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
1ATJ 1997-08-14T00:00:00+0000 2.15 RECOMBINANT HORSERADISH PEROXIDASE C1A
1GW2 2002-03-03T00:00:00+0000 2.15 RECOMBINANT HORSERADISH PEROXIDASE C1A THR171SER IN COMPLEX WITH FERULIC ACID
1GWO 2002-03-20T00:00:00+0000 2.07 Recombinant horseradish peroxidase C1A ALA170GLN
1GWT 2002-03-25T00:00:00+0000 1.7 RECOMBINANT HORSERADISH PEROXIDASE C1A PHE221MET
1GWU 2002-03-25T00:00:00+0000 1.31 RECOMBINANT HORSERADISH PEROXIDASE C1A ALA140GLY
1GX2 2002-03-26T00:00:00+0000 2.2 Recombinant horseradish peroxidase Phe209Ser complex with benzhydroxamic acid
1H55 2001-05-18T00:00:00+0000 1.61 STRUCTURE OF HORSERADISH PEROXIDASE C1A COMPOUND II
1H57 2001-05-20T00:00:00+0000 1.6 Structure of horseradish peroxidase C1A compound III
1H58 2001-05-20T00:00:00+0000 1.7 STRUCTURE OF FERROUS HORSERADISH PEROXIDASE C1A
1H5A 2001-05-21T00:00:00+0000 1.6 STRUCTURE OF FERRIC HORSERADISH PEROXIDASE C1A IN COMPLEX WITH ACETATE

Relevant UniProtKB Entries

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