An investigation of the peroxidase activity of Vitreoscilla hemoglobin.
In order to investigate the ability of the Vitreoscilla hemoglobin (VHb) to act as a peroxidase, the protein was overexpressed in Escerichia coli and purified using a 6xHis-tag. The peroxidase activity of VHb was studied using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferrocene carboxylic acid (FcCOOH) dopamine and L-dopa as substrates. The effects of external agents such as pH, salt concentration/ionic strength, and the thermal stability of VHb on the catalytic activity were assessed. The optimum pH for VHb using ABTS as a substrate was estimated to be 6-7. The VHb protein proved to be stable up to 80 degrees C, as judged by its peroxidase activity. Furthermore, NaCl concentrations up to 100 mM did not exert any significant effect on the activity. The catalytic activity against ABTS and FcCOOH was similar to that measured for horseradish peroxidase, whereas in the case of the phenolic substrates dopamine and L-dopa the activity was several orders of magnitude lower. The Michaelis constants, KmH2O2, were in good agreement with the data for human and bovine hemoglobin. No activity could be detected for the negative controls lacking VHb. These results demonstrate that VHb exhibits peroxidase activity, a finding in line with the hypothesis that VHb has cellular functions beyond the role as an oxygen carrier.
Kvist
Malin
M
Center for Chemistry and Chemical Engineering, Pure and Applied Biochemistry, Lund Institute of Technology, Box 124, 221 00, Lund, Sweden.
Ryabova
Ekaterina S
ES
Nordlander
Ebbe
E
Bülow
Leif
L
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D052160:Benzothiazoles / Q000737:chemistry; D002384:Catalysis; D004298:Dopamine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005296:Ferrous Compounds / Q000737:chemistry; D006454:Hemoglobins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D007980:Levodopa / Q000737:chemistry; D000075163:Metallocenes; D010544:Peroxidases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000737:chemistry; D054793:Truncated Hemoglobins
D001426:Bacterial Proteins; D052160:Benzothiazoles; D005296:Ferrous Compounds; D006454:Hemoglobins; D000075163:Metallocenes; D011994:Recombinant Proteins; D013451:Sulfonic Acids; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; C032949:ferrocenecarboxylic acid; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D007980:Levodopa; D010544:Peroxidases; D004298:Dopamine