A step towards understanding the folding mechanism of horseradish peroxidase. Tryptophan fluorescence and circular dichroism equilibrium studies.


Abstract

The guanidinium chloride denaturation/renaturation of the holo- and apo-horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies. A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo-HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem. The denaturation of the holo-HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed. Ca2+ appears to be important for the stability of the protein as the apo-HRP is more resistant to denaturation in the presence of Ca2+. The free-energy change during unfolding of the apo-HRP was determined in the absence and presence of Ca2+ and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Ca2+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin-IX-apo-HRP complex. Study holds ProTherm entries: 7575, 7576 Extra Details: equilibrium intermediate; secondary structure content;,native conformation; protoporphyrin-IX-apo-HRP complex

Submission Details

ID: rfYL5BvX3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:34 p.m.

Version: 1

Publication Details
Pappa HS;Cass AE,Eur. J. Biochem. (1993) A step towards understanding the folding mechanism of horseradish peroxidase. Tryptophan fluorescence and circular dichroism equilibrium studies. PMID:8444158
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1GWU 2003-03-28 1.31 RECOMBINANT HORSERADISH PEROXIDASE C1A ALA140GLY
7ATJ 2000-01-14 1.47 RECOMBINANT HORSERADISH PEROXIDASE C1A COMPLEX WITH CYANIDE AND FERULIC ACID
1W4W 2005-01-19 1.55 Ferric horseradish peroxidase C1A in complex with formate
1HCH 2002-07-19 1.57 Structure of horseradish peroxidase C1A compound I
1H57 2002-06-17 1.6 Structure of horseradish peroxidase C1A compound III
1H5E 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (11-22% dose)
1H5K 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (78-89% dose)
1H5L 2002-06-21 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (89-100% dose)
1H5G 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (33-44% dose)
1H5A 2002-06-18 1.6 STRUCTURE OF FERRIC HORSERADISH PEROXIDASE C1A IN COMPLEX WITH ACETATE
1H5H 2002-06-21 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (44-56% dose)
1H5J 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (67-78% dose)
1H5I 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (56-67% dose)
1H5D 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (0-11% dose)
1H5M 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (0-100% dose)
1W4Y 2005-01-19 1.6 Ferrous horseradish peroxidase C1A in complex with carbon monoxide
1H5F 2002-05-27 1.6 X-ray induced reduction of horseradish peroxidase C1A Compound III (22-33% dose)
1H55 2002-06-18 1.61 STRUCTURE OF HORSERADISH PEROXIDASE C1A COMPOUND II
1H5C 2002-06-18 1.62 X-ray induced reduction of horseradish peroxidase C1A Compound III (100-200% dose)
2YLJ 2012-06-13 1.69 Horse Radish Peroxidase, mutant S167Y
1GWT 2003-03-28 1.7 RECOMBINANT HORSERADISH PEROXIDASE C1A PHE221MET
1H58 2002-06-18 1.7 STRUCTURE OF FERROUS HORSERADISH PEROXIDASE C1A
1KZM 2002-10-09 2.0 Distal Heme Pocket Mutant (R38S/H42E) of Recombinant Horseradish Peroxidase C (HRP C).
6ATJ 2000-01-14 2.0 RECOMBINANT HORSERADISH PEROXIDASE C COMPLEX WITH FERULIC ACID
2ATJ 1998-01-28 2.0 RECOMBINANT HORSERADISH PEROXIDASE COMPLEX WITH BENZHYDROXAMIC ACID
1GWO 2003-03-28 2.07 Recombinant horseradish peroxidase C1A ALA170GLN
1GW2 2003-03-28 2.15 RECOMBINANT HORSERADISH PEROXIDASE C1A THR171SER IN COMPLEX WITH FERULIC ACID
1ATJ 1998-02-04 2.15 RECOMBINANT HORSERADISH PEROXIDASE C1A
1GX2 2003-03-28 2.2 Recombinant horseradish peroxidase Phe209Ser complex with benzhydroxamic acid
3ATJ 1999-04-28 2.2 HEME LIGAND MUTANT OF RECOMBINANT HORSERADISH PEROXIDASE IN COMPLEX WITH BENZHYDROXAMIC ACID
4ATJ 2002-10-02 2.5 DISTAL HEME POCKET MUTANT (H42E) OF RECOMBINANT HORSERADISH PEROXIDASE IN COMPLEX WITH BENZHYDROXAMIC ACID

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Peroxidase C2 P17179 PER2_ARMRU
90.8 Peroxidase C1A P15232 PER1B_ARMRU
91.2 Peroxidase C1A P15233 PER1C_ARMRU
91.2 Peroxidase C1A P24101 PER33_ARATH
93.1 Peroxidase C1A Q9SMU8 PER34_ARATH
100.0 Peroxidase C1A P00433 PER1A_ARMRU