rubtsova, AM Egorov: Recombinant horseradish peroxidase: production and analytical applications. In: Biochemistry. Biokhimiia. Volume 80, Number 4, April 2015, pp. 408-416, doi : 10.1134 / S0006297915040033 , PMID 25869357 .
  12. ^ O. Ryan, MR Smyth, CO Fágáin: Horseradish peroxidase: the analyst's friend. In: Essays in biochemistry. Volume 28, 1994, pp. 129-146, PMID 7925315 .
  13. ^ Edward A. Greenfield: Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratories, 1988. ISBN 0-87969-314-2 . Pp. 346-348.
  14. ↑ Beyzavi K, Hampton S, Kwasowski P, Fickling S, Marks V, Clift R: Comparison of horseradish peroxidase and alkaline phosphatase-labeled antibodies in enzyme immunoassays . In: Annals of Clinical Biochemistry . 24 (Pt 2), Mar 1987, pp. 145-52. PMID 3035992 .
  15. ↑ SS Deshpande: Enzyme Handbook 7: Class 1.5-1.12: Oxidoreductases . Springer, 1994, ISBN 978-3-642-48964-8 . Pp. 169-171.
  16. ↑ Salehe Ghasempur, Seyed-Fakhreddin Torabi, Seyed-Omid Ranaei-Siadat, Mehdi Jalali Heravi, Nasser Ghaemi, Khosro Khajeh: Optimization of peroxidase-Catalyzed Oxidative Coupling Process for Phenol Removal from Wastewater Using Response Surface Methodology . In: Environmental Science & Technology . 41, No. 20, October 1, 2007, ISSN  0013-936X , pp. 7073-7079. doi : 10.1021 / es070626q .
  17. ↑ YL Kapeluich ,. Rubtsova MYu, AM Eg: Enhanced chemiluminescence reaction applied to the study of horseradish peroxidase stability in the course of p-iodophenol oxidation. In: Journal of bioluminescence and chemiluminescence. Volume 12, Number 6, 1997 Nov-Dec, pp. 299-308, doi : 10.1002 / (SICI) 1099-1271 (199711/12) 12: 6 <299 :: AID-BIO459> 3.0.CO; 2-S , PMID 9509338 .
  18. ^ A b C. Haan, I. Behrmann: A cost effective non-commercial ECL solution for Western blot detections yielding strong signals and low background. In: Journal of immunological methods. Volume 318, Number 1-2, January 2007, pp. 11-19, doi : 10.1016 / j.jim.2006.07.027 , PMID 17141265 .