Buffer-anion-dependent Ca2+ leaching from horseradish peroxidase at low pH.
Despite highly conserved active-site structures, members of the plant peroxidase superfamily exhibit a wide range of pH optima. Horseradish peroxidase isozyme C (HRPC) is an ideal peroxidase to investigate the structural determinants of pH stability and activity in superfamily members. Conflicting reports exist on the low-pH stability of HRPC and consequently the pKa of the catalytic distal histidine, which is neutral in active peroxidases. Towards resolving such discrepancies, acid-induced changes in HRPC from two popular commercial suppliers were systematically analyzed. Specifically, FTIR v(CO) and Soret-CD spectra of HRPC-CO and Soret absorption of ferric HRPC were recorded to probe time-dependent heme-pocket changes at pH 3.0 in phosphate, citrate and formate buffers, while the FTIR amide I' and far-UV CD spectra were examined to probe changes in secondary structure. Both HRPC-CO samples exhibited identical pH 7.0 v(CO) bands at 1934 and 1905 cm-1. In the pH 3.0 spectrum of sample A, the 1934 cm-1 band was dominant while a broad 1969 cm-1 band appeared in sample B. The intensity of this band, which is assigned to solvent-exposed heme, was greater in citrate than phosphate buffer, but in formate the 1934 cm-1 band remained dominant. Other spectral changes mirrored the v(CO) trends. No time- or buffer-anion-dependent conformation changes were detected in 1 mM CaCl2, revealing that buffer-anion-dependent leaching of stabilizing Ca2+ from HRPC occurs at pH 3.0. Since the N-glycans present in HRPC are of the flexible protein-surface-shielding type, the variation in low-pH conformational stability of the HRPC samples could be attributed to heterogeneous glycosylation, which was detected by SDS-PAGE. It is further proposed that glycosylation patterns may affect the low-pH stability of class II and III plant peroxidases.
Wright
P J
PJ
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec H3G 1M8, Canada.
English
A M
AM
D000838:Anions; D002021:Buffers; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002122:Calcium Chloride / Q000737:chemistry; D002942:Circular Dichroism; D019343:Citric Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005561:Formates / Q000737:chemistry; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010710:Phosphates / Q000737:chemistry; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
D000838:Anions; D002021:Buffers; D005561:Formates; D010710:Phosphates; C030544:formic acid; D019343:Citric Acid; D006735:Horseradish Peroxidase; D002122:Calcium Chloride; D002118:Calcium