Detailed circular dichroism and fluorescence studies at different pHs have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme c (HRPc). The change in CD in the 222 nm region corresponds to changes in the overall secondary structure of the enzyme, while that in the 400 nm region (Soret region) corresponds to changes in the tertiary structure around the heme in the enzyme. The temperature dependence of the tertiary structure around the heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme. The results suggested that melting of the tertiary structure to a pre-molten globule form takes place at 45 degrees C, which is much lower than the temperature (T(m) = 74 degrees C) at which depletion of heme from the heme cavity takes place. The melting of the tertiary structure was found to be associated with a pK(a) of approximately 5, indicating that this phase possibly involves breaking of the hydrogen-bonding network of the heme pocket, keeping the heme moiety still inside it. The stability of the secondary structure of the enzyme was also found to decrease at pH below 4.5. A 'high temperature' unfolding phase was observed which was, however, independent of pH. The stability of the secondary structure was found to drastically decrease in the presence of DTT (dithiothreitol), indicating that the 'high temperature' form is possibly stabilized due to interhelical disulfide bonds. Depletion of Ca(2+) ions resulted in a marked decrease in the stability of the secondary structure of the enzyme. Study holds ProTherm entries: 7900 Extra Details: DTT (30 mM) was added in the experiment secondary structure; tertiary structure; heme cavity;,interhelical disulfide bonds
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:35 p.m.
|Number of data points||1|
|Proteins||Peroxidase C2 ; Peroxidase C1A|
|Assays/Quantities/Protocols||Experimental Assay: Tm|
|Libraries||Mutations for sequence QLTPTFYDNSCPNVSNIVRDTIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESACPRTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRVVNS|