Chimeric antibody-binding Vitreoscilla hemoglobin (VHb) mediates redox-catalysis reaction: new insight into the functional role of VHb.
Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric Vitreoscilla hemoglobin (VHb). Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain) were successfully constructed and expressed in E. coli strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 10(7) and 49.1 x 10(7) per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (approximately 7.0) in the presence of high concentration (20-40 mM) of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of Vitreoscilla hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications.
Suwanwong
Yaneenart
Y
Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand.
Kvist
Malin
M
Isarankura-Na-Ayudhya
Chartchalerm
C
Tansila
Natta
N
Bulow
Leif
L
Prachayasittikul
Virapong
V
D000907:Antibodies, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D003001:Cloning, Molecular; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D006418:Heme / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D020349:Surface Plasmon Resonance; D054793:Truncated Hemoglobins / Q000235:genetics / Q000276:immunology*; D020589:Vitreoscilla / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D006418:Heme