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Buffer-anion-dependent Ca2+ leaching from horseradish peroxidase at low pH
1Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec H3G 1M8, Canada.
Summary
Heterogeneous glycosylation in horseradish peroxidase isozyme C (HRPC) affects its low-pH stability and activity. Buffer anions influence calcium ion leaching, impacting structural integrity and enzyme function.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Plant peroxidases display diverse pH optima despite conserved active sites.
- Horseradish peroxidase isozyme C (HRPC) is a model for studying pH stability.
- Conflicting data exists regarding HRPC's low-pH stability and distal histidine protonation.
Purpose of the Study:
- To investigate the structural basis for pH stability differences in HRPC.
- To resolve discrepancies in HRPC's low-pH behavior.
- To analyze acid-induced changes in HRPC from different commercial sources.
Main Methods:
- Fourier-transform infrared (FTIR) spectroscopy (v(CO) and amide I' bands).
- Circular dichroism (CD) spectroscopy (Soret and far-UV regions).
- Analysis of HRPC from two commercial suppliers at pH 3.0 in various buffers.
Main Results:
- HRPC samples showed distinct spectral profiles at pH 3.0, indicating varied heme-pocket accessibility.
- Buffer anions (citrate, phosphate, formate) influenced spectral changes, correlating with Ca2+ leaching.
- Heterogeneous N-glycosylation patterns, detected by SDS-PAGE, were linked to differences in low-pH conformational stability.
Conclusions:
- Buffer anions promote Ca2+ leaching from HRPC at low pH, affecting stability.
- Variations in HRPC glycosylation are a key factor in its differential low-pH conformational stability.
- Glycosylation patterns may similarly influence the pH stability of other plant peroxidase classes.