Introduction of a specific binding domain on myoglobin surface by new chemical modification.
A new myoglobin, reconstituted with a modified zinc protoporphyrin, having a total of four ammonium groups at the terminal of the two propionate side chains was constructed to introduce a substrate binding site. The protein with a positively charged patch on the surface formed a stable complex with negatively charged substrates, such as hexacyanoferrate(III) and anthraquinonesulfonate via an electrostatic interaction. The complexation was monitored by fluorescence quenching due to singlet electron transfer from the photoexcited reconstituted zinc myoglobin to the substrates. The binding properties were evaluated by Stern-Volmer plots from the fluorescence quenching of the zinc myoglobin by a quencher. Particularly, anthraquinone-2,7-disulfonic acid showed a high affinity with a binding constant of 1.5 x 10(5) M(-1) in 10 mM phosphate buffer, pH 7.0. In contrast, the plots upon the addition of anthraquinone-2-sulfonic acid at different ionic strengths indicated that the complex was formed not only by an electrostatic interaction but also by a hydrophobic contact. The findings from the fluorescence studies conclude that the present system is a useful model for discussion of electron transfer via non-covalently linked donor-acceptor pairing on the protein surface.
Hayashi
T
T
Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka, Japan. thayatcm@mbox.nc.kyushu-u.ac.jp
Ando
T
T
Matsuda
T
T
Yonemura
H
H
Yamada
S
S
Hisaeda
Y
Y
D000880:Anthraquinones / Q000378:metabolism; D001665:Binding Sites; D005295:Ferrocyanides / Q000378:metabolism; D005453:Fluorescence; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D011524:Protoporphyrins / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D000880:Anthraquinones; D005295:Ferrocyanides; D009211:Myoglobin; D011524:Protoporphyrins; D000644:Quaternary Ammonium Compounds; C116383:ferrocyn; D015032:Zinc