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Relationship between the heme active site of Arthromyces ramosus peroxidase and its oxidation activity for luminol
HEME IRON AND METALLOPORPHYRINS 83
Journal of Inorganic Biochemistry
D06
R E L A T I O N S H I P B E T W E E N THE H E M E ACTIVE SITE OF ARTHROMYCES
R A M O S U S P E R O X I D A S E AND ITS O X I D A T I O N
ACTIVITY FOR L U M I N O L M. Tanaka, K. Ishimori and I. M o r i s h i m a
Deapartment of Molecular Engineering, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-01, Japan Horseradish peroxidase (HRP), a prototypical heme peroxidase, catalyzes oxidation of small aromatic molecules by use of hydrogen peroxide as an oxidant. The oxidation reaction for luminol by HRP has an important implication in analytical chemistry.
HRP has been extensively utilized in
immunoassays to quantitate its conjugating analytes such as digoxin, rubella virus-specific antibody and thyroxine by chemiluminescence of oxidation product of luminol. Recently, it has been revealed that
Arthromyces ramosus peroxidase (ARP) has much high oxidation activity for luminol by more than 500 factors relative to HRP [ 1], indicating avaliability to detection of a smaller amount of analytes. Although X-ray crystal structure of ARP has been solved2), it has not clarified what factors enhance the oxidation activity for luminol. Thus, in order to elucidate the relationship between the heme active site of ARP and its high oxidation activity for luminol, we examined heine environmental structure in a solution with or without luminol (0.8 mM) by CD and 1H-NMR spectroscopies and compared the structural properties of ARP with those of HRP. By addition of luminol, the molar ellipticity for the Soret region was decreased in ferric ARP, whereas HRP exhibited no significant changes. The decreased molar ellipticity indicates that the chirality around the heme was perturbed by luminol, suggesting that luminol can approach to the heme in ARP. To get further insight into the effect of lunimol on the heme environmental structure, we measured 1HNMR spectra of ARP.
In the presence of luminol, the positions of resonances from heme peripheral
groups in ferric ARP were shifted by 1 - 2 ppm, compared to those in the absence of luminol. 1H-NMR spectrum of cyanide-bound ARP also showed that addition of luminol induces a small but significant upfield shift (0.5 ppm) of the signal from the heme 8-methyl group.
In the X-ray crystal structure of
ARP [2], the 8-methyl group is located near the putative access channel of substrate to the heme. These observations suggest that luminol would be located near the 8-methyl group and interact with the heme edge around the 8-methyl group in ARP. Therefore, we can conclude such a accessibility of luminol to the active site could improve the oxidation activity for luminol in ARP. 1.
K. Akimoto, Y. Shinmen, M. Sumida, S. Asami, T. Amachi, H. Yoshizumi, Y. Saeki, S. Shimizu and H. Yamada, Anal. Biochem., 189, 182 (1990).
2.
N. Kunishima, K. Fukuyama, K. Matsubara, H. Hatanaka, Y. Shibano and T. Amachi, J. Mol.
Biol., 235, 331 (1994).
Journal of Inorganic Biochemistry
D06
R E L A T I O N S H I P B E T W E E N THE H E M E ACTIVE SITE OF ARTHROMYCES
R A M O S U S P E R O X I D A S E AND ITS O X I D A T I O N
ACTIVITY FOR L U M I N O L M. Tanaka, K. Ishimori and I. M o r i s h i m a
Deapartment of Molecular Engineering, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-01, Japan Horseradish peroxidase (HRP), a prototypical heme peroxidase, catalyzes oxidation of small aromatic molecules by use of hydrogen peroxide as an oxidant. The oxidation reaction for luminol by HRP has an important implication in analytical chemistry.
HRP has been extensively utilized in
immunoassays to quantitate its conjugating analytes such as digoxin, rubella virus-specific antibody and thyroxine by chemiluminescence of oxidation product of luminol. Recently, it has been revealed that
Arthromyces ramosus peroxidase (ARP) has much high oxidation activity for luminol by more than 500 factors relative to HRP [ 1], indicating avaliability to detection of a smaller amount of analytes. Although X-ray crystal structure of ARP has been solved2), it has not clarified what factors enhance the oxidation activity for luminol. Thus, in order to elucidate the relationship between the heme active site of ARP and its high oxidation activity for luminol, we examined heine environmental structure in a solution with or without luminol (0.8 mM) by CD and 1H-NMR spectroscopies and compared the structural properties of ARP with those of HRP. By addition of luminol, the molar ellipticity for the Soret region was decreased in ferric ARP, whereas HRP exhibited no significant changes. The decreased molar ellipticity indicates that the chirality around the heme was perturbed by luminol, suggesting that luminol can approach to the heme in ARP. To get further insight into the effect of lunimol on the heme environmental structure, we measured 1HNMR spectra of ARP.
In the presence of luminol, the positions of resonances from heme peripheral
groups in ferric ARP were shifted by 1 - 2 ppm, compared to those in the absence of luminol. 1H-NMR spectrum of cyanide-bound ARP also showed that addition of luminol induces a small but significant upfield shift (0.5 ppm) of the signal from the heme 8-methyl group.
In the X-ray crystal structure of
ARP [2], the 8-methyl group is located near the putative access channel of substrate to the heme. These observations suggest that luminol would be located near the 8-methyl group and interact with the heme edge around the 8-methyl group in ARP. Therefore, we can conclude such a accessibility of luminol to the active site could improve the oxidation activity for luminol in ARP. 1.
K. Akimoto, Y. Shinmen, M. Sumida, S. Asami, T. Amachi, H. Yoshizumi, Y. Saeki, S. Shimizu and H. Yamada, Anal. Biochem., 189, 182 (1990).
2.
N. Kunishima, K. Fukuyama, K. Matsubara, H. Hatanaka, Y. Shibano and T. Amachi, J. Mol.
Biol., 235, 331 (1994).